NONPOLAR CONTRIBUTIONS TO CONFORMATIONAL SPECIFICITY IN ASSEMBLES OF DESIGNED SHORT HELICAL PEPTIDES
Chandra Lynn Boon
A thesis subrnitted in conformity with the requirernents for the degree of Master of Science Graduate Department of Medicai Biophysics University of Toronto
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Chandra Lynn Boon
Master of Science 2000
Graduate Department of Medical Biophysics
University of Toronto
Abstract
A series of designed short helical peptides was used to study the effect of nonpolar interactions on conformational specificity. A consensus sequence was designed to contain a heptad repeat (abcdefg), yielding short helices (17 residues), with minimal interhelical polar interactions. Heptad positions a and d were occupied by al1 possible combinations of the hydrophobie residues Leu, Ile, or Val, and positions e and g were occupied by Ala, yielding a series of nine peptides. An experimental methodology was developed to characterize the nine peptides and their pairwise mixtures; the results indicated that a vast arny of structural states were formed possessing different degrees of conformational specificity. In other work, the control of specificity has been linked to polar interactions.
This work demonstrated that subtie changes in the configuration of nonpolar interactions can have a significant effect on the conformational specificity of oiigomenc short helices. Acknowledgements
1 am very grateful to my supervisor, Dr. Avijit Chakrabartty, for taking the chance with me and for teaching me how to pipette! 1 have really enjoyed "brainstorming" sessions with him and 1 find his enthusiasm for thinking and learning very contagious. 1 look fonvard to many interesting discussions in the future (even though these discussions tend io draw out lab meetings!).
T would like to thank the members of my supervisory cornmittee: Dr. David Rose, Dr. Gil Pnve, and Dr. Dwayne Barber for their interest, guidance, and challenging questions.
1 want to thank Kevin Galley and Thomas Goldthorpe from Research Information Systems for al1 their patience and assistance with my thesis and my defense presentation.
Past and present members of the Chakrabartty lab have been a great help. Thank you to Xiao Fei Qi for his help with the NMR data. 1 want to thank Paul Gorman for his friendship and support, and for being so much fun! 1 owe infinite thanks to Sandy Go for her patience while teaching me ALL of my lab techniques (1'11 never forget the pasteur pipette incident!). 1 also appreciate her friendship, sense of humour, and her talent for organizing surprise parties! 1 am very gnteful to Cynthia Qum for being a good friend and for al1 her support, especially during the wnting process. We still have to go for bubble tea! Thank you to JoAnne McLaunn for her support and guidance early in my Master's and for her friendship.
During the course of my Master's, 1 experienced some difficulties that I could never have overcome without the help of my fnenâs. 1 would like to thank (in no particular order) Tim Davison, Brenda Rutherford, Linda Mark, Rey Interior, Tarek Harb, Kevin Laiiberte, Jason Maydan, Barbara Guinn, Seishi Shimizu, Shireen Khi, Sandy Batten, George Jones, Jim and Nancy Kost, and Jason Hinek for being so supportive and encouraging. 1 feel extremely forninate to have such amazing fnends! 1 would also like to Say a special thank you to Elyssa Elton and Judi Bechard. E!yssa, 1 can't properly express how much your friendship has meant to me. Your kindness and suppon helped so much. You are a fabulous friend and 1 have great respect for you. Judi, what can 1 say? Thank you for being such a true friend, dl these yean! Your encouragement and your belief in me were a great help, especially right before my defence.
Lastly, 1would like to thank my family. 1want to thank my parents, Henry and Mary, for their love and support through al1 these years of school. 1 am so grateful to them for allowing me to explore my own interests and for always encounging me to pursue whatever 1 wanted. 1 also want to thank my sister, Khrista, for that 230 am phone conversation two weeks before my defence - thank you for being there for me. TABLE OF CONTENTS
LIST OF TABLES ...... v LIST OF FIGURES ....,...... t.....l...... t...... v
.. LEXICON .." ... t.w.C....we..~"....~U...... œ...... H...... VU
CHAPTER 1: INTRODUCTION ...w...w.HH...... mHc.t,..-m." ...... -...... -. 1 THE CL-HELIX ...... -3 ISOLATED HELR FORMATION...... 3 DESIGNOF CX-HELICALBUNDLES ...... 10
Asm~cr...... 19 INTRODUCTION ...... 20 TERIALS AND METHODS...... 23 Peptide Synthesis and Purification...... 23 Peptide Concentration Determination ...... 33 Circulrir Dichroism Spectmscopy...... 24 Fluorescence Specuoscopy...... 24 SolubiIity Measurements ...... 3 Light Scattering...... 3 Sedimentation Equilibrium Ultracentrifugation ...... 36 Assessrnent of Pimile1 venus Antiparallei Alignment of Heiices in Helical Bundles Through Disuifide Cross-linking ...... 77 Amide Proton Exchmge Rate Mesisurernents ...... 28 RESULTS ...... 29 Peptide Design ...... 29 Determining Presence of SpeciîÏc Oligomen in Peptide Mixtures ...... 33 OIigornenzrition Strrte ...... 39 Stability of the adeg Oiigornen ...... -Ml Assessrnent of Pimilel versus Antipdlel iüignment of Helices in rideg Oligomers ...... 44 Amide Proton Exchange Kinetics of vlaa ...... 51 Discuss~o~...... 55 CONCLUSIONS...... 60
CHAPTER 3: FUTURE WORK- ...... -.t...cmM«.....m+...mmm..mtm+.t...t,-..HHIH.m+H..... STRU~RALBASIS FOR CONFORMATIONALSPECF~C~ EN THE adeg PEJlïDES ...... 62 THENEXT DESIGN ...... 68 REFERENCES ...... -..-...... -...... -. .-70
APPENDIX: ADAPTATION OF THE HILL EQUATION TO OLIGOMERIZATION OF a- HELICES -....- ...... U.*.«-..WII...... W...... rm -- 78 List of Tables TABLE: SEQUENCES OF THE adeg PEPTIDES...... 30 TABLE2: SEQUENCESOF PEPTIDES USEDFOR ALIGNMENT DETERM~NATION...... 50 TABLE3: ASSESSWG PARALLELVERSUS ANTIPARALLELALIGNMENT OF adeg HELICrU. BUNDLES...... 52 TABLE4: COMPARISONOF SPECIFICITYPA~RNS ...... 58 TABLE5: SEQUENCEALENMENT OF iiaa AND ONE HELK OF THE 4HB 1 PROTEiN ...... 66 List of Figures FIGURE1. HELICALWHEEL DIAGRAM OF hl...... 31 FIGURE2 . SCREYC:::~ Tws...... 30 FIGURE3. CIRCUWRD KHROISM SPECTW OF THE rideg PElTiDES...... 38 FIGURE4 . SEDIMWTATIONEQU~LIBRIUM ULTRACENTRINGATION OF iiriâ, lia, AND vlari...... *...... 41 FIGURE5 . CONCENTRATIONDEPENDENCE OF THE HELLX CONTENT OF THE adeg PEfl'IDES ...... 43 FIGURE6. STABIL~TYOF iim, dari. AND hari OLIGOMERS ...... 45 FIGURE7 . ASSESSMENT OF HELICAL ALIGNMENT ...... 48 FIGURE 8. AMIDE PROTON MCHANGE KiNETICS OF ...... 54 FIGURE9 . RIDGE GEOME~RYOF Ilriri. viari. AND iim...... 67 Abbreviations
ANS 1-anilinonapthalene-8-sulfonic acid
CD Circular Dichroism
DMF,
ES-MS
Fmoc
HATU O-(7-arabenzotriazol-1-y1)-1,1,3,3-tetramethyluronium hexafluorophosphate
HP H: hydrophobic, P: polar
HPLC High Performance Liquid Chromatopphy
PAL-PEG-PS 5-(arninomethyl-3',5'-dirnethoxyphenoxy)valeric acid polyethylene glycol polystyrene-graft copolymer
ultraviolet LEXICON amide proton exchange: CONHl + 2D -> CONDz + 2H, where D represents a deuteron; chemical exchange of hydrogens on amide groups with the solvent molecules. conformation: Three-dimensional arrangement of atoms; usually the energetically most stable arrangements, that are separated by distinct energy bamen. are defined as individual conformations. conformational entropy (ASmd): Therrnodynamic representation: AS,,, = R ln N, where N is the number of conformations possible. Conformational entropy is reduced when a polypeptide chah adopts a single conformation. conformational specificity: Specificity rnay be defined by the number of products of a particular reaction, e.g., in the case of peptide association, the presence of multiple oligomeric States corresponds to low specificity. Alternatively, stales with high conformational specificity have low conformational entropy. molten globule: A partly organized compact state which has most of the secondary structure of a native state; it is less compact than the native state and lacks proper packing interactions in its intenor. It may be viewed as an ensemble of related structures that are rapidly interconverting nther than a single structural state. number average molecular weight:
&MI
i where ni = c,/Mi which is the number of molecules or the mole concentration, and ci is the concentration of molecules. protection from amide proton exchange: Hydrogen atoms on amide groups in the intenor of exchange a protein do not readily undergo chemical exchange with solvent molecules and are thus considered to be "protected" from exchange. protection factor: The protection against exchange irnposed by protein structure is often expressed by a factor comparing the hydrogen exchange rate measured in a protein with the rate expected in a random coi1 k):P = k, 1 b. Rop: Repressor of primer; small four helix bundle protein whose function is to bind RNA and is involved in plasmid replication. sedimentation equilibriurn equation: A form of the Lamm equation which is a partial differential equation that describes mass transport in the ultracentrifuge (Le., in the presence of a centrifuga1 force). At equilibnum conditions there is no net movement of solute, so the Larnm equation may be simplified. For a single ided solute at equilibrium the movement of solute is descnbed by v al- --.~(t-;~).(r' - F') Cr = CF - e 2RT T where Cris the solute concentration at radius r. CF is the solute concentration at a reference distance F (the choice of F is arbitrary). o is the angular velocity (o= (2ir/6O)-RPM), R is the gas constant, and T is the temperature in Kelvin. By knowing Cras a function- of r at equilibrium, one usually seeks to estimate the buoyant rnolecular weight. ~(1-vp) . torsion angles: These angles are denoted by @, y, and w; angles about which the atoms in a polyppetide backbone may rotate. Rotation about the N-Ca bond is denoted by @; rotation about the Ca-C=O bond is denoted by W; land the rotation about the O=C-N bond is given by o.
viii CHAPTER 1: INTRODUCTION
The study of protein folding offen a scientist the opportunity to investigate one of the most intriguing problems of the last several decades. The protein folding problem is defined by the question, "How does the amino acid sequence of a prorein determine its three dimensional structure". Although Our understanding of the intricacies of this problem has grown, a general solution has not yet been found. One approach to investigating the protein folding problem involves the use of de novo designed proteins, whose sequences are not found in nature. Many interrelated parameters contribute to the structure of a natunlly occumng protein; since the sequence of a novel protein may be designed to contain a limited number of interactions, protein design offers an attractive means of simplification. Thus, protein design may be used to study the way one or more interactions contribute to protein folding (Kamtekar et al., 1993; Xiong et al., 1995:
Munson et al., 1996; Fairman et al., 1996; Zhu et al., 1993).
The most farnous element of secondary structure is the a-helix, in fact it "... is the classic element of protein structure" (Branden & Tooze, 1999). The fundamentals of a- helix formation in peptides have been studied extensively in order to gain insight into one aspect of the protein folding problem (Baldwin, 1995). In this work, the problem has been extended to helical assemblies. A simple system of de novo designed peptides was used to study the way nonpolar interactions affect conformational specificity in assemblies of short a-helices. In order to place the present work in context, this chapter reviews some of the previous studies on a-helix formation and designed helicai bundles, highiighting what has been leamed about protein folding. The achelix
In 1951, the a-helix was predicted by Pauling and Corey to be a stable structure. energetically favoured by arnino acid sequences. This prediction was Iater confirmed by x-ray diffraction experiments on protein crystals. Cantor and Schimmel eloquently descnbe the importance of the Pauling and Corey prediction. "In one of the most impressive feats of structural chemistry, Linus Pauling and Robert Corey postulated the exact structure of the cc-helix...six years before an a-helix was seen at molecular resolution for the fint time in the crystal structure of myoglobin." (Cantor & Schimmel,
1980).
The torsion angles @,y completely specify the backbone conformation of a protein. In a protein. a-helices occur when consecutive amino acids al1 have torsion angle values of (+,y)= (-60°,-50'). The average length of a-helices in proteins is approximately ten residues (Branden & Tooze, 1999). Further chancteristics of an a- helix include 3.6 residues per turn and a rise per residue of 1.5 A dong the helix mis, with a resulting pitch of 3.6 x 1.5 = 5.4 A. The helix diameter is 6 A, neglecting the amino acid sidechains. In addition, the a-helix contains an intricate hydrogen bonding pattern such that the carbonyl oxygen of residue i is bonded to the amide hydrogen of residue i4(Cantor & Schimmel, 1980). Al1 the hydrogen bonds (H-bonds) point in the same direction since al1 the H-bond donors have the same orientation (Darne11 et al.,
1995). Moreover, al1 the amide and carbonyl groups are H-bonded except for the first three peptide amide groups of the fmt turn of the helix and the last three carbonyl groups of the last tum. As a result, an a-heiix has a net dipole with a partial positive charge at the N-terminus and a partial negative charge at the C-terminus (Branden &Tooze, 1999). Recognition of the ubiquity of the a-heiix in protein structure led to extensive studies aimed at understanding al1 aspects of helix formation. The next section sumrnarizes w hat has been learned about a-helix formation in the last few decades.
Zsohed HelU: Formation
Factors important for protein stability have been elucidated through the study of peptide helices or isolated helices. Using peptide helices, it is possible to separate particulx interactions from other interactions present in a protein. Thus, peptide helices serve as an excellent system for quantitatively studying the factors that contribute to stability in protein helices and proteins in genenl (Scholtz & Baldwin. 1995). Early studies on poly- amino acid peptides revealed that poly-Lys (pH 11) and poly-Glu (pH 3) adopted stable helical conformations in aqueous media (Cantor & Schirnmel. 1980; Doty et al., 1957).
Doty et al. postulated that perhaps the helical conformation is generally a stable configuration for polypeptides of sufficient chah Iength, composed of L-amino acids in
the proper environment (Doty et al., 1957). In addition, in the 1960s it was found that tendencies of poly-amino acid peptides to form a-helices were correlated with amino acid
frequencies in helix-containing proteins (Chakrabartty & Baldwin, 1995). The idea that amino acids have an inherent predisposition toward helix formation arose from the above
work and inspired extensive expenmental work.
The structurai transition that occurs when an a-helix unfolds into a random coi1 is referred to as the helix-coil transition. Several models for the helix-coi1 transition were
developed in the 1950s and early 1960s using statistical mechanics to treat the transition
in terms of populations of molecules. When the entire peptide chain is taken into consideration, the helix-coi1 transition is not a two-state process. The transition occurs from a population of helical molecules with frayed ends to the random coi1 state, rather than from a completely helical conformation to a random coil (Scholtz & Baldwin, 1995).
Theoretical treatments of the transition were developed by Zimrn and Bragg in 1959, and by Lifson and Roig in 1961. These theoretical approaches, although similar, are distinguished by the criteria used to decide the structural state of a residue (i.e., helix or random coil). In Zimm-Bragg (ZB)theory the peptide group (which is shared by two amino acids) is the basic unit of helix formation, whereas for Lifson-Roig (LR) theory the basic unit of helix formation is the amino acid residue (Chakrabartty & Baldwin, 1995).
In both models helix formation is descnbed in terms of three parameters, namely an initiation parameter, a propagation parameter, and the chain length. The propagation parameter is defined as "an equilibrium constant for the addition of a residue ont0 an existing helical segment" and this value represents the intnnsic helix propensity of an amino acid (Scholtz & Baldwin, 1995). In LR theory the propagation parameter is denoted by w, however the ZB theory has been most commonly used, where the propagation parameter is denoted by s (Baldwin, 1995).
An experimental investigation began in the 1970s, whose goal was to measure the helix-forming tendencies of the 20 amino acids. The influence of a sidechain on the conformation of a peptide backbone is gauged by the helix propensity of the arnino acid.
The helix propensity arises from sidechain-backbone, sidechain-solvent, backbone- backbone, and backbone-solvent interactions. Based on the value of its heiix propensity, an arnino acid may be labelled as helix forming (s > l), helix breaking (s c l), or helix indifferent (s = 1) (Chakrabartty & Baldwin, 1995). It was necessary to detennine experimentally helix propensities in order to use them to predict helical regions in proteins, and to understand their contribution to protein stability. Using the host-guest system, Scheraga et al. sought to measure expenmentally the ZB parameter s as a representation of the helix propensity of each amino acid (Wojcik et al. 1990). The host- guest system used a copolymer of hydroxybutyl- or hydroxypropyl-L-glutamine (HBLG or HPLG, respectively) as the host, and each of the 20 arnino acids as the guest residue
(Scholtz & Baldwin, 1995). Using a random distribution of the guest residue in the copolymer, the authors intended to measure structurai effects solely due to the influence of the guest residues (Wojcik et al., 1990). The resuits of the host-guest studies indicated thlit the helix propensities of the amino acids are approximately one (helix indifferent) with the exception of Pro and Gly. Pro and Gly did not favour helix formation
(Chakrabartty & Baldwin, 1995). Moreover, the host-guest studies implied that helix- formation in water should be minimal for short peptides (5 13 residues) regardless of the sequence or temperature (Shoemaker et al.. 1985).
Some key expenmental work perfomed in the late 1960s seemed to be consistent with the host-guest helix propensities. Taniuchi and Anfinsen showed that two peptide fragments from staphylococcal nuclease were unstructured in isolation (Taniuchi and
Anfinsen, 1969). Moreover, Epand and Sheraga studied apomyoglobin, a predominantly helical protein. It was obsewed that cyanogen-bromide peptides from apomyoglobin did not retain helical structure in isolation (Epand & Scheraga, 1968). However, in 1971 work by Brown and Klee on nbonuclease A seemed incongnious with the host-guest studies. Brown and Klee showed that a peptide fragment consisting of residues 1-13 from the amino terminus of bovine pancreatic ribonuclease A (C-peptide) rernained monomeric and had a pax-tly helical conformation in solution. Furthemore, it was observed that the helix content of C-peptide was dependent on temperature and pH
(Brown & Klee, 197 1). This discrepancy remained unresolved until the 1980s.
To reconcile the results of Brown and Klee with predictions from the host-guest studies, Baldwin and coworkers conducted further investigations using C-peptide. This work was motivated by the hypothesis that C-peptide was most likely stabilized by sidechain interactions that were not incorporated in the helix propensity measurements using the host-guest system. In 1982, Bierzynski et al. reported that C-peptide is stablilized by a salt bridge between Glu' at position 9 (Glu9') and His' at position 12
(His 12'). These results had important implications for protein folding: (i) short a-helices with stabilizing sidechain interactions may acquire sufficient stability in water to function as early folding intermediates; and (ii) the effects of particular intrahelix interactions on the stability of a-helices in water may be factored using peptides with controlled sequences (Bierzynski et al., 1982). In addition, expenments using C-peptide analogs revealed that another charged-group effect played an important role in the stability of isolated C-peptide. Shoemaker et al. (1987) obtained results suggesting that Glu2 and
Hisl2' participated in favourable interactions with the helix dipole, thereby stabilizing the helix. It is clear that studies using C-peptide facilitated a qualitative look at the role of sidechain interactions in isolated helix formation. However, the C-peptide system was too complicated to use as a mode1 system (Scholtz & Baldwin, 1995). Peptides with controlled sequences were designed de novo, thus providing a simpler system for quantitative studies of the energetics of sidechain interactions. Marqusee and Baldwin (1987) designed a series of Ala-based peptides, de novo, for an in-depth study of Glu--Lys' salt bridges and helix stability. The study looked at the effect of spacing of Glu'-Lys' ion pairs, i.e. (i, i+3) and (i, i+4), and position (Glu-Lys versus Lys-Glu) as a function of both pH and salt concentration (Scholtz & Baldwin.
1995). The effect of position was investigated with respect to the interaction of charged sidechains with the helix dipole. The results indicated that peptides containing the i+4 ion pairs (in both orientations) showed more helix formation that those with the i+3 ion pairs, where the best helix former was the i+4 peptide with the Glu-Lys orientation.
Also, the orientation of the Glu and Lys residues affected stability as expected, in terms of an interaction with the helix dipole (Scholtz & Baldwin, 1995; Marqusee & Baldwin,
1987).
An in teresting empirical study performed by Richardson and Richardson (1988) also contributed to understanding the determinants of stability in isolate a-helices. This investigation looked at the empirical preferences of arnino acids for locations at the ends of 215 a-helices from 45 globular proteins. The interface residues were referred to as the
N-cap (N-terminus) and C-cap positions (C-terminus), and these positions were defined as being "half in and half out of the helix". At the C-cap, the stmngest preference was for
Gly, which terminated 34% of helices. At the N-terminus, the strongest preference was to have Asn in the N-cap position, and Pro in the N-cap+l position. It was noted that amino acid preferences for the ends of helices were dominated by hydrogen bonding patterns. In addition, negatively charged amino acids were preferred in the first turn and positively charged residues in the Iast tum. This work had important implications for improving helix prediction algonthms and for studying the effect of substitution at N- and C-cap positions on the stability of isolated helices and proteins (Richardson &
Richardson, 1988).
Another signi ficant development, w hich contributed to an understanding of the factors involved in isolated helix formation, occurred in 1989 when Marqusee et al. observed that alanine itself can stabilize short helices. A series of short alanine-based peptides was designed to contain three or more residues of a single charge type (Lys or
Glu) such that salr bridge and sidechain-dipole interactions were absent. Based on the helix propensities measured using the host-guest system, it was predicted that a 16- residue peptide containing Ala and Glu or Lys should not show measutable helix formation. Moreover, the results could not be explained due to the hydrophobic interaction between Ala residues. Thus. the observations suggested that Ala inherently favours helix formation. Marqusee et al. further proposed that perhaps the helix propensity of Ala is actually higher than the values measured in the host-guest system. It was also speculated that sidechain interactions involving the hydroxyalkyl groups of
HBLG and HPLG in the copolymers affected the propensity measurements. This was later confirmed by Padmanabhan et ai. (1994). It was clear that a new system needed to be devised to remeasure the helix propensities of the amino acids.
Several groups redetermined helix propensities using designed peptide systems.
Although the numeric values for the helix propensities differed among the various studies, they were highly correlated in tems of the rank order of the amino acids. One study that made use of a monomenc peptide system, where Ala was the major constituent, will be highlighted here. The Ala-based system was designed such that specific sidechain interactions, known to be helix-stabilizing, were absent. Thus, the values of the helix propensities were not affected by these interactions (Chakrabartty et al., 1994). In this system a wide range of values for the helix propensities were measured. It was found that "Ala is a strong helix former, Leu and Arg are helix indifferent, and al1 other amino acids are helix breakers of varying seventy"
(Chakrabartty et al., 1994). These results did not correlate with the host-guest results but they did correlate well with other peptide systems for detennining helix propensity. In addition, the expenmental results were analyzed by a modified version of LR theory. In the original manifestation of LR and ZB theories, the N-cap and C-cap positions are defined as being outside of the helix, so they are treated as having a coi1 or unfolded conformation. This treatment implies that the N- and C-cap position amino acids are inconsequentiai to helix stability, but it has been shown experimentally that the N-cap residue contributes to stability in peptide helices (Baldwin, 1995). nus, Chakrabartty et al. (1994) used a form of LR theory, modified to include helix capping effects. An analysis of the data was perfomed by comparing the predicted and observed values of the helix contents of al1 the peptides studied (Baldwin, 1995). They found that use of the modified LR theory produced a better fit to the data than the unmodified version which neglects the effect of helix capping (Chaknbartty et al., 1994).
The development of accurate helix-coi1 theones is important in order to apply them for the purpose of prediction. One goal has been the prediction of the locations of protein helices using helix propensities and knowledge of stabilizing sidechain interactions; there has been some success in meeting this goal (Baldwin, 1995:
Chakrabartty & Baldwin, 1995). Another objective is the prediction of aqueous helix contents for peptide fragments whose sequences correspond to helical regions in proteins. This second application impacts on protein folding studies. If a section from a protein sequence is predicted to favour the formation of an isolated helix in water, it may function as an early folding intermediate (Baldwin, 1995). In other words, "...such regions may represent initiation sites for protein folding" (Chakrabûrtty et al., 1994). In
1994, Munoz and Serrano developed an empirical method for predicting the behaviour of helical peptides in solution. They deveioped an algorithm that used a simplified version of ZB theory and included contributions from helix propensities, sidechain interactions and helix capping effects. This algorithm was applied to a large set of published data and was used to predict the stability of isolated helices. By incorporating contributions from the various interactions known to stabilize helices in isolation, the algorithm of Munoz and Serrano was able to predict successfully the helix content of a large number of isolated peptides in solution (Munoz & Serrano, 1994).
The various studies mentioned above contributed to an increased understanding of the factors involved in stabilizing isolated helices. This insight proved invaluable in the de novo design of helical peptides.
Design of ~HelicolBundles
Helical bundles are a recumng structural motif in naturally occumng, functionally diverse proteins such as cytokines, enzymes, and viral coat proteins. The simplicity and approximate symmetry which are characteristic of heiical bundle structures have made them a natural target for de novo design studies (Bryson et al., 1995). A major contribution to the field of helical bunde design has been made by DeGrado and coworkers using a "hierarchic" or "incremental", minirnalist approach to protein design (DeGrado, 1988; DeGrado et al., 1989; Bryson et al., 1995). In other words, they started with a minimal or simple sequence having few interactions and introduced more complex interactions incrementally, thereby building up a hierarchy of sequences with increasing complexity and stability. One motivation for the DeGrado group has been to cntically test and expand their understanding of the forces goveming protein folding, with the achievement of a designed, uniquely folded. functional protein as the ultimate goal. The incremental approach used by DeGrado and coworkers consisted of three main steps, and was fint applied toward the design of a series of 4-helix bundle proteins called the "a- series". The fint step was the design of a simple sequence containing amino acids with high helix propensities, which would Form an amphipathic helix in order to promote oligomerization (Ho & DeGrado, 1987). The stability of the tetramer relative to the monomer was assessed by determining the dissociation constant for tetramerization. In order to optimize the sequence it was modified in an iterative manner to maxirnize the siability of the tetramer (DeGrado, 1988). The next step was the design of loops that were inserted between two optimal, identical helical sequences. The helix-loop-helix structure favoured dimerization, resulting in a Chelix bundle. The loop sequence was optimized by evaluating the effects of sequence modifications on the stability of the 4-
helix bundle. The last step was the construction of the Chelix bundle structure from four optirnized identical helices connected by three optimized identical loops (DeGrado,
1988).
Characterization of the various a-series peptides has been an on-going study since the mid 1980s. The nomenclature for the a-senes is as follows. The initial sequence of the monomer was called a,. Modified a, sequences were denoted by lettem. e-g.. a,A, a$. etc.. When the monomer sequence was connected by a loop to form a dimer, trimer, or tetramer the sequence was labelled a? , a, , or a, respectively. In 1987, Ho and
DeGrado optimized the initial a, sequence to obtain a,B, which had only Leu in the hydrophobic core. Although a,B formed tetramers, they behaved like molten globules according to their ID and 7D NMR spectra (Osterhout et al., 1992; Betz et al., 1995).
The peptide azB was the next peptide in the hierarchy, which was made by connecting two identicai a,B copies with the Ioop sequence Pro-Arg-Arg. The sequence of CL$ was modified to contain a heme binding site, in an attempt to induce more native-like charactenstics, such as a specific tertiary structure, and as a means of introducing functionality into the design. Although the designed peptides bound heme, the heme group was solvent exposed. The results were different than expected due to the molten globule nature of the structure (Choma et al., 1994). In Iater work, the sequence of u2B was incrementally optimized to obtain a,D, whose solution structure was determined by
NMR spectroscopy in 1998 by Hill and DeGndo. The results indicated that a,D formed a 4-helix bundle with native-like properties, Le., it had thermodynarnic characteristics typical of a native structure, it had a well dispeoed NMR spectrum, and it did not show
ANS binding (Betz et al., 1995). Furthemore, the helical dimers packed against each other in a parallei manner, and associated into a tetramer with a novel Ioop topology called a "bisecting U" (Hill & DeGrado, 1998). The improved conformational specificity of a,D was attributed to the incorporation of interhelix polar interactions at the hydrophobic interface of qD (Raleigh et al., 1995; Betz et al., 1995). Another variation on the a,B sequence was the peptide a,, which contained four copies of the a,B sequence connected by three Ro-Arg-Arg loops (Regan & DeGrado, 1988). Regan and DeGrado successfully cloned and expressed a gene in E. coli, which encoded the sequence of a,, and found that it formed a Chelix bundle with molten globule properties.
Another member of the a-senes was not based on the original a, sequence. The a, peptides were based on the sequence of coil-Ser, which is a designed 3-helix bundle whose crystal structure was determined in 1993. Initially, it was intended that coil-Ser would fom a double-stranded parallel coiled-coil, but the crystal structure revealed that coil-Ser formed an antiparallel trimer due to the presence of a bulky Trp residue at the N- terminus (Lovejoy et al., 1993). However, in solution coil-Ser existed in a monomer- dimer-trimer equilibnum. Thus coil-Ser was used as a basis for the design of a 3-helix bundle whose structure was specific both in solution and in the solid state (Bryson et al.,
1998). Using the hierarc hic or incremental approac h described earlier, DeGrado and coworkers iterativel y modi fied the sequence of coi1Ser to final1y obtain the sequence of a,D. This designed protein was cloned and expressed in E. coli and its solution structure was determined by NMR spectroscopy. The structure of a,D showed that it formed a 3- helix bundle with thermodynarnic and spectroscopie propenies comparable to that of a native protein (Walsh et al., 1999).
Using the hierarchic minimalist approach, DeGrado and coworkers have successfully designed native-like helical bundles. Throughout this work, DeGrado and coworkers have stressed the importance of the concept of "negative design", i.e., the destabilization of dtemate fol& while simultaneously stabilizing the desired fold.
The design of a 4- helix bundle protein was undertaken in two ways by Hecht and coworkers. Firstly, in 1990, Hecht et al. designed Felix, a 4-helix bundle with a novel
"native-like sequence", meaning it was nomepetitive and contained 19 of the 20 amino acids. The peptide was 79 amino acids long and the sequence design was based on helix propensities and amino acid preferences for specific locations in helices as determined by a statistical analysis of naturally occurring proteins. Structurai characterization revealed that Felix was predorninantly helical and monomenc, but it behaved like a molten globule
(Hecht et al., 1990).
As an introduction to the second approach taken by the Hecht group, it is necessary to consider work by DeGrado and Lear from 1985. DeGrado and Lear Iooked at the importance of hydrophobic periodicity in detennining the structure of peptides.
Three peptides were designed, each having the same ratio of Leu to Lys. and therefore the same helix propensities. However, the three peptides differed in their LeuLys periodicities and their chain lengths. The peptides were synthesized and their secondary structures were characterized by CD spectroscopy in aqueous solution. It was found that peptide 1 was unfolded, peptide II showed a concentration dependent association of four helices, and peptide III tended to precipitate but seemed to fom B-sheets in certain conditions. The results of the experiments suggested that the periodicity of polar and nonpolar amino acids in a peptide sequence is an important factor affecting secondary struc tue. DeGrado and Lear further speculated that the minimum structural requirement for formation of a Chelix bundle motif is a sequence hydrophobic periodicity that matches the repeat period (i.e., the tum period) of an a-helix @eGrado & Lear, 1985).
An investigation of the minimum requirements for obtaining the 4-helix bundle fold was not undertaken in a general way until the early nineties. In 1993, Hecht and coworkers produced a collection of protein sequences, intended to fold into 4-helix bundles, by designing a library of synthetic genes. Each gene produced a different amino acid sequence. but al1 the sequences possessed the same periodicity of polar and nonpolar arnino acids (Kamtekar et al., 1993). Their strategy was based on the assumption that designing an arnphipathic structure, Le., specifying the positions of polar and nonpolar amino acids in a sequence rather than sequence identity, was sufficient to obtain a compact, native-like structure. Thus, the design positioned random polar arnino acids on the surface of a Chelix bundle and random nonpolar amino acids in the core. After expression of the genes, 29 soluble proteins were found to be appropriate for further characterization (Kamtekar et al., 1993). Several of the soluble proteins possessed the 4- helix bundle topology but did not have a specific tertiary structure. They concluded that the binary code does not explicitly design tertiary packing but the proteins can be designed w i th nati ve-li ke properties, w hic h re fers to specific secondary structure and overall topology (Roy et al., 1997). DeGrado and coworkers have asserted that periodicity of polar and nonpolar amino acids is important for specificity as well as breaks in these regular patterns. They suggest that these breaks don't necessarily serve to stabilize a particular fold but to a greater degree they serve to destabilize alternate fol&
(Betz et al., 1995).
Another successful helical bundle design was developed by Schafrneister et al. in
L993 to maintain the solubility of membrane proteins for crystailization. A 34-amino acid peptide called PD, was designed to form arnphipathic helices that self-associate and subsequently interact with a transmembrane protein as a detergent. The hydrophobic face contained Leu and Ala residues, and Glu-Lys (i, i+4) salt bridges were also incorporated into the design to confer stability to the helices. It was expected that PD, alone would dimerize however, when its structure was deterrnined by x-ray crystailography to a resolution of 2.5 A, it was revealed that PD, formed an aniiparallel Chelix bundle with a helix crossing angle of -20' (Schafmeister et al., 1993). It was speculated that connecting the four helices with short loops would result in the formation of a specific, well packed structure. On this basis, the 108 amino acid protein DHP,was designed to fom a Chelix bundle using four copies of the PD, sequence connected by three short Gly loops. The crystal structure was determined to a resolution of 2.9 À and it showed that DHP, formed a 4-helix bundle. In addition, 'H exchange expenments indicated that DHP, had high protection factors, on the order of 106 and it possessed thermodynarnic properties charactenstic of a natural protein (Schafmeister et al., 1997). It is interesting to note that this specific native-like protein was designed with a sequence consisting of a combination of only seven amino acids.
Although the focus of this section has been the contribution of protein design studies to the protein folding problem, designed peptides may also have exciting applications as catalysts. In 1993, Johnsson et al. designed helical peptides, called oxaldies (oxaldie i and oxaldie 2). that catalyzed the decarboxylation of oxaloacetate through an imine intermediate. CD spectra of the oxaldies indicated that both peptides were helical, and that oxaldie 1 formed a Chelix bundle in a concentration-dependent manner, while oxaldie 2 formed Iarger aggregates. In the oxaldie-catalyzed reaction, the consumption of oxaloacetate was three to four orders of magnitude faster than through the use of simple amine catalysts (Johnsson et al., 1993).
In surnmary, impressive progress in the field of de novo protein design has been
made in the last twenty years. The goal of designing proteins with well-defined native-
like structures has been successfully achieved (Schafmeister et al., 1997; Walsh et ai., 1999; Hill & DeGrado, 1998). Also, the goal of designing functional proteins has exciting potential for success. However, despite these practical successes, the need for a fundamental understanding of the forces governing protein folding remains. The current work was undertaken in an attempt to contribute io this understanding. In particular, the effect of nonpolar interactions on conformational specificity was studied in a system of designed assemblies of short helices. A unique experimental rnethodology was developed for this study which may have a more general application. Furthemore, the system was designed to rninirnize the presence of interhelical polar interactions so as not to obscure the contribution of nonpolar interactions to conformational specificity. CHAPTER 2: NONPOLAR CONTRIBUTIONS TO CONFORMATIONAL SPECIFICITY IN ASSEMBLIES OF DESIGNED SHORT HELICAL PEPTIDES
This chapter appears as the manuscript accepted for publication by the journal Protein Science. Reprinted with the permission of Cambridge University Press. Abstract A senes of designed short helical peptides was used to study the effect of nonpo interactions on conformational specificity. The consensus sequence was designed obtain short helices (17 residues) and to minimize the presence of interhelical po interactions. Furthemore, the sequence contained a heptad repeat (abcdefg), where positions a and d were occupied by hydrophobic residues Leu, Ile, or Val. and positions e and g were occupied by Ala. The peptides were named according to the identities of the residues in the adeg positions, respectively. The peptides Ilaa, ha, ilaa, iiaa, ivaa, viaa,
Ivaa, v laa, and vvaa were synthesized and their characterization revealed marked differences in specificity. An experimental rnethodology was developed to study the nine peptides and their painvise mixtures. These peptides and their mixtures formed a vast amy of structurai States, which may be classified as follows: helical tetramers and pentamers. soluble and insoluble helical aggregates, insoluble unstructured aggregates. and soluble unstructured monomers. The peptide liaa formed stable helical pentamers. and iiaa and vlaa formed stable helical tetramers. Disulfîde cross-linking experiments indicated the presence of an antiparallel helix alignment in the helical pentamers and tetramers. Rates of amide proton exchange of the tetrameric form of vlaa were 10-fold slower than the calculated exchange rate for unfolded vlaa. In other work, the control of specificity has been attributed to polar interactions, especially buned polar interactions; this work demonstnted that subtle changes in the configuration of nonpolar interactions resulted in a large variation in the extent of conformational specificity of assemblies of designed short helical peptides. Thus, nonpolar interactions cm have a significant effect on the conformational specificity of oligomenc short helices. Introduction
The hydrophobic interaction is considered to be a dominant force in stabilizing proteins
(Dill, 1990). However, the contribution of h ydrophobic interactions to conformational
specificity or structurai uniqueness is under debate. Early atternpts at protein design were
based on the belief that the hydrophobic interaction could encode both stability and
specificity in protein structure. The first design targets were helical bundles; and the
design principle used a simple sequence pattern to produce a bundle of arnphipathic
helices, with an exclusively hydrophobic core and minimal exposure of nonpolar amino
acids to solvent (Ho & DeGrado, 1987). Synthesis and characterization of the early
designed peptides revealed that they were very stable to thermal and chemical
denaturation. however, the sidechains were not close-packed and thus lacked the
conformationd specificity typical of a native protein (Handel et al., 1993). After a series
of design modifications, where an increasing number of polar interactions were
introduced into the core, a four-helix bundle with native4ke properties was produced
(Hill & DeGrado, 1998). Thus, while hydrophobic interactions imparted stability to the
designed proteins, they were insufficient for the formation of a specific structure. These
protein design studies have Ied to the idea that buned polar interactions in the
hydrophobic core contribute more to conformational specificity than hydrophobic
interactions.
The ability of buned polar interactions to cause an increase in conformational
specificity has also been highlighted in studies of coiled-coils. In the dimeric state of the
occurring coiled-coi1 motif of GCN4? there is an Asn residue that lies in the
dimer interface. This Asn foman interhelicai, sidechain-sidechain hydrogen
1 bond with the other corresponding Asn sidechain in the dimer (O'Shea et al., 1991).
Mutation of the Asn to a nonpolar amino acid resulted in a loss of conformational specificity (Harbury et al., 1993; Lumb & Kim, 1995). Similar results were obtained in studies of peptide Velcro, a designed coiled-coil peptide based on the sequence of GCN4
(O'Shea et al., 1993). Substitution of Leu for Asn at position 14 in the hydrophobic core of peptide Velcro led to an increase in stability, but a loss of specificity. From these results, it was concluded that nondirectionai hydrophobic interactions contribute to protein stability, but do not irnpart conformational specificity (Lumb & Kim, 1995).
An opposing view anses frorn theoretical approaches, employing HP models, which predict that structural uniqueness can be encoded by hydrophobic interactions (Di Il et al., 1995). Experimental support for this idea cornes from the work of Hecht, Regan, and coworkers. The Hecht group designed a protein with native-like properties, whose sequence was based on a "binary pattern" of polar and nonpolar amino acids with a predominantly hydrophobic core (Roy et al., 1997). The Regan group redesigned the hydrophobic core of Rop to contain Leu and Ala residues, exclusively (Munson et al.,
1996). The redesigned Rop had native-like properties. These studies demonstrate that it is possible to construct proteins having exclusively hydrophobic cores and also possess conformational specificity.
Many of the studies mentioned above made use of coiled-coils whose helicai segments were relatively long (approximately 30 residues). However, helices in globular proteins are usually less than 20 residues long, with an average length of 11 residues
(Schulz & Schirmer, 1979). Current data suggsts that the behaviour of coiled-coi1 assembiies may differ from short helical bundles. For example, while helices of coiled- coi1 proteins are usually parallel (exceptions: Monera et al., 1993; Oakley & Kim, 1997), to date the crystal structures of al1 bundles of short helices indicate an antiparallel alignment (Taylor et al., 1996; Schafmeister et al., 1993; Prive et al., 1999). Another difference is that short helical assemblies are not dimeric, but usually contain three or more helices. Therefore, the mechanism through which conformational specificity is conferred may be different for shorter helices. Consequently. we have chosen to investigate how nonpolar interactions contribute to specificity in assemblies of short helices (17 residues) and compare the results with coiled-coi1 studies.
A further distinction of al1 the above mentioned studies is that they use mode1 proteins which contain numerous polar interactions involving hydrophilic sidechains.
The presence of these interactions may mask the contributions of nonpolar interactions to conformational specificity. To avoid this complication we have designed a simplifieci system for examining the specificity and stability of helix associations. This system contains minimal interhelical polar interactions so that helix association is driven by nonpolar interactions involving relatively few arnino acid residues. Our major finding is that subtle changes in the number and type of nonpolar interactions resulted in a distribution of states ranging from unstructured aggregates to specific oligomeric states.
While we did not find stnicturally unique species within this distribution, we did observe states with vast ciifferences in their conformational specificity. Materids and Methods
Peptide Synthesis and Purifcation
The peptides were synthesized by the solid phase method on a 9050 Plus PepSynthesizer
(Perseptive Biosystems) as peptide amides. Fmocldimethy lfomarnide chemistry was used with the Fmoc-PAL-PEG-PS resin. The deprotection step (removal of the Fmoc group) was performed using 20 % piperidine in dimethylformamide and active ester coupling reactions were performed using the coupling reagent HATU. After synthesis and final deprotection step, the N-terminal a-amino group was acetylated using 0.5 M acetic anhydride and 0.5 M pyridine. The peptides were cleaved from the resin in 95% trifluoroacetic acid, 5% ethanedithiol, and 2% triisopropylsilane for four hours. The cleaved peptides were separated from the resin by filtration, precipitated in diethyl ether,
and washed twice in diethyl ether. After washing, the peptides were dissolved in water
and purified by reverse phase HPLC using a Cl8 Radial-Pak cartridge on the Waters
KPLC system. A wavelength of 230 nm was used for detection. Peptide identity was confirmed by electrospray mass spectrometry (ES-MS).
Peptide Concentration Deteminarion
Since al1 peptides contained one tyrosine residue in their sequence, their concentration could be deterrnined by measuring tyrosine absorbance at 275 nm. Aliquots of stock
solutions were dissolved in 6.5 M guanidine hydrochloride, absorbance at 275 nm was
measured using a Milton Roy Spectronic 3000 Arny UV Spectrophotometer, and the concentration was detennined using Beer's Law and an extinction coefficient of e = 1450 M%rn'' (Brandts & Kaplan, 1973).
Ckcrt lai- Dich roism Spectroscopy
CD measurements were performed on an Aviv 62A DS Circular Dichroism Spectrometer.
Al1 CD measurements are reported in degcm' dmol". Spectra were obtained using a 1 mm quartz cuvette at 25 'C in 10 mM Na,HPO,, 120 rnM NaCI. pH 7. Peptide concentrations were in the range 69-76 pM and the wavelength range was 195-260 nm.
Concentration dependence experiments and the CD screening experiment were performed at a wavelength of 221 nm using a 1 cm quartz cuvette in 10 mM Na,HPO,,
120 mM NaCI, pH 7 at 25 OC. A total peptide concentration of 50 pM was used for the
CD screening experiment.
Fliiorescence Spectroscopy
Steady-state fluorescence was measured at room temperature using a Photon Technology
International QM-1 fluorescence spectrophotometer equipped with excitation intensity correction and a magnetic stirrer. Emission spectra of the ANS dye were collected from
450 nrn to 600 nrn using an excitation wavelength of 372 nm, at 1 sechm, and a bandpass of 4 nm for excitation and emission. Sarnples consisted of 50 pM peptide and 50 pM
ANS in 10 mM Na,HPO,, 120 mM NaCl, pH 7 at 25 OC. Another set of measurements was also made on sarnples containing 50 pM peptide, 50 ANS, and saturated benzene in 10 mM NamO,, 120 mM NaCI, pH 7 at 25 OC. Solubility Measurernents
Solubility of the peptide mixtures was measured by monitoring UV absorbance before and after centrifugation. Each sample was prepared in a total volume of 500 PL consisting of 50 pM total peptide in 10 rnM Na,HPO,, 120 rnM NaCl, pH 7 ai 25 OC. In order to rernove insoluble aggregates, the samples were centrifuged in an IEC MicroMax microfuge for 30 minutes at 13 200 rpm; the supernatant from each sample was then transferred to a new tube. Since each peptide contained one tyrosine in its sequence, the absorbance of the supernatant was measured at 275 nrn, (A,,), using a Milton Roy
Spectronic 3000 Amy W Spectrophotometer. A 50 pM solution of soluble peptide containing a single tyrosine should have A,, = 0.0695. An absorbance value less than
0.0695 indicated that the peptide or peptide mixture formed insoluble aggregates which sedimented dunng centrifugation. Samples where A,, = 0.060-0.075 were considered
100% soluble; samples where A,, = 0.025-0.045 were 50% soluble; and samples where
A,, < 0.025 were 0% soluble.
Liglir Scanering
Light scattering experiments were performed on a DynaPro Dynamic Light Scattering
Instrument with DYNAMICS software from Protein Solutions. Eac h sample was prepared in a total volume of 500 consisting of 50 pM total peptide in 10 mM
Na,HPO,, 120 rnM NaCl, pH 7 at 25 'C. In order to eliminate the presence of dust and insoluble aggregates, the samples were centrifuged in an IEC MicroMax microfuge for
30 minutes at 13 200 rpm; the supernatant from each sample was then transferred to a new tube. Measurements of scattered light intensity, or counts, were made using a sarnple volume of 20 pL in a cuvette. The average scattering intensity of ddH20 and buffer (10 rnM NalHPO,, 120 mM NaCI, pH 7) was 5 x 10) counts/sec over a period of 5 minutes with minimal Ructuations; this value was used as a control since neither dM20 nor buffer would contain any deteciable pmicles.
Sedimentatiun Eqicilibrium llltracent~fugation
The molecular weights of the adeg oligomers and cys-vlaa+vlaa-cys peptides were determined by sedimentation equilibrium on a Beckman Optima XL-1 analytical ultracentrifuge. Experiments were performed on the peptides vlaa, liaa, and iiaa in a six- sectored ce11 with quartz windows in buffer containing 10 mM Na,HPO,, 120 mM NaCI, pH 7 at 20 OC with total peptide concentrations of 50 PM, 75 FM, and 100 FM. The
SedNterp software was used to calculate the solvent density and the partial specific volumes of the peptides. A value of 1.00454 g/mL was calculated for the soivent density and the partial specific volumes were calculated to be 0.7599 mUg, 0.7541 mUg, and
0.7599 mg,for iiaa, vlaa, and liaa, respectively. Measurements were made every
0.001 cm using 50 replicates. For the experirnents using liaa and vlaa, data were collected after 20 hours at a wavelength of 275 nm, using rotor speeds of 26 500,29 000, and 31 000 rpm. Data fitting and analysis were performed with Microcal Origin 4.1 using a self-association model. Global data fits were obtained by fitting nine sets of data simultaneously to an equation for a single ideal species. The experiment using iiaa was performed at a wavelength of 230.5 nm, using a rotor speed of 25 000 rpm and data was collected after 20 hours; a global data fit was performed by fitting 3 sets of data simuItaneously to an equation for a single ided species. In addition, an experiment was performed on cys-vlaa+vlaa-cys in a six-sectored ce11 with quartz windows in buffer containing 50 mM CH,COONH, with total peptide concentrations of 30 pM, 40 PM, and 50 pM. Using the SedNterp software, the solvent density was calculated to be 0.99815 g/mL and the partial specific volume of cys- vlaa+vlaa-cys was 0.7415 mL/g Measurements were made every 0.001 cm using 50 replicates; data were collected after 20 houn at a wavelength of 230.5 nm using rotor speeds of 33 000, 37 000, and JO 000 rpm. Global data fits were obtained by fitting nine sets of data to a monomer-dimer-tetramer equilibrium using Microcal Origin 4.1.
Another experiment was performed with cys-vlaa+vlaa-cys in slightly denaturing conditions using buffer containing 20 mM CH,COONH, and 4M Gdn-HCI, with total peptide concentrations of 20 PM, 17 pM, and 14 pM. A solvent density of 1.09632 g/mL was obtained from the SedNterp software. Data collection and rotor speeds were the same as above. Global data fits were obtained by simultaneousiy fitting nine sets of data to a monomer-dimer equilibrium using Microcal Origin 4.1.
Assessrnent of Parallel versus Antiparallel Alignmenr of Helices in Helical Brtndk
Throitgh Disulfide Cross-linking
Each sample consisted of two cysteine-containing peptides: one with an N-terminal cysteine and the other with a C-terminai cysteine. In each sample, the two peptides had different molecular weights (see Table 2) so that after cross-linking the different species were distinguished by their molecular weights using mass spectrometry. The samples were prepared in a total volume of 8 mL consisting of 50 pM peptide in 6.5 M Gdn-HC1,
1% p-mercaptoethanol, pH 8. Al1 pH tintions were perfonned using WOH. On day one, the sample was dialyzed ovemight in a dialysis buffer that contained distilled, deionized water and 1% B-mercaptoethanol, pH 8. On day two, the dialysis buffer was changed to distilled, deionized water, pH 8, in order to remove the P-mercaptoethanol and dlow disulfide bond formation. The dialysis buffer was changed 3 times at 3 hour intervais. On day the. the sample was lyophilized. Once dry. the sample was dissolved in a buffer containing 50% acetonitde, 0.1% acetic acid, and 2 mM CH,COONH, to a final concentration of 0.5 rnM. The sample was then analysed by ES-MS.
As a control, cys-vlaa+vlaa-cys was prepared as above, for analysis using sedimentation equilibnum ultracentrifugation. The final equilibrium conditions were in dialysis buffer containing 50 rnM CH,COONH, and a total peptide concentration of
50 FM.
Amide Proton Erchange Rate Measwernenrs
Amide proton exchange was measured by 'H-NMR spectroscopy. An aqueous solution of vlaa (1 rnM, 700 w)was adjusted to pH 3.26 and lyophilized. At t = O min. vlaa was dissolved in 700 PL of D,O containing 5 mM NaH2P0,, pH* 2.26 (the glass electrode reading at room temperature, uncorrected for isotope effects). Multiple 1-D proton spectra were acquired using a Varian 500 Unity Plus spectrometer at a proton frequency of 500.053 MHz. Data were collected using a 7000.4 Hz spectral width and a 90" pulse.
Presaturation was used to suppress residual HDO signal. The peaks in the amide region were integrated and normalized using one of the nonexchanging tyrosine sidechain resonance peaks. The proton occupancy was determined by dividing the normalized amide peak areas by the extrapolated area at t = O min. Peptide Design
Table 1 shows the sequences of the peptides designed for this study. The consensus sequence, given by Ac-DdeQgaKQdeEgaQKdeGGY-amide, was designed to obtain helices 17 residues long containing a heptad repeat, i.e. seven residues (labelled a. b, c, d, e, f, g) that recur in the sequence. A heptad repeat is cornmonly found in the sequence of many naturally occurring coiled-coil- and a-helix bundle-containing proteins (Cohen &
Pany, 1990). Usually, heptad positions a and d are occupied by hydrophobic arnino acids, whereas positions e and g are occupied by charged amino acids. In this work, the name of each peptide was denved from the identities of the arnino acids in the a, d, e, g positions, respectively. For exmple, the peptide vlaa has Va1 in position a, Leu in position d, and Ala in positions e and g. Because of this nomenclature, the peptide series as a whole is cdled the adeg senes. Upon folding, the presence of the heptad repeat in the consensus sequence will result in the formation of amphipathic helices, thereby favouring oligomerization. Figure 1 shows a helical wheel diagram of the peptide llaa and illustrates the arnphipathic nature of the adeg peptides.
In order to study the contribution of nonpolar interactions to conformational specificity of short helical bundles. the peptides were designed to minimize the impact of polar interactions on specificity. Interhelicd polar interactions occumng between amino acids occupying e and g heptad positions are known to affect specificity and, to a lesser extent, stability (O'Shea et al., 1993; Kohn et ai., 1995; Nautiyal et al., 1995; Zeng et al.,
1997). Thus, Ala was chosen to occupy the e and g heptad positions in order to prevent
interhelicai polar interactions at these positions. The a and d positions, comprising the Table 1: Sequences of the adeg' Peptides
Name Sequence and Hep tad Positions
cdefgabcdefgabcde i IilaaID L AQALKQLAEALQKLAGGYI liaa DIAQALKQIAEALQKIAGGY ilaa DLAQA 1 KQLAEAIQKLAGGY iiaü DIQIQIAEQKXGGY ivaa DVAQAIKQVAEAIQKVAGGY viaa DIAQAVKQIAEAVQKIAGGY lvaa DVAQALKQVAEALQKVAGGY vlaa DLAQAVKQLAEAVQKLAGGY vvaa DVAQAVKQVAEAVQKVAGGY -- . nie n&e of eachpeptideis derived f&& the id&Ïtity ofthe residues in the a. d. e. g. heptiid positions, respectively. The narne adeg is used to refer to the peptides collectively. LLL
LL
Figure 1. Helical wheel diagram of llaa. The view is from the N-terminus, down the helical ais. The heptad positions are contained in circles labelled a-g, and they span the fint two tums of the helix. The
sequence of llaa begins at heptad position c. The other adeg peptides differ only by the
identity of the residues in the a and d heptad positions (see Table I for sequences of the adeg peptides). hydrophobie core, were occupied by the nonpolar arnino acids Leu, Ile, or Val in al1
possible combinations (nine peptides in total). In addition, Asp was chosen for the N-
terminus in order to stabilize helix formation, since (i) it is known to have a preference
for the N-cap position in naturally occumng helices (Richardson & Richardson, 1988)
and (ii) it pmvides a favourable electrostatic interaction with the helix dipole (Shoemaker
et al., 1987). The interna1 heptad positions b, c, f are occupied by Gln because it is a
polar uncharged amino acid with a high helix propensity (Chakrabartty & Baldwin.
1995), features which will aid in peptide solubility, formation of an amphipathic helix,
and helix stability. Moreover, an (i. i+4) Glu-Lys salt bridge was designed into the
sequence since it has been shown to stabilize helix formation in short peptides (Marqusee
and Baldwin, 1987).
Lastly, Tyr appears in the consensus sequence in order to facilitate concentration detemination by UV absorbante. Two Gly residues link the Tyr to the rest of the
sequence in order to prevent a contribution from Tyr to the circular dichroism (CD)
signal of the peptides (Chaknbartty et al., 1993). In addition, the two Gly residues, by
virtue of their helix breaking activity (Chakrabartty & Baldwin, 1995), position the Tyr
outside of the helical segment. Moreover, in previous work by Lovejoy et al., a Trp
residue was incorponted into the sequence of a designed coiled-coi1 named coil-Ser, at
the N-terminus, in order to facilitate W spectroscopie studies. However, the crystal
structure of coil-der indicated that it formed antiparallel tnmers because the bullcy
sidechain of Trp was best accommodated in this structure (Lovejoy et al., 1993). Thus,
the presence of Trp within the helix actively affected the oligomenzation of coil-ber. Since the Gly linkers in the consensus sequence of the adeg series leave Tyr out of the helix, it is unlikely that Tyr interfered with oligomerization in this system.
Determining Presence of Specifc Oligomers in Peptide Mirrures
We examined two types of oligomen in the adeg series: (i) homo-oligomers of each of the adee-- ~e~tides - 19 in total) and (ii) hetero-oligomen that would mise from al1 possible mixtures of the adeg peptides (36 in total). In order to improve the efficiency of characterization of the 45 sarnples, we devised 5 experiments to rapidly screen the samples. Since we want to study specific oligomers, these tests allowed us to eliminate, from further analysis, those peptides and mixtures that were predominantly unfolded and those which formed nonspecific aggregates. Furthemore, the tests provided insight into the secondary structure and conformational specificity of the peptides. Al1 the screening tests were performed using 50 yM total peptide concentration in buffer (10 mM
NaJIPO,, 120 mM NaCl, pH 7) and the results of each test are given in matrix fom.
The first test examined the solubilities of the adeg peptides and mixtures. The samples were centrifuged and then the UV absorbantes of the supernatants were measured. This experiment showed which peptides and mixtures formed insoluble nonspecific aggregates, and eliminated them from further characterization. Figure 2A is a summary of the results of the solubility test. This experiment indicated that the peptides Ilaa, ivaa, and viaa were the only peptides that formed insoluble aggregates. The peptides liaa, ilaa, iiaa, lvaa, vlaa, vvaa, and their mixtures were 100% soluble.
After UV absorbante measurements were performed, the same samples were
studied using light scattenng. This method allowed us to identify large soluble
aggregates present in the peptide solutions. Since the samples had peptide concentrations 5 50 W. only large aggregates would scatter enough light to be detected. The matnx in
Figure 2B summarizes the results of the light scattering experiment. The peptides llaa and ilaa had high scattering intensities due to the presence of large aggregates in solution.
Quantitative analysis of dynamic light scattering data from the ilaa stock sample (1.5 mM) indicated that > 90 % of the molecular species detected had a hydrodynamic radius of -20 nm. The results shown in Figure 2A indicate that llaa also formed insoluble aggregates, whereas ilaa was 100% soluble. Therefore, ilaa fomed large soluble aggregates and llaa formed both soluble and insoluble aggregates. The remaining adeg peptides showed insignificant scattering intensities.
After establishing the solubility and relative sizes of the adeg homo- and hetero- oligomers, their structure was investigated using CD spectroscopy. CD spectroscopy was used to distinguish between folded and unfolded structures, and to determine the nature of the folded States of the adeg peptides. Figure 3A shows that the peptides llaa, liaa, ilaa. iiaa, and vlaa had spectra characteristic of a-helices with minima at the wavelengths
222 nm and 208 nm. Figure 3B shows that ivaa, viaa, lvaa, and vvaa had spectra characteristic of the random coil or unfolded state. Since the peptides seemed to be either helical or random coil, a wavelength of 222 nm was chosen to compare the secondary structures of al1 the peptides and mixtures.
Figure 2C shows the matrix that summarizes the results of the CD screening experiment. The results indicate that liaa had the highest helix content, followed by ilaa and llaa; iiaa and vlaa had an intermediate helix content, and ivaa, viaa, Ivaa, and vvaa had the lowest heiix content. Furthemore, these data are consistent with the resuIts of the light scattenng expenment. The peptide llaa formed insoluble nonspecific helical aggregates; ilaa formed soluble nonspecific helical aggregates; lvaa and vvaa had a low scattering intensity because they were unfolded; liaa was soluble and helical, but may fom some large aggregates. However, iiaa and vlaa may fom specific helical oligomers since they did not form soluble or insoluble aggregates.
After the CD screen, the same samples were used for an ANS (1- anilinonapthalene-8-sulfonic acid) dye binding experiment, whose results are summarized in Figure 2D. ANS is a dye whose fluorescence increases when it is sequestered in a hydrophobic environment and has been used to identify exposed
hydrophobic surfaces in proteins. Furthemore. molten globule structures also show ANS binding (Kuwajima, 1989). The helical peptide liaa showed a large increase in ANS
fluorescence, illustrating that this folded peptide had an exposed hydrophobic region in
which ANS was bound. The peptides iiaa and vlaa, respectively, showed an intermediate and smdl increase in ANS fluorescence, which may be indicative of a more native-like character than that possessed by liaa. The peptides Ilaa, ilaa, ivaa, and viaa, which form
nonspecific aggregates, showed an increase in ANS fluorescence indicating that there are exposed ciusters of hydrophobic residues in the aggregates. Lastly, the unfolded peptides
lvaa and vvaa did not show an increase in ANS fluorescence.
Using the screening tests, we eliminated peptides that were unstructured,
insoluble, and prone to aggregation, and we identified peptides that may form specific
helical oligomers. The four tests (i) proved to be an efficient means of screening for
particular properties from a large number of similar peptides, and (ii) provided important
information about the structure and specificity of the peptides. Based on the results of the
screening tests we have identified liaa, iiaa, and vlaa as peptides that potentially form Figure 2. Screening Tests.
(A) SoIubility of adeg peptide mixtures. The 45 samples were centrifuged and the UV absorbance of the supematants was measured at 275 nrn (A,,) to determine peptide solubility. AI1 sarnples consisted of 50 pM total peptide in buffer containing 10 mM
Na2HP0,, 120 mM NaCl, pH 7 at 25 OC. A soluble peptide solution at a concentration of
50 pM should have A,, = 0.0695. The peptides were considered to be 0% soluble when
A,, < 0.025 (square containing an "X"), 50% soluble when A,, = 0.025-0.U45 (square containing a " \ "), and 100% soluble when A,, = 0.060-0.075 (empty square). (B)Light scattering properties of adeg peptide mixtures. The right angle scattering intensity was measured and normalized to obtain the relative scattenng intensity using (xi - x,J(x,, - x,,), where xi is the scattenng intensity of peptide mixture i, xrmo= 4 x ld countsls, and x, = 2490 x 10' counts/s. The relative scattenng intensity values were then applied to a grey scale for shading. (C) Helix content of adeg peptide mixtures. Al1 measurements were performed in buffer containing 10 mM Na,HPO,, 120 mM NaCl, pH 7 at 25 "C.
[el, was used an indication of helix content. The shading indicates the relative helix content of the adeg peptides and mixtures, and was calculated as in Figure 2B. In this case x,, = 2k and x,, = 26k (where k = 10) deg-cm2/dmol). @) ANS binding of adeg peptide mixtures. The relative fluorescence (FR)of each peptide mixture was calculated from the fluorescence of a peptide sample containing ANS (F,,,ANs), and the fluorescence of ANS alone (FA,), using FR= FW+,/FANs. These values were then nomalized to fdl between zero and one as in Figure 2B and applied to a grey scale. PEPTIDE 2 190 200 210 220 230 240 250 260 Wavelength (nm)
O, I t I J l 1 i -2.5 104 190 200 210 220 230 240 250 260 Wavelength (nm) Figure 3. Circular dichroism spectra of the adeg peptides. Al1 measuremenis were perforrned in buffer cmtaining 10 mM Na,HPO,, 130 mM NaCl, pH 7 at 25 OC. The helical peptides are (A) +, vlaa 75 pM;O, llaa 75 w; V, liaa 76 pbf; 0, ilaa 75 pM; O,iiaa 75 W. The unfolded peptides are (B) 0, viaa 75 pM; 0,waa 69 pM; +, ivaa 100 pM;V, lvaa 75 pM. specific helical oligomers. In addition, the tests indicated that viaa and ivaa formed insoluble, unstructured aggregates; llaa and ilaa formed insoluble and soluble helical aggregates. respectively; and Ivaa and vvaa formed soluble, unstnictured monomen.
Information regarding the presence of specific stable hetero-oligomers was obtained using the helix content data (Figure 3C), ANS fluorescence data (Figure 2D), and the foollowing rnethod. If hetero-oligomers did not form, then the helix content (or
ANS fluorescence) of any given painvise mixture would be equal to the average of the helix content (or ANS fluorescence) of the two constituent peptides. We measured the percent deviation of the observed helix content (or ANS fluorescence) of peptide mixtures from the calculated average. Percent deviations greater than 10% of both the helix content and the ANS fluorescence were deemed significant. Using these criteria, the only peptide mixture that seemed to forrn hetero-oligomers was Ilaa+iiaa. However, the mixture llaa+iiaa contained some insoluble material and consequently it was not investigared further. This method would not highlight mixtures containing similar proportions of homo- and hetero-oligomers. However, we were only interested in identifying mixtures solely containing hetero-oligomers, which would be highlighted by this method.
Oligomerization State
The screening tests identified the peptides iiaa, vlaa, and liaa as candidates for specific oligomer formation. Subsequently, the oligomerization States of the peptides were determined using sedimentation equilibrium ultracentrifugation. Sedimentation equilibrium analysis was performed for each peptide by simultaneously fitting dl the data taken at different concentrations and different rotor speeds to the sedimentation equilibrium equation for a single ideal species. For the peptides iiaa, vlaa, and liaa
(Figure 4), the analysis indicated a number average molecular weight of 8277 Da, 8603
Da, and 11275 Da, respectively. The corresponding 95% confidence intervals were
7755-8806 Da for iiaa, 7561-9666 Da for vlaa, and 10073-12469 Da for liaa. These data suggested that both iiaa and vlaa form tetnrners, while liaa forrns pentamers. The percentage difference between the number average molecular weight and the calculated oligomeric molecula. weight is 3%. 2%,and 5%, respectively, for iiaa, vlaa, and liaa. To confirm our assignrnent of the oligomeric state of the peptides, the data for each peptide were also fit to various association models. The goodness of the different fits were assessed by comparing the randornness of the residuals, the magnitudes of the variance estimates, and whether the association constants seemed physicdly plausible. Based on these criteria, the simplest mode1 that best descnbed the data was a stable tetramer for iiaa and vlaa, and a stable pentarner for liaa. However, the presence of a small amount of oligomeric species smaller than a tetramer cannot be ruled out.
Stability of the odeg Oligomers
To further characterize the specific adeg peptides, the variation of helix content with concentration of liaa, iiaa, and vlaa was examined using CD spectroscopy. In addition, the concentration dependence of the helix content of Ilaa, ilaa, and vvaa was investigated in order to compare the stabilities of the specific and nonspecific adeg peptides. Figure 5 shows the concentration dependence of the ellipticity of liaa, Ilaa, ilaa, iiaa, vlaa, and Figure 4. Sedimentation equilibrium ultracentrifugation of ha, Uaa, and vlaa.
Data shown is at 100 pM total peptide concentration in buffer containing 10 mM
Na2HP04, 120 mM NaCl, pH 7 at 20 'C for the peptides (A) iiaa: 25 000 rpm, 230.5 nm;
(B) liaa: 26 500 rpm, 275 nm;and (C)vlaa: 31 000 rpm, 275 nm. The curves represent the best fit of the data to the sedimentation equilibrium equation for a single ideal species. 6.90 6.95 7.00 7.05 7.1 0 Radius (cm) O 2 4 6 8 Concentration (M x 10')
Figure 5. Concentration dependence of the helix content of the adeg peptides. [O], was used as an indication of helix content. The [O]= of (a) ilaa was obtained in dm0at 25 'C. pH 3.15. The spectra of peptides (+) ha, (v) iiaa, (0)vvaa. (+) vlaa, and (a) liaa were obtained in 10 mM Na&IPO,, 120 mM NaCl. pH 7 at 35 'C. vvaa at 222 nm, which was used as an indication of helix content (as described earlier).
In order to quantitate the dissociation constants of the specific adeg peptides, the data in
Figure 5 for liaa, vlaa, and iiaa were fit to a version of the Hill equation, modified to model oligomerization (DeGrado & Lear, 1985), using a nonlinear least squares fit (see
Appendix for derivation of modified Hill equation). The goodness of the fits was determined by minimizing on the sum of the squares of the residuals given by
Z(eobS- Since the sedimentation equilibrium results indicated that iiaa and vlaa form tetramers, a monomer-tetramer model was used to fit the data from Figure 5 for iiaa and vlaa. The curve fitting results for iiaa and vlaa are shown in Figure 6A and B, respectively. The concentrations at which iiaa and vlaa are 50% unfolded are 36 pM and
50 pM. respectively. Since liaa was found to form pentarnen using sedimentation equilibrium analysis, a monomer-pentamer model was used to fit the CD data for liaa from Figure 5. The curve fit results for liaa are shown in Figure 6C. Moreover, the concentration at which liaa was 50% unfolded was 16 PM. The results obtained frorn the curve fitting suggest that liaa formed the most stable oligomers followed by iiaa. and vlaa.
Assessrnent of Paralle! versus Antipardlel Alignment of Helices in adeg Oligomers
The finai step in the characterization of the adeg oligomers was to assess whether the helical bundles contained helices in a parailel or antiparallel orientation. The alignment of the helices was determined through a disulfide cross-linking experïment employing two cysteine-containing peptides: one with an N-terminal cysteine and the other with a C- terminai cysteine. In order to ensure that the cysteine was extemal to the helix, the Figure 6. StabiIity of iiaa, vlaa, and liaa oligomers.
The concentration dependence of the helix content of iiaa, vlaa, and liaa were fit to a modified version of the Hill equntion. The data for the peptides (A) iiaa and (B) vlaa were fit to the equation for a tetramer model. The dissociation constants for iiaa and vlaa were calculated to be 1.80 + 0.48 x 10"' M3and 1.22 -c 0.43 x 10'" M', respectively. The data for (C) liaa were fit to a pentamer model, and the dissociation constant was calculated to be 6.15 -e 0.97 x Ma.
O 2 4 6 8 Concentration (M x 105) 2 4 6 Concentration (M x 105) cysteine residues were appended ont0 the sequences of liaa, iiaa, and vlaa using a
diglycine linker. The new peptides were called cys-adeg or adeg-cys, according to
whether the cysteine residue was located at the N- or C-terminus, respectively. In
addition, the adeg-cys peptides contained an extra glycine residue after the cysteine so
that each member of an adeg-cys+cys-adeg pair had a different molecular weight, as
shown in Table 2. Thus, after disulfide bond formation, each species present in solution
would be distinguished by its molecular weight using mass spectrometry.
Figure 7 shows the strategy used in the cross-linking experiment. If the adeg
peptide of interest forms 01-11y paraIlel. Chelix bundles then. after disulfide cross-linking . there would only be two molecular weight species present in solution. The two
molecular weight species would correspond to two helices having a parailel orientation
with a disulfide bond between two N-terminal cysteines (denoted NN) and also between
two C-terminai cysteines (denoted CC). However, if the adeg peptide of interest forms
only antiparallel 4-helix bundles, three molecular weight species would be detected
corresponding to the NN and CC species mentioned above, as well as two antiparallel
helices with a disulfide link between an N-terminai and a C-terminal cysteine (denoted
NC) .
Table 3 shows the results of mass spectrometry performed after the disulfide
cross-linking of the peptides cys-iiaa+iiaa-cys, cys-vlaa+vIaa-cys, and cys-liaa+liaa-cys.
In al1 cases, NC species were detected which is consistent with antiparallel alignment. In
addition, monomers were detected in the case of liaa (see Table 3) which is consistent
with pentamer formation. In a five-helix bundle, two disulfide-bonded pairs would Figure 7. Assessrnent of helical alignment.
Strategy used in the assessrnent of the parallel venus antiparallel alignrnent of helices in the adeg oligomen. Ml represents the molecular weight of the cys-adeg peptide and M2 represents the molecular weight of the adeg-cys peptide. If the alignment of the adeg oligomer is (A) parailel, only 3 molecular weight species will be detected (Ml+Ml and
M2+W).whereas if the alignment is (B) antiparallel, 3 molecular weight species will be detected (M l+M 1, M2+M2. and M1+M2) (Harbury et al., 1993).
Table 2: Sequenees of Peptides Used for Alignment Determination
Molecular Locatio Name Sequence Weight n of (g/mol) Cy sa cys-iiaa CGGDIAQAIKQIAEAIQKIAGGY 2361 N iiaa-cys YGGDIAQAIKQIAEAIQKIAGGCG 2418 C cys-vlaa CGGDLAQAVKQLAEAVQKLAGGY 2333 N vlaa-cys YGGDLAQAVKQLAEAVQKLAGGCG 2390 C cys-lia CGGDIAQALKQIAEALQKIAGGY 236 1 N liaa-cys YGGDIAQALKQIAEALQKIAGGCG 3418 C N = N-terminus: C = C-terminus. provide four helices, while the fifth helix would be excluded from a disulfide pair and remain monomeric.
To determine whether disulfide bond formation was compatible with the tetrameric structure of vlaa, we perfonned sedimentation equi li bnum anal ysis on the disulfide-bonded mixture of cys-vlaa+vlaa-cys. We found that in 50 mM CH,COONH, the data did not fit to a model for a single ideal species, and was best descnbed by an association model for a monomer-dimer-tetramer equilibrium. In this case, the monomer corresponds to the disulfide-linked species: NN, CC, and NC which are shown in Table
3: these species are likely to be unfolded. The dimer corresponds to noncovalent association of two monomers described above, and is likely to possess four-helix bundle structure. The tetramer is likely to be compnsed of two four helix bundle structures
linked by disulfide bonds. In buffer containing 20 mM CH,COONH, and 4M Gdn-HCI sedimentation analysis indicated that the peptides formed a monomer-dimer equilibrium
mixture. Therefore, under these conditions the unfolded NN, CC, and NC species
(monomer) were in equilibrium with four-helix bundle structures (dimer), and the
disuifide-linked pair of four-helix bundles (tetramer) was absent. Therefore, disulfide
bonded mixtures of cys-vlaa+vlaa-cys are capable of forming antiparallel four-helix
bundles, dong with other oligomenc States, however the antiparallel four-helix bundle
state is the most stable thermodynamic state.
Amide Proton Erchange Kinetics of vlaa
Another method for assessing conformational specificity in protein design studies is the
measurement of amide proton exchange &umb & Kim, 1995). Under conditions where Table 3: Assessing Paralle1 Versus Antiparallel Alignment of adeg Helical Bundles
MoIecuIar Weight of Species Peptide Mixture Detected by ES-MS
I (dmol)5' cys-iiaa + iiaa-cys 1 47 18 (NN)", 4774 (NC)b,483 1 (CC)' cys-vlaa + vlaacys 4662 (NN), 47 18 (NC),4776 (CC) cys-Iiaa + liaa-cys 47 17 (NN), 4775 (NC),4832 (CC), 2360 (N). 24 16 (Cl " NN = panllel orientation with disulfide link between two N-termini. NC = antiparallel orientation with disulfide link between N- and C-termini. CC = parallel orientation with disulfide link between two C-termini. the EX2 amide proton exchange mechanism dominates, NH groups in folded structures
with high conformational specificity exchange at rates that are significantly slower than those in unfolded States. The expected exchange rate for MI groups in an unfolded state, defined as the intrinsic exchange rate, can be calculated from model cornpound data (Bai et al., 1993). The high conformational specificity of the designed coiled-coi1 ACID- p l/BASE-p 1 was demonstrated by amide proton exc hange measurements, which revealed three MI groups that exchange 10'-fold slower than the intrinsic exchange rate (Lumb &
Kim, 1995). Amide proton exchange measurements have dso established the high conformational specificity of the de novo designed peptide a,D, which contains slowly exchwging amide groups that exchange between ~O~-l~~-foldslower than the intrinsic exchange rates (Walsh et al., 1999).
We examined the amide proton exchange rates of vlaa because it showed the highest degree of conformational specificity arnong the adeg peptides. In particuiar, vlaa had the lowest ANS binding characteristics and formed soluble tetramen with stable helical structure. The exchange kinetics of vlaa were too fast to measure at pH 7, but at pH 2.26 the exchange was slow enough to make accunte measurements. Sedimentation equilibrium ultracentrifugation of the pH 2.26 NMR sarnple indicated that under the
NMR conditions (1 mM peptide concentration), vlaa remained predominantly tetrameric however a small amount (-10%) of a much Iarger aggregate was dso present (data not shown). The pH-dependence of the exchange kinetics indicated that the NH groups of
vlaa exchanged by the EX2 exchange mechanism (Englander & Kallenbach, 1984). At pH 2.26, the simplest model that best described the exchange kinetics was a double exponential model in which there was one fast and one slow exchanging class of MI O 0.5 1 1.5 2 2.5 Time (sec x 10~)
Figure 8. Amide proton exchange kinetics of vlaa. Amide proton exchange was monitored by 'H-NMR using 1 mM total peptide
concentration at pH 2.26, 5 OC. Fractional proton occupancy is determined as described in Matenals and Methods. The decay in fractional proton occupancy was fit to the equation: fractionai occupancy = A exp(-k,t) + B exp(-k,t). The best fit was obtained with the values: A = 0.412, B = 0.608,k, = 3.70 x 104 sec-', k, = 3.69 x 10" sec". groups (Figure 8). The rate constants and fractional concentrations for the fast- and slow- exchanging NH groups were 3.70 x 104 sec-' and 3.69 x sec-', respectively, and 40% and 60%. respectively. The intrinsic exchange rates of the individual NH groups of vlaa, calculated using the method of Bai et al., ranged between 2.2 x 104 to 5.78 x 10' (mean value = 3.27 x 104 sec"). The rate constants for the NH groups in the fast exchanging class corresponded closely to the intrinsic exchange rates, suggesting that these groups are unprotected. Conversely, the rate constant for the MI groups in the slow exchanging class was 10-fold slower than the intnnsic rates. Thus, the NH groups in the slow exchanging class show protection similar to that of a molten globule (Hughson et al..
1990), rather than a native protein. One plausible explanation for the structural origins of the fast and slow exchanging class of protons is that the fast exchanging protons onginate
from unstructured or non-hydrogen bonded NH groups, and the NH groups that are hydrogen bonded within the helix make up the slow exchanging class. The vlaa peptide contains 21 NH groups. If we assume that the helix ends at the C-terminal -Gly-Gly-Tyr sequence, then 8 out of the 21 (or 38%) MI groups would not be hydrogen bonded within the helix. This percentage is very close to the fractional concentration (40%) of the fast exchanging class of NH groups, thus reinforcing the proposa1 that the fast exchanging class is comprised of NH groups that are not hydrogen bonded.
Discussion
In a system designed to study assemblies of short helices, in which the potential for
interhelical polar interactions has been rninimized, we used a unique methodology to
differentiate specific oligomen korn stnicniral States with low conformationai specificity. We observed that subtie changes in the configuration of the nonpolar interactions resulted in a large variation in the extent of conformational specificity. In particula., permutations of Leu, ne, and Val in the a and d heptad positions of the adeg consensus sequence caused the formation of the following structures: helical tetramers and pentamers, soluble and insoluble helical aggregates, insoluble unstructured aggregates, and soluble unstructured monomers. Both iiaa and vlaa fonned helical tetramers, and liaa formed helical pentamers. The stability of the specific oligomers was assessed by the concentration dependence of their helix content and this data indicated that the rank order of stability was liaa > iiaa > vlaa. The ANS dye binding experiment indicated that the same rank order exists in terms of exposed hydrophobie surface area: liaa > iiaa > vlaa.
A disulfide cross-linking experiment was used to assess the alignment of the helices in the tetramers and pentamers. This experiment indicated that anùparallel alignment of helices was present in the tetramers and pentamers. However, this method cannot eliminate the possibility that there is a mixture of both parallel and antipanllel helical bundles. Interestingly . no specific hetero-oligomers were formed in the adeg series. One possible reason for this may be the asyrnrnetry in the sequence, i.e., there are unequal numbers of a and d heptad positions in the consensus sequence. Altematively, the lack of hetero-oligorner formation may be due to the absence of interactions between amino acids in the e and g heptad positions. Previously it has been shown that, in both naturally occurring and designed coiled-coils, interhelical electrostatic interactions between amino acids in the e and g heptad positions drive hetero-oligomer formation (O'Shea et al.,
1989; O'Shea et al., 1993). The method we developed for evaiuating conformationai specificity was capable of dealing with the large number of samples generated by the peptide senes, and the array of possible structures fomed. The procedure assessed the solubility, size, and secondary structure of peptide assernblies using an effective combination of common analytical techniques. This method may be adapted for use in other systems, for example, identifying soluble, folded proteins from expression Libraries.
A number of other systematic studies have been performed on helical peptides with a heptad repeat in their sequences, whose a and d positions are occupied by Ile, Leu, or Val, exclusively. The properties of these systems are compared in Table 4. There are two major differences between our adeg peptides and the other peptide systems listed in
Table 4. First, the identities of the e and g residues in the adeg peptides are Ah, whereas in the other systems the polar residues Glu, Gln, or Lys occupy e and g positions.
Second, the length of the helical segments in the adeg series is significantly shorter (17 residues) than those in the other systems (29-35 residues). One cornmon feature of al1 the systems listed is that peptides with Leu in the a and d positions do not possess conionnational specificity. In contrast, the peptides in the adeg system, which formed specific oligomers. have oligomeric states that are one higher than the corresponding peptides in the other systems. For example, in the adeg system iiaa and vlaa formed helical tetramers, while the analogous peptides in the other systems formed trimers and dimerhimer mixtures. respectively. Furthemore. while liaa formed helical pentamers, the analogous peptides in the other systems formed helical tetramers. The srnall size of the adeg peptides could account for the higher oligomeric states observed in the adeg system compared to the coiled-coi1 systems. The smaller hydrophobic surface area of the Table 4: Cornparison of Specifcity Patterns
1 System 1 Peptide a,d,e,g Oligomerization State in Solution Name residues vIaa V.L.AbA tetramer Peptides iiaa ITI,AvA tetramer lia L.1,A.A pentamer heiix = ilaa 1L.A.A soluble helical aggregate llaa LLAA insoluble helical aggregate M viaa V.1.A.A insoluble unsfiuctured aggregate ivaa 1.V.A.A insoluble unstructured aggregate lvaa L.V,A,A soluble unstructured monomer vvaa V.V.ATA soluble unstnictured monomer I GCN4 P-VL nonspecific Analogs" P-iI trimer P-LI tetmmer heiix = PL dimer 33 aa P-LL nonspecific nonspeci fic nonsoecific
trimer helix = 34 aa
1 coü- 1 V&,P CoiI- trimer VL helix = 29 aa
" Harbury et al., et al, 1999; ' Kohn et al., 1995; et al., 1997; ' Lovejoy et al., 1993 adeg peptides may require formation of higher order oligomers for stability. Another difference between the adeg system and the other systems listed in Table 4 (except coil-
Ser) is that the adeg oligomers have an antiparallel alignment whereas the helical assemblies in the other systems are parallel. This alignment difference may be a result of the shorter chain length of the adeg peptides. While there are numerous examples of antiparallel assemblies of short helices (< 23 residues) (Hill & DeGrado, 1998;
Schafmeister et al., 1993; Prive et al., 1999; Taylor et al., 1996). we are unaware of any examples of parallel assemblies of short helices less than 23 residues in length (Lumb et al., 1994).
Our finding that liaa forms pentamenc helical bundles is notewonhy because this is a relativeiy uncommon structure. Designed five helix bundles include PEN COL-1 which is an amphiphilic helical peptide containing penodic prolines (Kitiikuni et al.,
1994), and a "peptoid" pentamer which is a polymer of N-substituted glycines (Armand et al., 1998). Naturdly occumng five helix bundles include cartilage oligomenc matrix protein (COMP)(Malashkevich et al., 1996), phospholamban (Simmerman et al., 1996), acetylcholine receptor (Opella et al., 1999), and shiga-like toxin (Stein et al., 1992). One feature of dl these five helix bundles is that they possess a central pore. The peptide liaa may form a pore, as well. As mentioned earlier, it was observed in the ANS dye binding experiment that liaa showed a large increase in ANS fluorescence (see Figure 2D).
Moreover, we observed that addition of benzene to saturation (18 mM final concentration) to the liaa+ANS sample caused a 30% decrease in the ANS fluorescence; the ANS fluorescence of al1 other adeg peptides did not decrease upon adding benzene (data not shown). It is possible, therefore, that the benzene was bound by the liaa oligomer in a cenual cavity.
Conciusions
The extensive work done on GCN4 analogs (O'Shea et al.. 1991: Lumb & Kim. 1995:
Gonzalez et al., 1996) and from de novo designed helical bundles (Handel et al., 1993;
Hill & DeGrado, 1998) has led to a working definition for structurai uniqueness based on the following cntena: cooperative thermal unfolding, dispersion in NMR spectra, a high level of protection from amide proton exchange, the existence of a single oligomeric state, and low ANS binding. Protein structures that do not meet al1 the above criteria are not considered to be structurally unique and are designated as nonspecific. Moreover. the above work has led to the hypothesis that hydrophobic interactions contribute stability, but not necessarily specificity to protein structure. The present work used a system in which interhelical polar sidechain interactions have been minimized and buned polar interactions are absent; therefore, effects on specificity can be solely attributed to nonpolar interactions. We found that conservative sequence changes entailing either a rearrangement or deletion of methyl groups produced a distribution of States that differ in conformational specificity, dthough they are not stnicturally unique. At one end of this distribution we found hydrophobic interface sequences that yielded peptides with extremely low conformational specificity in that they formed helical aggregates that precipitated. Adjacent in this distribution were polymeric assemblies of helices with
hydrodynamic radii of 20 m. At the other end of the distribution, we found particular sequences yielding peptides that possessed a relatively high degree of conformationai specificity, in that they hedunique tetrameric and pentamenc States. However, amide proton exchange indicated that vlaa, the adeg peptide with the greatest conformationai specificity, still lacked structural uniqueness. Our results indicate that nonpolar interactions can make a significant contribution to conformational specificity albeit the contribution may be less than that of buried polar interactions. It appears that a quantitative method is required to factorire the contributions of different interactions to conformational specificity.
Acknowledgments
This work was supported by a gnnt from the Medical Research Council of Canada. C.B. ac knowledges financial support from the Ontario Student Opportunity Tram fer Fund.
We thank Robert Fairman and Alan Davidson for valuable suggestions, Gene Merutka and Thomas Leung, for experimental contributions early in this work. and Xiao-Fei Qi,
Sandy Go, Jackson Huang, and JoAnne McLaurin For technical assistance. CHAPTER 3: FUTURE WORK
Structural basis for Conformational Specificity in the adeg peptides
It has been shown that peptides in the adeg series possess various degrees of conformational specificity. The differences are due solely to changes in the identities of the nonpolar amino acids at specific positions in the adeg consensus sequence. A structural basis for differences in conformational specificity may be elucidated by a three- dimensional structure as detemùned by NMR spectroscopy or by x-ray crystallography.
Without a crystal structure or an NMR solution structure, 3D-computer modelling may be used to predict answers to structural puzzles. The program Swiss PDB Viewer (SPDBV) was used to build models of selected adeg peptide monomers. Using the "ridges-into- grooves" scheme of helix association (Chothia et al., 1981) as a frarnework, the peptide models were examined in an attempt to gain insight into the structural underpinnings of conformationai specificity in the adeg system.
The peptides iiaa, vlaa, and llaa were chosen for mode1 building because they exemplify the diversity of structural States observed in the adeg series: iiaa and vlaa formed specific helical tetramers, while llaa formed nonspecific helical aggregates.
Using the program SPDBV, models of the sequences of ha, iiaa, and vlaa were generated as ideal a-helices, where = -65' and w = -40'. In order to generate plausible sidechain conformations of the Leu, ne, and Val residues, the peptide models were superimposed ont0 the crystal structure of a de novo designed antiparallel4-helix bundle protein called DHP,, whose PDB accession code is 4HB 1. The "Magic Fit" and
"Iterative Magic Fit" functions of the software were used to superimpose the adeg peptide models onto one helix in the 4HB1 structure through a built-in 3D structure alignment procedure. In the resulting structure alignrnent, the Leu, Ile, and Val residues of the adeg models coincided with the helical interface region of 4HB 1 (see Table 5). Furthemore, the software-generated default sidechain conformations of the Leu, Ile, and Val residues in the adeg models closely approximated the sidechain conformations of the corresponding residues in the 4HB 1 structure. Although sampling a11 the sidechain rotamers available in the software library altered the sidechain conformations of the Leu,
Ile, and Val residues in the adeg models, this process did not appear to improve the structure alignment with 4HB 1.
In order to speculate on the reasons for differences in oligomerization among the adeg peptides, the adeg peptide models were examined using the "ridges-into-grooves" modeI of helix association (Chothia et al., 1981) as a frame of reference, In this rnodel, the surface of an a-helix is cornposed of rows of sidechains usually separated by 3 or 4 residues in the sequence (Branden & Tooze, 1999). The sidechain rows form the
"ridges", where adjacent mws are separated by shallow indentations or "grooves".
Moreover, associating helices are characterized in terms of the angle between helical axes, denoted by B. The most cornmon ridges are called the t 4n rows and the i 3n rows, so-named because they are formed by amino acid sidechains separated by four and three residues in the sequence, respectively (Branden & Tooze, 1999). According to the scheme of Chothia et al., helix association occurs when ridges on the surface of one helix fit into the grooves of another helix in the assembly. The iiaa, vlaa, and llaa peptide models were inspected to identify potential "ridges" and "grooves". In each of the peptide models, the + 4n ridges formed by residues 2.6 and 9, 13 were readily observed. Moreover, in the adeg rnodels these positions corresponded with the a and d heptad positions, occupied by the nonpolar amino acids Ile (iiaa), ValLeu (vlaa), and Leu (llaa).
The Ile sidechains in the iiaa model were arranged in the rnost uniform ~4nndges, while the ValLeu sidechains in the vlaa mode] and the Leu sidechains in the llaa model formed less uniform iIn ridges (Figure 9). The 13n ndges were not as visually clear as the +4n ridges, but sidechains in positions 6, 9 and 13, 16 could fom +3n ridges composed of nonpolar amino acids. However, the differences in the ridges formed by vlaa versus llaa were too subtle to make a reasonable speculation as to why vlaa formed specific oligomers while llaa did not. Moreover, although iiaa and vlaa both formed tetramers, the iiaa oligomers were more stable than the vlaa oligomers. Examination of the iiaa and vlaa peptide models did not provide an obvious reason for this stability difference.
According to Chothia's model, the association of two helices, each containing the k4n ridges, occurs such that the helices pack with R = -50' (4-4 packing) (Chothia et al.,
1981). Furthermore, if a helix containing i4n ridges associates with a helix containing gnridges, the helices pack such that R = 20' (3-4 packing) (Chothia et al., 198 1). It has been shown that the a-helices in Chelix bundles pack together with R = 20' (Branden &
Tooze, 1999), corresponding to the 3-4 packing model. The appearance of the GIIndges in the iiaa, vlaa, and llaa peptide models suggests that their sequences did not lead to the formation of optimal +3n ridges. It was not clear that a proper groove existed between the fin ridges in order to accommodate the 3-4 packing. The ndge geometry observed in the adeg peptide models rnay favour the 4-4 packing, where SZ = -50'. Perhaps the adeg sequence design contains an inherent geometric conflict that may affect stability, since nanirally occurring 4-helix bundles seem to favour 3-4 packing. In summary. the structural differences visible in the cornputer-generated models of iiaa, vlaa. and llaa did not lead to a clear explmation for the resulting differences in oligomenc states. Without an actual structure determination by x-ray crystallography or
NMR spectroscopy, it was not possible to develop a plausible mode1 for the oligomenc state preferences of the adeg peptides. However, the model-building exercise elucidated some of the inherent geomevical features of the adeg design, narnely the formation of obvious k4n ridges. This information will be useful in order to improve future designs of short helical assernblies. Table 5: Sequence alignment of üaa and one helix of the 4HB1 protein.
Peptide Sequence
iiaa DIAQAI KQIAEAI QKI A CEELLKQALQQAQQLLQQAQ Figure 9. Ridge Geometry of Maa, vlaa, and iiaa. The amino acids fonning the * 4x1 ndges are shown in the peptide models for ha, vlaa, and iiaa. The helix backbone is represented as a nbbon. The sidechains in red correspond to sequence positions 2 and 6, while the residues in green correspond to sequence positions 9 and 13. The highlighted amino acids are the nonpolar amino acids ne (iiaa), VaVLeu (vlaa), and Leu (llaa) which correspond to heptad positions a (positions
6 and 13) and d (positions 2 and 9). The Next Design
Using the adeg system it has been show qualitatively that nonpolar interactions make a significant contribution to conformational specificity. It would be useful to have a quantitative method for studying the effects of nonpolar and other interactions on conformational specificity and stability. In other studies on helical systems (GCN4, Rop,
V,L,, etc.) the presence of multiple interactions in these systems has made it complicated to resolve the contributions of individual interactions to stability and specificity. The adeg system will be adapted and simplifieci to serve as a framework to quantitate the contribution of individual interactions to stability and specificity in helical assemblies.
Although the adeg consensus sequence was designed to minimize the presence of interhelical polar interactions, the new system will eliminate the possibili ty of polar interactions affecting specificity. Interhelical H-bonds and salt bridges will be completely avoided by placing lysine in the b, c, and f heptad positions (i.e., al1 sequence positions other than the a, d, e, g heptad positions). Using vlaa as a model, the a, d, e, g heptad positions will be occupied by Val, Leu, Ah, Ala, respectively. The peptide vlaa possessed marginal stability and conformational specificity. Thus, small sequence changes in the new system will have a marked and measurable effect on both stability and specificity. Furthemore, the possibility of forming a mixture of parallel and antiparallel helicai bundles will be removed by synthesizing helical hairpin structures using the loop structure (Pro-Arg-Arg) employed by DeGrado et al., in the designed protein (DeGrado, et al., 1998). The stability of the helical bundes will be assessed by meauring the association and dissociation constants of the helical hairpin structures by analytical ultracentrifugation and CD spectroscopy, respectively. The conformational specificity of the system will be assessed using analytical ultracentrifugation by measuring the molecular weight species present at equilibrium. Moreover. Hl-exchange using mass spectrometry will also be performed and the protection factor will be used as a quantitative index of specificity. Once the new system has been characterized, different interactions will be engineered into the hydrophobie helical interface, e.g., aromatic- aromatic interactions. salt bridge, sidechain-sidechain H-bonds, etc., and their effect on stability and speci ficity wi 11 be quantitative1y determined using analytical ultracentrifugation and mass spectrometry. It will be interesting to observe how the effects of the various interactions on stability and specificity correlate, and it may be possible to identify emerging themes. Armand P, Kirshenbaum K, Goldsmith RA. Farr-Jones S. Barron AE, Truong KT. Dill
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For cooperative ligand binding, the Hill equation may be used to relate the fraction of binding sites filled to the ligand concentration and dissociation constant of the reaction P+nA- PA, where P represents a macromolecule with n binding sites, and A represents the ligand molecule. The dissociation constant for the above reaction is given by
The fraction bound is given by
The rbove is commonly written as follows and is cailed the Hill Equation
where n is referred to as the Hill coefficient. A system in which a peptide, A. oligomerizes or self-assembles, A,, may be represented by the reaction A-%, where n is the order of oligomerization. For the above reaction, the dissociation constant is given by
Furthemore, the total peptide concentration, [AT], is given by [41= [Al+n[q,l so that the monomer concentration rnay be expressed as [Al = MI-1- n[41- Substitute 2 into 1 to obtain K, in terms of the total peptide concentration In this system, the "fraction oligomer" is andogous to the "fraction bound in ligand binding. The fraction oligomer, denoted by r', is given by
For a helical assembly, the fraction oligomer may also be expressed in tenns of the ellipticity of the peptide at 222 nrn:
r'= 6,s - 'mon Cs1 6" -%ln The oligomer concentration may be expressed in terms of the ellipticity by equating 4 and
Substitute 6 into 3 and solve for [AT]to obtain a modified version of the Hill equation for oligornerized helices:
la)" - n[AT (1 a)" Kd = ([AT 1 - 14 - ln-'- a
Solving for [AT]we obtain
= a(i - a)"' Resubstitute 7 into 8 to obtain the modified Hill equation: