DOCSLIB.ORG

This Article Appeared in a Journal Published by Elsevier. the Attached

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
This Article Appeared in a Journal Published by Elsevier. the Attached This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Archives of Biochemistry and Biophysics 531 (2013) 4–13 Contents lists available at SciVerse ScienceDirect Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi Review The stability of 2-state, 3-state and more-state proteins from simple spectroscopic techniques... plus the structure of the equilibrium intermediates at the same time ⇑ Javier Sancho Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza. Pedro Cerbuna 12, 50009 Zaragoza, Spain Biocomputation and Complex Systems Physics Institute (BIFI), Joint Unit BIFI-IQFR, CSIC, Universidad de Zaragoza. Mariano Esquillor, Edificio I+D, 50018 Zaragoza, Spain article info abstract Article history: Protein stability is not just an academic matter. Biotechnology, Cell Biology and Drug Design are some of Available online 8 November 2012 the fields where both theoretical and practical knowledge of protein stability is required. Simple equip- ment and chemicals, such as a thermostated fluorimeter and common denaturants, suffice to determine the conformational stability of a protein. To this end, the most important experiments are the preliminary ones done to establish the minimum number of species (conformations) accumulating in the equilibrium. For proteins with non-functional equilibrium intermediates, determining the relevant stability of the pro- tein (the free energy difference between the native conformation and the intermediate) is most important, and it allows very valuable structural information on the intermediate to be derived when protein variants are compared to wild type using equilibrium /-analysis. The principles, tricks and equations involved in the analysis of denaturant induced or temperature induced equilibrium unfolding curves by the linear extrapolation method or using the integrated Gibbs–Helmholtz equation, respectively, will be discussed, and a brief outline of challenges and frontiers in the protein stability field will be presented. Ó 2012 Elsevier Inc. All rights reserved. The different types of protein stability a low chemical or biochemical stability is that the population of ac- tive protein molecules will be progressively reduced in an irrevers- Proteins are the molecules that do most things in living beings, ible manner because the reactions involved tend to be irreversible and the wonders they do are almost always related to their molec- and tend to compromise function. Increasing their chemical stabil- ular surfaces and intrinsic dynamics. These, in turn, are shaped by ity is an issue for proteins that are used in biotechnological pro- folding reactions that lead, for each protein, to stabilization of one cesses, which sometimes require extreme conditions such as high specific tridimensional structure among the inconceivably large temperature, low pH, presence of cosolvents, etc. [2]. In contrast, number of possible alternative conformations. In apparent con- the biochemical stability of any given protein, whether high or trast, there are proteins that display unfolded conformations under low, is in principle a functional property that might be thought of native solution conditions [1]. Yet, some of these proteins have as being best left unmodified. However, the increasing availability been observed to become folded when they bind to appropriate of biologics as therapeutic agents [3] makes the rational tailoring molecular targets, which indicates the stabilization of those pro- of biological stability an important goal. teins into defined structures is coupled to binding events. Being The conformational stability of a protein is something different. conformationally stable at some time seems to be a general requi- A protein molecule that leaves the ribosome and becomes folded site of functional proteins. can still experience the inverse process of becoming unfolded It should be noticed that ‘‘protein stability’’ is an ambiguous term and then folded again for as long as its chemical integrity is main- that may refer to several different things (Fig. 1). The chemical or tained. Therefore, for any population of identical protein molecules biochemical stability of a protein is related to its resistance to expe- there is an equilibrium constant (Kf) that governs the fraction of rience changes in its covalent structure, i.e., have some covalent molecules that are folded (vf) or unfolded (vu). bonds cleaved or some atoms replaced. The consequence of having Kf ¼ vf =vu ð1Þ Such an equilibrium constant can be determined using many ⇑ Address: Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza. Pedro Cerbuna 12, 50009 Zaragoza, Spain. different techniques and it can be mathematically transformed into Fax: +34 976762123. a free energy difference of folding (DGf) which is usually referred to E-mail address: [email protected] as the conformational stability of the protein. 0003-9861/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.abb.2012.10.014 Author's personal copy J. Sancho / Archives of Biochemistry and Biophysics 531 (2013) 4–13 5 DGf ¼RT ln Kf ð2Þ etc. Mechanical stability refers to the resistance of native proteins to be unfolded by external forces, and it is studied with atomic It is easy to get confused with the sign of this magnitude be- force microscopes or optical tweezers [8]. Mechanical unfolding cause there is a tendency in the literature to state that if the free is usually reversible and the magnitude of the force needed to un- energy of folding of a protein is, say, À10 kcal/mol, then its confor- fold a particular protein changes with the orientation of the force. mational stability is 10 kcal/mol. This is in part explained by the This indicates that different transition states may be sampled fact that what is determined and reported in most cases is the free when the force is applied at different points. Thus the mechanical energy of unfolding, which for a stable protein bears, of course, a stability of a protein will be related to the specific energy barrier positive sign. that has to be crossed, given the force applied, to convert native Equilibrium constants are ratios of kinetic constants, and two molecules into unfolded ones. It seems that b proteins tend to dis- proteins with the same value for their folding equilibrium constants play higher mechanical stabilities than a proteins. may differ much in their folding and unfolding rate constants. This It is common that in the folding/unfolding equilibrium of small is not without practical consequences because the reactivity of proteins the only conformations that populate to measurable molar folded and unfolded states [4] may not be the same. A protein rap- fractions are the native, folded state and the unfolded state (U M F) idly sampling the folded and unfolded states may often get a chance [9,10]. In contrast, for most proteins, additional intermediate con- to engage in reactions only possible for or just much faster in the formations (less folded than the native state but more folded than unfolded conformation. If these are irreversible reactions leading the denatured state) accumulate as the solution conditions become to loss of function, a much greater fraction of such a protein may be- more destabilizing of the native conformation, yet not fully stabiliz- come inactive in a given time period than in a protein with the same ing of the denatured state [11]. The number of species that will ap- conformational stability but with slow unfolding kinetics. Proteins pear as the solution conditions are shifted from native to denaturing whose native conformation is preserved from experiencing inacti- will depend on the specific shape of the folding landscape in native vation related to reactions of its unfolded conformation due to their conditions and on the changes that the specific denaturing agent slow unfolding kinetics are considered to display a high kinetic sta- used to probe the equilibrium will induce in that shape. The more bility [5]. It seems now that aggregates rich in b-strands may be for complex the landscape, the more likely a higher number of interme- many, perhaps most, proteins more stable than their corresponding diates will be observed. On the other hand, a particular denaturing native conformations [6]. It is thus possible that proteins have agent (say, urea) may not reveal the presence of a potential interme- evolved to protect their native conformations from transformation diate while other (say, temperature) may do so [12]. For proteins into more stable aggregated conformations such as those mani- displaying equilibrium intermediates, the global equilibrium be- fested in aggregative diseases [7]. A high energy barrier between tween the native and the unfolded states can be divided
Load more

Recommended publications
  • Protein Unfolding (Model Peptide Helix/Helix-Coil Transition/Denaturant Effects) J
    Proc. Natl. Acad. Sci. USA Vol. 92, pp. 185-189, January 1995 Biochemistry Urea unfolding of peptide helices as a model for interpreting protein unfolding (model peptide helix/helix-coil transition/denaturant effects) J. MARTIN SCHOLTZ*tt, DOUG BARRICK*§, EUNICE J. YORK0, JOHN M. STEWART$, AND ROBERT L. BALDWIN*: *Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 97305-5307; tDepartment of Medical Biochemistry and Genetics, Center for Macromolecular Design, Texas A&M University, College Station, TX 77843-1114; and 1Department of Biochemistry, University of Colorado Health Sciences Center, Denver, CO 80262 Contributed by Robert L. Baldwin, August 29, 1994 ABSTRACT To provide a model system for understand- The peptides examined here consist primarily of alanine;"1 ing how the unfolding of protein a-helices by urea contributes thus side-chain interactions are minimal, and the primary to protein denaturation, urea unfolding was measured for a structural and thermodynamic changes during helix unfolding homologous series of helical peptides with the repeating should result from the peptide backbone. Because these helical sequence Ala-Glu-Ala-Ala-Lys-Ala and chain lengths varying peptides lack hydrophobic cores and long-range tertiary in- from 14 to 50 residues. The dependence of the helix propa- teractions, the thermodynamics of solvent denaturation of gation parameter of the Zimm-Bragg model for helix-coil secondary structure can be measured directly. Although sec- transition theory (s) on urea molarity ([urea]) was deter- ondary structure formation must be an integral part of the mined at 0°C with data for the entire set of peptides, and a protein folding reaction, these other factors (hydrophobic core linear dependence ofIns on [urea] was found.
    [Show full text]
  • State Folding of Globular Proteins
    Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 20 February 2020 doi:10.20944/preprints202002.0290.v1 Peer-reviewed version available at Biomolecules 2020, 10, 407; doi:10.3390/biom10030407 Review The Molten Globule, and Two-State vs. Non-Two- State Folding of Globular Proteins Kunihiro Kuwajima,* Department of Physics, School of Science, the University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, and School of Computational Sciences, Korea Institute for Advanced Study (KIAS), Seoul 02455, Korea ; [email protected] * Correspondence: [email protected] Abstract: From experimental studies of protein folding, it is now clear that there are two types of folding behavior, i.e., two-state folding and non-two-state folding, and understanding the relationships between these apparently different folding behaviors is essential for fully elucidating the molecular mechanisms of protein folding. This article describes how the presence of the two types of folding behavior has been confirmed experimentally, and discusses the relationships between the two-state and the non-two-state folding reactions, on the basis of available data on the correlations of the folding rate constant with various structure-based properties, which are determined primarily by the backbone topology of proteins. Finally, a two-stage hierarchical model is proposed as a general mechanism of protein folding. In this model, protein folding occurs in a hierarchical manner, reflecting the hierarchy of the native three-dimensional structure, as embodied in the case of non-two-state folding with an accumulation of the molten globule state as a folding intermediate. The two-state folding is thus merely a simplified version of the hierarchical folding caused either by an alteration in the rate-limiting step of folding or by destabilization of the intermediate.
    [Show full text]
  • Acid Dissociation Constant - Wikipedia, the Free Encyclopedia Page 1
    Acid dissociation constant - Wikipedia, the free encyclopedia Page 1 Help us provide free content to the world by donating today ! Acid dissociation constant From Wikipedia, the free encyclopedia An acid dissociation constant (aka acidity constant, acid-ionization constant) is an equilibrium constant for the dissociation of an acid. It is denoted by Ka. For an equilibrium between a generic acid, HA, and − its conjugate base, A , The weak acid acetic acid donates a proton to water in an equilibrium reaction to give the acetate ion and − + HA A + H the hydronium ion. Key: Hydrogen is white, oxygen is red, carbon is gray. Lines are chemical bonds. K is defined, subject to certain conditions, as a where [HA], [A−] and [H+] are equilibrium concentrations of the reactants. The term acid dissociation constant is also used for pKa, which is equal to −log 10 Ka. The term pKb is used in relation to bases, though pKb has faded from modern use due to the easy relationship available between the strength of an acid and the strength of its conjugate base. Though discussions of this topic typically assume water as the solvent, particularly at introductory levels, the Brønsted–Lowry acid-base theory is versatile enough that acidic behavior can now be characterized even in non-aqueous solutions. The value of pK indicates the strength of an acid: the larger the value the weaker the acid. In aqueous a solution, simple acids are partially dissociated to an appreciable extent in in the pH range pK ± 2. The a actual extent of the dissociation can be calculated if the acid concentration and pH are known.
    [Show full text]
  • Global Analysis of Protein Stability by Temperature and Chemical Denaturation
    bioRxiv preprint doi: https://doi.org/10.1101/2020.04.20.049429; this version posted April 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Global analysis of protein stability by temperature and chemical denaturation Louise Hamborg, Emma Wenzel Horsted, Kristoffer Enøe Johansson, Martin Willemoës, Kresten Lindorff-Larsen and Kaare Teilum* Structural Biology and NMR laboratory and the Linderstrøm-Lang Centre for Protein Science, Department of Biology, University of Copenhagen, Ole Maaloes Vej 5, 2200 Copenhagen N, Denmark * Corresponding author Phone: +45 35 32 20 29 Postal Address: Ole Maaloes Vej 5, 2200 Copenhagen N, Denmark Email: [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.04.20.049429; this version posted April 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Abstract The stability of a protein is a fundamental property that determines under which conditions, the protein is functional. Equilibrium unfolding with denaturants requires preparation of several samples and only provides the free energy of folding when performed at a single temperature. The typical sample requirement is around 0.5 – 1 mg of protein. If the stability of many proteins or protein variants needs to be determined, substantial protein production may be needed.
    [Show full text]
  • Revisiting Absorbance at 230Nm As a Protein Unfolding Probe
    Analytical Biochemistry 389 (2009) 165–170 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio Revisiting absorbance at 230 nm as a protein unfolding probe Pei-Fen Liu a,b, Larisa V. Avramova c, Chiwook Park a,b,c,* a Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, Heine Pharmacy Building, Room 224A, 575 Stadium Mall Drive, West Lafayette, IN 47907-2091, USA b Purdue University Interdisciplinary Life Science Program (PULSe), Purdue University, West Lafayette, IN 47907-2091, USA c Bindley Bioscience Center, Purdue University, West Lafayette, IN 47907-2057, USA article info abstract Article history: Thermodynamic stability and unfolding kinetics of proteins are typically determined by monitoring pro- Received 19 February 2009 tein unfolding with spectroscopic probes, such as circular dichroism (CD) and fluorescence. UV absor- Available online 24 March 2009 bance at 230 nm (A230) is also known to be sensitive to protein conformation. However, its feasibility for quantitative analysis of protein energetics has not been assessed. Here we evaluate A230 as a structural Keywords: probe to determine thermodynamic stability and unfolding kinetics of proteins. By using Escherichia coli Protein stability maltose binding protein (MBP) and E. coli ribonuclease H (RNase H) as our model proteins, we monitored UV absorbance their unfolding in urea and guanidinium chloride with A . Significant changes in A were observed 230 nm 230 230 with both proteins on unfolding in the chemical denaturants. The global stabilities were successfully Conformational change Unfolding kinetics determined by measuring the change in A230 in varying concentrations of denaturants. Also, unfolding kinetics was investigated by monitoring the change in A230 under denaturing conditions.
    [Show full text]
  • Kinetic Evidence for a Two-Stage Mechanism of Protein Denaturation by Guanidinium Chloride
    Kinetic evidence for a two-stage mechanism of protein denaturation by guanidinium chloride Santosh Kumar Jhaa,1 and Susan Marquseea,b,2 aCalifornia Institute for Quantitative Biosciences and bDepartment of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3220 Edited by Robert L. Baldwin, Stanford University, Stanford, CA, and approved February 14, 2014 (received for review August 14, 2013) Dry molten globular (DMG) intermediates, an expanded form of hydrophobic core, resulting in global structural disruption. Ex- the native protein with a dry core, have been observed during perimental support for this model has, however, been scarce and denaturant-induced unfolding of many proteins. These observa- indirect (4, 6, 13, 26). tions are counterintuitive because traditional models of chemical Here, we demonstrate that the well-studied protein Escherichia denaturation rely on changes in solvent-accessible surface area, coli ribonuclease HI (RNase H) (28–30) also rapidly populates and there is no notable change in solvent-accessible surface area a DMG state when exposed to denaturant and use this observation during the formation of the DMG. Here we show, using multisite to explore the denaturant dependence of the DMG. We use fluorescence resonance energy transfer, far-UV CD, and kinetic multisite fluorescence resonance energy transfer (FRET) and thiol-labeling experiments, that the guanidinium chloride (GdmCl)- kinetic thiol-labeling measurements to dissect the temporal or- induced unfolding of RNase H also begins with the formation of der of protein expansion, side-chain disruption, and water sol- the DMG. Population of the DMG occurs within the 5-ms dead time vation during GdmCl-induced unfolding.
    [Show full text]
  • I. Equilibrium Unfolding Studies of Cytochrome C. II. the Role of Helix 1 Aspartates in the Stability and Conversion of Prion Protein
    University of Montana ScholarWorks at University of Montana Graduate Student Theses, Dissertations, & Professional Papers Graduate School 2003 I. Equilibrium unfolding studies of cytochrome C. II. The role of helix 1 aspartates in the stability and conversion of prion protein Jonathan O'Connor Speare The University of Montana Follow this and additional works at: https://scholarworks.umt.edu/etd Let us know how access to this document benefits ou.y Recommended Citation Speare, Jonathan O'Connor, "I. Equilibrium unfolding studies of cytochrome C. II. The role of helix 1 aspartates in the stability and conversion of prion protein" (2003). Graduate Student Theses, Dissertations, & Professional Papers. 9470. https://scholarworks.umt.edu/etd/9470 This Dissertation is brought to you for free and open access by the Graduate School at ScholarWorks at University of Montana. It has been accepted for inclusion in Graduate Student Theses, Dissertations, & Professional Papers by an authorized administrator of ScholarWorks at University of Montana. For more information, please contact [email protected]. Maureen and Mike MANSFIELD LIBRARY The University of Montana Permission is granted by the author to reproduce this material in its entirety, provided that this material is used for scholarly purposes and is properly cited in published works and reports. **Please check "Yes" or "No" and provide signature** Yes, I grant permission ___ No, I do not grant permission ___ Author's Signature:__ Date: ____ 3 Any copying for commercial purposes or financial gain may be undertaken only with the author's explicit consent. 8/98 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
    [Show full text]
  • Microcalorimetric Techniques
    Microcalorimetric techniques Isothermal titration calorimetry (ITC) Differential scanning calorimetry (DSC) Filip Šupljika [email protected] Laboratory for the study of interactions of biomacromolecules Division of Organic Chemistry and Biochemistry Ruđer Bošković Institute, Zagreb, Croatia Isothermal titration calorimetry (ITC) - characterization of binding reactions between proteins and ligands or other macromolecules - enzyme kinetics Differential scanning calorimetry (DSC) - characterization of the thermal stability of proteins and other macromolecular assemblies Isothermal titration calorimetry (ITC) Isothermal titration calorimetry (ITC) VP-ITC PEAQ-ITC ITC – binding ITC – enzyme kinetics • based on simple Michaelis-Menten mechanism kcat - constant that describes the turnover rate of an enzyme-substrate complex to k k ES 1 kcat cat tot E + S ES E + P v product and enzyme k1 Km S KM - Michaelis constant that describes the • Two experimental methods: amount of substrate needed for the enzyme - single injection method (SIM) to obtain half of its maximum rate of reaction - multiple injection method (MIM) ITC – enzyme kinetics - SIM 10 START STOP 9 8 SINGLE CONTINUOUS INJECTION µcal/sec 100 ul 2.2 mM 2'CMP injected in 16 minutes into .06 mM RNase 7 6 0.00 5.00 10.00 15.00 20.00 Time (min) ITC – enzyme kinetics - MIM dP[] 1 dQ v dt Vr H dt dQ/dt cal/sec) µ P( K = 80 M M Rate (millimoles/l/sec) k = 215 s-1 cat Time(sec) [S] (mM) Differential scanning calorimetry (DSC) The Nano DSC Cutaway views of the Nano-DSC New Nano DSC • Platinum capillary cells • New USB connection to computer • Innovative sensor design • Unmatched sensitivity The Nano DSC Cell Geometry .
    [Show full text]
  • Doc 6259.Indd
    SCIENTIFIC CORRESPONDENCE For these reasons, it is unlikely that the does not affect our analysis of unfolding as chaperones, whereas unfolding does not unfolding approach will provide new in­ it occurs after the rate-determining step depend on these factors. Far from the sight into the still enigmatic process of in the unfolding direction and accumu­ unfolding approach being unlikely to protein folding. lates only slightly in the transition region provide new insight into protein folding , it JOHANNES BUCHNER (unpublished data). should be clear from the above that un­ lnstitut fiir Biophysik und Physikalische Third, we stated clearly that micro­ folding studies are an essential element in Biochemie, scopic reversibility applies under identical the understanding of folding, and are just Universitat Regensburg, reaction conditions and for a reversible as important as refolding studies. Universitatstrasse 31< process (ref. 1; see also ref. 7, p. R9-90). It A.R. FERSHT D-8400 Regensburg, holds not just for "strongly denaturing J.T. KELLISJR FRG conditions" but for all concentrations of A.T.E.L. ~ATOUSCHEK urea, because folded and unfolded protein l. SERRANO THOMAS KIEFHABER always exist in equilibrium even though MRC Unit for Protein Function and Laboratorium fiir Biochemie, the equilibrium constant may be far from Design, Universitat Bayreuth, unity. At any one of these concentrations, University Chemical Laboratory, Postfach 10 12 51, the transition states for folding and un­ Lensfield Road, D-8580 Bayreuth, folding are the same. The question is Cambridge CB2 1EW, FRG whether or not the transition state changes UK with change of denaturant. Creighton" FERSHT ET AL.
    [Show full text]
  • Unravelling the Complex Denaturant and Thermal-Induced Unfolding Equilibria of Human Phenylalanine Hydroxylase
    International Journal of Molecular Sciences Article Unravelling the Complex Denaturant and Thermal-Induced Unfolding Equilibria of Human Phenylalanine Hydroxylase María Conde-Giménez 1 and Javier Sancho 1,2,* 1 Departamento de Bioquímica y Biología Molecular y Celular, Biocomputation and Complex Systems Physics Institute (BIFI)-Joint Units: BIFI-IQFR (CSIC) and GBsC-CSIC, University of Zaragoza, 50009 Zaragoza, Spain; [email protected] 2 Aragon Health Research Institute (IIS Aragón), 50009 Zaragoza, Spain * Correspondence: [email protected] Abstract: Human phenylalanine hydroxylase (PAH) is a metabolic enzyme involved in the catabolism of L-Phe in liver. Loss of conformational stability and decreased enzymatic activity in PAH variants result in the autosomal recessive disorder phenylketonuria (PKU), characterized by developmental and psychological problems if not treated early. One current therapeutic approach to treat PKU is based on pharmacological chaperones (PCs), small molecules that can displace the folding equilib- rium of unstable PAH variants toward the native state, thereby rescuing the physiological function of the enzyme. Understanding the PAH folding equilibrium is essential to develop new PCs for different forms of the disease. We investigate here the urea and the thermal-induced denaturation of full-length PAH and of a truncated form lacking the regulatory and the tetramerization domains. For either protein construction, two distinct transitions are seen in chemical denaturation followed by fluorescence emission, indicating the accumulation of equilibrium unfolding intermediates where Citation: Conde-Giménez, M.; the catalytic domains are partly unfolded and dissociated from each other. According to analytical Sancho, J. Unravelling the Complex centrifugation, the chemical denaturation intermediates of either construction are not well-defined Denaturant and Thermal-Induced species but highly polydisperse ensembles of protein aggregates.
    [Show full text]
  • Characterization of the Folding and Unfolding Reactions of Single-Chain Monellin: Evidence for Multiple Intermediates and Competing Pathways† Ashish K
    Biochemistry 2007, 46, 11727-11743 11727 Characterization of the Folding and Unfolding Reactions of Single-Chain Monellin: Evidence for Multiple Intermediates and Competing Pathways† Ashish K. Patra and Jayant B. Udgaonkar* National Centre for Biological Sciences, Tata Institute of Fundamental Research, GKVK Campus, Bangalore 560065, India ReceiVed June 11, 2007; ReVised Manuscript ReceiVed August 7, 2007 ABSTRACT: The mechanisms of folding and unfolding of the small plant protein monellin have been delineated in detail. For this study, a single-chain variant of the natively two-chain monellin, MNEI, was used, in which the C terminus of chain B was connected to the N terminus of chain A by a Gly-Phe linker. Equilibrium guanidine hydrochloride (GdnHCl)-induced unfolding experiments failed to detect any partially folded intermediate that is stable enough to be populated at equilibrium to a significant extent. Kinetic experiments in which the refolding of GdnHCl-unfolded protein was monitored by measurement of the change in the intrinsic tryptophan fluorescence of the protein indicated the accumulation of three transient partially structured folding intermediates. The fluorescence change occurred in three kinetic phases: very fast, fast, and slow. It appears that the fast and slow changes in fluorescence occur on competing folding pathways originating from one unfolded form and that the very fast change in fluorescence occurs on a third parallel pathway originating from a second unfolded form of the protein. Kinetic experiments in which the refolding of alkali-unfolded protein was monitored by the change in the fluorescence of the hydrophobic dye 8-anilino-1-naphthalenesulfonic acid (ANS), consequent to the dye binding to the refolding protein, as well as by the change in intrinsic tryptophan fluorescence, not only confirmed the presence of the three kinetic intermediates but also indicated the accumulation of one or more early intermediates at a few milliseconds of refolding.
    [Show full text]
  • Lipid Bilayer Composition Modulates the Unfolding Free Energy of a Knotted Α-Helical Membrane Protein
    Lipid bilayer composition modulates the unfolding free PNAS PLUS energy of a knotted α-helical membrane protein M. R. Sandersa, H. E. Findlaya, and P. J. Bootha,1 aDepartment of Chemistry, King’s College London, SE1 1DB London, United Kingdom Edited by David Baker, University of Washington, Seattle, WA, and approved January 12, 2018 (received for review August 18, 2017) α-Helical membrane proteins have eluded investigation of their gregation, most helical proteins are thought to insert directly into thermodynamic stability in lipid bilayers. Reversible denaturation the membrane cotranslationally with the assistance of the curves have enabled some headway in determining unfolding free translocon apparatus. Although the exact mechanism of mem- energies. However, these parameters have been limited to deter- brane protein insertion is unclear, the final stages of protein gent micelles or lipid bicelles, which do not possess the same me- folding are most likely to occur in the lipid bilayer (4, 5). Equally, chanical properties as lipid bilayers that comprise the basis of lipids can induce posttranslational repositioning of helices and natural membranes. We establish reversible unfolding of the protein orientation within the membrane (6, 7). If the unique membrane transporter LeuT in lipid bilayers, enabling the compar- folded state is an equilibrium structure, then it should be possible ison of apparent unfolding free energies in different lipid composi- to achieve the folded structure via other pathways, as has been demonstrated for example from coexpression (8), from reas- tions. LeuT is a bacterial ortholog of neurotransmitter transporters sembly of protein fragments (9), or from cell-free synthesis (10).
    [Show full text]