Molecular Recognition in Gas Phase: Theoretical Andexperimental Study of Non-Covalent Protein-Ligand Complexes by Mass-Spectrometry

Molecular recognition in gas phase: theoretical andexperimental study of non-covalent protein-ligand complexes by mass-spectrometry

Andrey Dyachenko

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$%/2&909 \ *)0{{=9~)(= {) {)90"= WY0V) WY0V'#+H *&+ =& * M' %6^%M 234^4. '11M $%31&= = = "WY0V) #=, =,+.=< Q.9=<.H+,+.=.=&.<.>/M+.<.>/:-/*+&* 43 . M.4^/%P'114 $%3%&+Q ,V QP "W 8WY0V: )) #=,+&=& > =,*+= +&+=<&- +*,/ 6 ' ''.^/3Q'116 $%3'&}} 0W }"\ 0Y9; #"9 =,:., +=R: :,+ *&+ =& M 3 /%M^'37'116 $%3.&, ~* 0 =V 9*" 0Y9) #==9< + O.HM+.&- +*,/ 46 3M%^41MP %22M $%3/&*("\ 0Y9 #2.9=< .HM+.&- +*,/ %.6 3 3/.^2Q'113 $%33&PQ77P=Q P,\*0Y~"7(*Y(Y7V997(Y+\ (+(+7;,9+9YQ7Y9#2.9=<.H .< &9<M+.<.>/ %' MM^%%M Q%243 $%34&Y} 0 V "\ K #+.&- +*,/ 3 % .43^M3 P %244 $%36& ) 0=9 }= * " ! (,) (\,)% YQ #,9 *,9&,9<R .< &9<M+.<.>/ %' . '..^6Q'113 , ! "#$%& Molecular Recognition at Protein Surface in Solution and Gas Phase: MolecularFive VEGF Recognition Peptidic Ligands at Protein Show Surface Inverse in Solution Afﬁnity When and Gas Studied Phase: by Five VEGFNMR Peptidic and CID-MS Ligands Show Inverse Afﬁnity When Studied by NMR and CID-MS

Andrey Dyachenko,1 Michael Goldﬂam,1 Marta Vilaseca,1 Ernest Giralt1,2 1Institute for Research in Biomedicine, Parc Cientı´ﬁc de Barcelona, Barcelona, Spain 2Department of Organic Chemistry, Universidad de Barcelona, Barcelona, Spain

Received 15 February 2010; revised 18 March 2010; accepted 18 March 2010 Published online 4 August 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.21462

ABSTRACT: Peptides were ranked on the basis of both solution and Protein-protein interactions comprise of collection of gas phase affinity to VEGF. The results indicate that the molecular recognition events that take place at protein ranking of peptides in terms of affinity in solution is surfaces. A better understanding of the mechanism reversed compared with the gas phase ranking. This behind these interactions would provide deeper insight observation opens up a vast field for the future study of into the nature of many diseases, caused by the the system, and the determination and characterization malfunction of protein networks, and contribute to design of factors, responsible for the change of stability of of molecules for efficient modulating of these interactions. noncovalent protein-ligand complexes upon complete or # One major factor in molecular recognition mechanism is partial removal of the solvent. 2010 Wiley Periodicals, interaction of reacting species with aqueous media. Thus, Inc. Biopolymers (Pept Sci) 94: 689–700, 2010. comparative study of noncovalent complex behavior in Keywords: protein-ligand interactions; molecular recognition; noncovalent interactions; gas-phase interactions; mass spectrom- solution and gas phase can provide valuable information etry; vascular endothelial growth factor (VEGF) about the role of the solvent. Here examined interactions of vascular endothelial growth factor (VEGF) protein with five peptidic ligands of the same molecular weight This article was originally published online as an accepted but with different affinities. Interactions of VEGF with preprint. The ‘‘Published Online’’date corresponds to the preprint version. You can request a copy of the preprint by emailing the ligands in solution were studied by ITC and NMR, and Biopolymers editorial office at [email protected] KDs were determined. Gas phase stability was addressed using CID-MS approach. The energy transfer model was INTRODUCTION taken and adapted for the calculation of binding energy. ne of the major highlights from biological sciences in this last decade has undoubtedly been the discov- ery that proteins inside the cells have a very rich ‘‘social life.’’1 They rarely work alone but interact with several others to form multi-protein com- Additional Supporting Information may be found in the online version of Oplexes. These complexes, which act as molecular machines, this article. Correspondence to: Ernest Giralt; e-mail: [email protected] are in charge of most of the multiple functions of proteins, Contract grant sponsor: MCYT-FEDER such as catalysis, transport, and signal transduction. In gen- Contract grant number: Bio2008-00799 Contract grant sponsors: Generalitat de Catalunya (XRB and Grup Consolidat) eral, protein-protein networks are topologically heterogene- VC 2010 Wiley Periodicals, Inc. ous in the sense that a few highly connected proteins-protein

PeptideScience Volume 94 / Number 6 689 690 Dyachenko et al. hubs mediate interactions between numerous less connected The difference with a conventional chemical reaction is proteins. The number of proteins per node varies greatly, that no covalent bond formation is involved. In contrast, A ranging from two or three to more than twenty. From a and B are held together by many noncovalent bonds. The dynamic point of view, the interactions vary from very tran- Gibbs energy in aqueous solution (DG) of this process can be sient to highly stable.2–6 decomposed in several terms18: The formation of holes or knots in the protein network of a DG ¼ DG þ þ nDG þ ADG þ RDG healthy cell normally leads to disease.1 By a hole, we mean the t r r h p loss-of-function of a protein hub. p53 is a typical example of where DGt+r (in fact, DSt+r) corresponds to the loss of trans- such a hub. Loss of function of p53 is strongly associated lational and rotational motion as a result of the transforma- with cancer. In fact, mutations of the gene coding for p53 tion of two molecular species (A and B) in one molecular have been identified in more than 60% of cancer patients. species (A:::B); nDGr (in fact, nDSr) is the entropic cost asso- We talk about a knot in the net when a series of new and ciated with restricted rotations upon binding; DGr is the unwanted protein-protein interactions appears. Protein entropic cost for each restricted rotation and n is the number self-assembly is a special case—‘‘narcissistic’’—of protein- of restricted rotations. ADGh (in fact, ADSh again) is the term protein interactions.7–9 In this regard amyloid diseases, dealing with the hydrophobic effect, where A is the area bur- such as Alzheimer’s or Parkinson’s, can also be considered ied in the binding site and inaccessible to water and DGh is to derive from the formation of knots in the net. In recent the Gibbs energy of hydrophobic effect per unit area. Finally, years we have been working on the design of therapeutic SDGp is the sum of the Gibbs energies of all the polar inter- strategies that modulate this socially ‘‘impolite’’ behavior. actions in the complex, where each DGp term has an Thus, we have developed cationic calixarenes able to rescue enthalpic (DHp) and an entropic (DSp) component. 10,11 the structure and stability of mutated versions of p53. Of all these terms, only nDGr and ADGh can be estimated in We have also designed peptides and peptide-decorated gold a relatively accurate way. In contrast, estimation—even a rough b nanoparticles that interfere with the formation of the - one—of DGt+r or SDGp presents severe problems. Estimation of 12–16 amyloid aggregates associated with Alzheimer’s disease. DGt+r can at first glance appear to be straightforward, namely, a Protein-protein interactions are the result of an ensemble simple calculation of the entropic loss associated with passing of exquisitely regulated molecular recognition events that from two free rigid bodies to a single rigid body. Nevertheless, take place at protein surfaces. This can be referred to as a things are not quite so simple, and true DGt+r values are prob- ‘‘protein recognition code.’’ To understand protein-protein ably at least one order of magnitude smaller. This may be interactions and to achieve the efficient design of molecules because the non-covalent interactions holding A and B together with the capacity to modulate these protein-protein interac- are in fact very weak. Thus, at room temperature one can expect tions, it is necessary to decipher this molecular recognition kinetic behavior with continuous formation and breakage of code, the language that proteins use to communicate. noncovalent bonds. This transient bonding scheme could be re-

Unfortunately, progress in this field is highly unsatisfactory. sponsible for this diminution of DGt+r. In other words, the A:::B Indeed, we are not completely illiterate, in the sense that we complex cannot be treated as a rigid body. Accurate estimations know the letters of this alphabet. They are the noncovalent of enthalpic and entropic components of SDGp are hindered by interactions, such as hydrogen bonds, electrostatic interac- difficulties in the following: to estimate the gain in entropy tions, p-cation interactions, Van der Waals forces, and the caused by the release of solvating water upon binding; to esti- others. However, we could be compared with a child who is mate changes of both entropy and enthalpy as a result of the learning to read and attempts Dickens’s Oliver Twist. release of counter-ions; and to estimate the effective dielectric Where do our difficulties arise from? As pointed out by permittivity in water-accessible regions of proteins. As discussed the seminal work of D. H. Williams,17,18 G. Whitesides,19 and below, modern mass spectrometry methods have opened the M. Gilson,20 dynamics—flexibility—and solvent effects are door to the quantitative study of binding in gas phase. Compari- the two villains in this field. Equation 1 represents two mole- son between binding in water and in gas phase appears as a cules, A and B, that form a noncovalent complex AB. Either promising approach shad light on this difficult field. A and B are proteins, or one is a protein and the other a Peptide molecules are wonderful tools with which to ligand (for example a peptide). examine protein-protein interactions21,22 and to modulate them.12,13 Here we present a comparative study of the inter- A þ B , A ::: B ð1Þ action of a family of diastereomeric cyclic peptides and a therapeutically relevant protein, the Vascular Endothelial Contract grant sponsor: Growth Factor A (VEGF-A).

Biopolymers (Peptide Science) Protein Surface Recognition in Solution and Gas Phase 691

VEGF-A is a member of the platelet-derived growth factor Table I Synthesized Cyclic Peptides, Arranged by the Predicted (PDGF)/vascular endothelial growth factor (VEGF) family, Decrease of Affinity to VEGF (From Top to Bottom) which comprises seven members (VEGF-A, VEGF-B, VEGF-C, Ligand Sequencea,b,c P-wt/P-mutd VEGF-D, VEGF-E, VEGF-F, and PlGF). VEGF-A is a globular water-soluble covalent homodimer, in which monomers are con- P-wt GGNECDIARMWEWECFERL 1 nected by two disulfide bridges.23 This growth factor is expressed P-18r GGNECDIARMWEWECFErLNA in the human body in at least seven isoforms, containing 121, P-7i GGNECDiARMWEWECFERL 25 145, 148, 165, 183, 189, and 206 residues per monomer.24 P-10m GGNECDIARmWEWECFERL 200 P-16f GGNECDIARMWEWECfERL >2000 The role of VEGF-A as a therapeutic target is extensively described in a number of excellent reviews (see, for example, a The lowercase bold letter represents the D amino acid. b Hoeben et al.24 or Ferrara and Kerbel25). VEGF-A is a major All the peptides were cyclized with C5-C15 disulfide bridge. c All the peptides have a carboxamide group at their C-termini. regulator of normal and abnormal angiogenesis, including d Decrease of binding strength after substitution of the 18th, 7th, 10th, that associated with tumors and several intraocular syn- and 16th amino acid residue with Ala. Data is taken from Ref. 23. dromes.26 It binds to two receptor tyrosine kinases (RTK), VEGFR-1 (Flt-1), and VEGFR-2 (KDR, Flk-1). In particular, VEGFR-2 is the major mediator of the mitogenic, angiogenic ligand, performed by Pan et al.23 (See Table I). Although sub- and permeability-enhancing effects of VEGF-A. stitution of the same amino acid with Ala and with D enan- Generally, VEGF-A up-regulation is associated with tumor tiomer should have a different effect on the structure of pep- growth, facilitating the spread of blood vessels and blood tide, Ala scan can provide a rough estimation of the relative supply of the tumor. Inhibition of VEGF-induced angiogene- importance of given amino acid side chain for binding.30 sis hinders this growth27 and increase the tumor response to The following four amino acids were chosen for the substitu- radiotherapy.28 In addition to tumor growth VEGF-A is tion: Arg18, Ile7, Met10, and Phe16 (Table I). involved in number of pathological conditions, including The purpose of designing the D analogs of peptides was to cardiovascular diseases, diabetic retinopathy, rheumatoid ar- introduce a local perturbation into the structure of the thritis and psoriasis. Furthermore, VEGF-A is required for ligand, leaving as many of its properties as possible intact. the normal functioning and development of mammals, thus The molecular weight of the ligand did not change, which making it an attractive target for both antiangiogenic and simplifies the interpretation of mass spectrometry data, and proangiogenic therapy. no new chemical groups were introduced. No rotatable

Here we used a 11-109 construct of VEGF-A121 (later in bonds were added or removed, thus leaving the number of the text referred to as VEGF). Although truncated, it was degrees of freedom of the ligand unchanged. However, the shown to retain its biological activity, because it still contains perturbation was strong enough to cause notable changes in the VEGFR-1 and VEGFR-2 binding domain.29 Thus, it can the strength of VEGF-ligand interactions. be used for the identification of inhibitors of VEGF- Chemical Shift Perturbation Nuclear Magnetic Resonance A:::VEGFR-1 and VEGF-A:::VEGFR-2 interactions. (CSP NMR) is a powerful technique that allows the mapping Phage-display-derived cyclic peptide,23 binds to VEGF in 1:2 of protein-protein and protein-ligand interactions31 and the 32 stoichiometry with a dissociation constant (KD)ofapprox.1 determination of the respective KDs. To determine the KD, lM, overlapping to a large extent with the VEGFR-1 binding protein is titrated with a ligand (or vice versa) and the HSQC site. It is a 2340 Da 19 amino acid peptide (P-wt in Table I), sta- spectrum is recorded for each point. Depending on the label- bilizedwithaC5-C15disulfidebond.Itispoorlyorderedinthe ing and acquisition strategy, changes in the protein or ligand unbound state, but in complex with VEGF it adopts a strongly spectrum can be monitored. Interactions induce an alteration amphipathic secondary conformation, positioning hydrophobic in the chemical shift of certain peaks (Dd), which can be corre- residues towards VEGF binding site and exposing hydrophilic lated with the fraction of observed species in the bound state. residues to the solvent. The NMR structure of VEGF in com- Plotting Dd versus the concentration of titrant and fitting the plex with P-wt (1KAT in PDB database) is shown in Figure 1. resulting plot to the respective model provides the KD value. A complex of VEGF with P-wt was chosen as a model for Mass spectrometry (MS) is a well-established method that this study. To gain further insight into the nature of noncova- allows the study of species in gas phase, including small or- lent interactions, we synthesized 5 peptide ligands to VEGF ganic molecules,33 large biomolecules,34 and even intact (Table I). Each ligand is a P-wt peptide with one L amino viruses.35 In particular, the development of so called ‘‘soft acid substituted with the D analog. The amino acids were ionization’’ methods, Electrospray Ionization (ESI) and Ma- selected on the basis of an Ala scan of the binding area of the trix Assisted Laser Desorption-Ionization (MALDI), capable

Biopolymers (Peptide Science) 692 Dyachenko et al.

FIGURE 1 P-wt peptide in complex with 11-109 construct of VEGF121.A:P-wt in the binding pocket. Amino acid residues, substituted by D-enantiomers in the present work, are shown as ball- and-stick models, with buried solvent accessible surface specified. B: 3D view of VEGF121 dimer in complex with P-wt peptide (1KAT PDB entry). of ionizing intact protein complexes, has facilitated the Collision induced dissociation (CID) is a ‘‘slow-heating’’ extensive study of noncovalent interactions by MS.36–38 dissociation method, used to provide the ion with enough Several features of MS make it an attractive approach to energy to fragment (brake covalent bonds) or dissociate address biological molecules. First, it is a sensitive method, (break noncovalent bonds).40 The ion is ‘‘heated’’ while mov- with the capacity to detect the samples at submicromolar ing through the collision cell, which is filled with inert gas, concentrations, and it requires, in the best of cases, several such as He, N2, Ar, Xe or CO2. Impacts with gas molecules nanograms of the sample for a single run. This spectacularly provide an ion with excess internal energy, which is used for low sample consumption of the method makes it highly dissociation. For more details, see the extensive review by suited for the screening of large libraries of compounds.39 Mayer et al.41 Another feature of the method is mass accuracy, which can reach about 1 ppm for the large molecules, thereby allowing straightforward assignment of the signals. Also, the method RESULTS is fast and, in many cases, it can be automated.36 Finally, the critical feature in the context of the present work is that MS Chemical Shift Perturbation NMR allows observations of biomolecules without solvation shells, The affinity of each of the five ligands to VEGF in solution because species are studied in the gas phase. Transfer from was determined with NMR by analyzing chemical shift per- solution to the gas phase affects structure and interaction turbations (CSP). Sample, containing VEGF in 100 lM con- forces of molecules. However, this gives valuable information centration, was titrated with a ligand. Concentrations of about the behavior of the system unaffected by the solvent. ligands were chosen so that the optimal range of titration curve was covered, on the basis of the expected values of re-

Biopolymers (Peptide Science) Protein Surface Recognition in Solution and Gas Phase 693

15 1 FIGURE 2 Determination of KD of VEGF:::P-7i complex. A: N- H HSQC spectra of a 100 lM 13 15 sample of (methyl C)-Met-all- N VEGF11-109 at 458C titrated with P-7i (600 MHz with cryoprobe). B: Zoom of Lys48 shifts.

15 1 spective KDs. N- H HSQC spectrum was recorded for each for each complex, and ligands were ranked on the basis of titration point during 40 min (see Figure 2). For each ligand, their afﬁnity to VEGF in solution (Table II). four peaks with the strongest displacement of chemical shift

(Dd) were used for the KD calculations. The curves for Dd versus total ligand concentration in the sample ([L0]) were CID Mass Spectrometry fitted to the analytical model, assuming that two binding All the MS experiments were performed using a Waters SYN- sites are equal and independent [Eq. (2)]. APT HDMS Time-of-flight (TOF) mass spectrometer. The exact mass of the recombinant protein construct was deter- ½ ½ L0 L ¼ mined under denaturating conditions (H O/CH CN/Acetic ½ 2 3 P0 qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi Acid 49/49/2). Convolution of the nanoESI spectra revealed 2 the exact mass of VEGF to be 23520 6 10 Da, which is in KD þ½L0þ2½P0 ðÞKD þ ½L0 þ 4½P0 ðÞKD ½þL0 ½P0 good agreement with the calculated mass, considering the 2 fact that the protein was 15N labeled for the NMR purposes. ð2Þ where [P0] is the total protein concentration and [L] is the Aqueous ammonium acetate (AmAc) solution is the buffer concentration of unbound ligand in solution (see Figure 3). most widely used for the MS experiments with noncovalent The fact that R2 values for most of the fittings were better complexes.42 This is because of the volatility of its components than 0.99 confirms that this model provides the acceptable and its near-physiological pH. These features allow the native

description of the system. The average value of KD was taken structure of many proteins to remain intact, and, in many

Biopolymers (Peptide Science) 694 Dyachenko et al.

peak was chosen for the study of gas-phase affinity of the ligands to VEGF. For this purpose, CID MSMS experiments were per- +10 formed for each complex. The [PL2] ions were isolated in the quadrupole section of mass-spectrometer and then transferred to the collision cell, which was filled with argon under precisely controlled pressure. Voltage bias, applied between the quadrupole and inlet electrode of collision cell (later referred to as collision voltage or Vc), was gradually increased, in order to augment the kinetic energy of the ions and provide them with internal energy, sufficient to disrupt the non-covalent bonds. The evolution of the MSMS spectrum of the VEGF:::P-18r complex with increase of Vc is shown in Figure 4. Dissociation of the 1:2 VEGF:ligand complex occurred in two steps. The Vc increased from its initial value to about 40 V. This caused the parent ion to gradually dissociate, emit- FIGURE 3 Relative amount of bound ligand plotted against the ting one ligand. The main pathway involved dissociation of +10 +8 +2 total ligand concentration and fitted to the ‘‘2 independent equal [PL2] into [PL] and [L] , and the secondary path- binding sites’’ model. Lys48 peak displacement in VEGF:::P-7i com- way—into [PL]+9 and [L]+1. In the case of VEGF:::P-18r plex spectrum was used. +10 complex dissociation of the [PL2] ion was complete when the voltage is around 44V. Upon further increase of Vc, it cases, to preserve noncovalent interactions. Concentrations of starts to be enough energy for two dissociation events, and AmAc up to 1M for noncovalent complexes42 and up to 8M [PL]+9 and [PL]+8 ions started to dissociate, releasing ligand for single proteins in the native state43 have been used without and VEGF. The Vc value of about 58 V corresponded to the negative impact on the spectrum. Moreover, in case of the total dissociation of the complex, and after this value only presence of nonvolatile adducts in the sample solution, addi- [P]+8, [P]+7, and [P]+6 ion peaks were present in the spec- tion of AmAc can substantially reduce the broadening of the trum. The most abundant one was [P]+6, corresponding to peaks, presumably, by replacing of adduct ions, such as Na+, the major dissociation pathway: + 43,44 with ammonium ions NH4 . þ10 þ8 þ2 þ6 þ2 In this study, we used BioRad BioSpin-6 columns two ½PL2 !½PL þ½L !½P þ 2½L times for each sample for effective desalting/buffer exchange, which resulted in very low concentration of Na+ ions in the sample. Thus, high concentrations of AmAc were not required. Nevertheless, comparison was made between 10 mM,50mM, and 200 mM AmAc buffer solutions, all containing VEGF in its native conformation. Although the samples showed a similar resolution and sig- Table II Left: Gibbs Energy and Dissociation Constants for the nal intensity, the 200 mM sample showed better signal Ligands in Solution. Right: Collision Voltage and Internal Energy stability, and thus this concentration was chosen for fur- Increase for the Ligands in Gas Phase. Ligands are Arranged by ther work. the Increase in Stability NanoESI TOF mass spectra of the five samples are shown in Supporting Information Figure S1. The intensities of peaks Solution Gas Phase corresponding to PL or PL2 species decreased in concordance Ligand KD, lM DG, kJ/mol Ligand Vc0,V DU,eV with a decrease in KDs of the respective ligands. However, in all the cases it was possible to detect the peak, corresponding P-10m 1810 6 147 16.7 6 5.1 P-wt 22.0 6 1.0 64.1 6 3.8 +10 6 6 6 6 to the [PL2] ion, with a m/z value around 2821 (here and P-16f 313 53 19.9 3.4 P-7i 24.0 1.0 68.1 4.5 later in the text, P, protein; PL, complex of protein with one P-7i 252 6 77 21.3 6 6.2 P-18r 25.5 6 1.0 75.1 6 4.1 6 6 6 6 ligand; PL , complex of protein with two ligands). Thus, this P-18r 3.50 0.60 33.0 2.5 P-16f 29.0 1.0 81.0 4.0 2 P-wt 1.02 6 0.18 36.4 6 13.0 P-10m 34.0 6 1.0 94.3 6 5.2

Biopolymers (Peptide Science) Protein Surface Recognition in Solution and Gas Phase 695

+10 FIGURE 4 Dissociation of [PL2] ion of VEGF:::P-18r, caused by increase in collision voltage from 10 to 60 V. The same peaks are highlighted with a gray stripe.

One exception was a [PL]+7 peak, which was still present atom into internal energy of the ion and distribution of this in the spectrum even under higher ion energies (Vc > 60 V). energy among its internal degrees of freedom. Presumably, this ion came from the minor pathway: Here we used low-energy CID, where the ion undergoes multiple collisions before dissociation. This method belongs ½þ10!½ þ9 þ þ !½ þ7 þ½ þ2 PL2 PL2 H PL L to the family of so-called slow-heating, or nonergodic meth- 40,41 In this case, the system underwent three dissociation ods, for which it is assumed that the time, required for events to reach a fully dissociated state. Thus, more energy the energy acquired in the collision to be distributed among was required to detach both ligands from the protein. internal degrees of freedom is negligibly small in comparison Complexes of VEGF with each of the five ligands were with the time needed for the dissociation of the complex. In examined this way, and a dissociation curve was made for the ICT transfer model, the energy is distributed inside the each one (see Figure 5). ion immediately after the second step. For more details see the Materials and Methods section. Ion internal energies, calculated with the ICT model, Internal Energy Calculation are listed in Table II. It is worth mentioning, that, strictly 45,46 The Impulsive Collision Transfer (ICT) model was used speaking, these energies are not ‘‘collisional’’ energies, that D to calculate the internal energy ( U) required for the detach- is to say they are not the energies of bonds, connecting ment of the ligand from the complex ion in the gas phase. the protein and the ligand. In fact, it is the amount of According to this model, it is assumed that the collision of a energy, transferred to the ion and distributed among its large macromolecule ion in the gas phase with a neutral mol- internal degrees of freedom, which appeared to be suffi- ecule of the buffer gas involves two separate events: first, elas- cient for disrupting these bonds. The question as to what tic (or partially inelastic) collision of a buffer gas molecule proportion of this energy was used for the dissociation is with an atom on the surface of the protein, and, second, a a subject for further study. fully inelastic interaction of the atom of the ion that underwent collision with the rest of the molecule. The second step results in the transformation of the kinetic energy of this

Biopolymers (Peptide Science) 696 Dyachenko et al.

the change in chirality of one amino acid induces strong alterations in the conformation the ligand adopts upon binding to the protein. This can produce entropic penalty, thereby also reducing the affinity of the ligand to VEGF. Thus, the decrease in binding affinity caused by the change in chirality of one amino acid can be attributed to both the perturbation of the hydrophobic surface and entropic penalty, caused by the loss of the pre-organized structure which may be present in the case of the wild-type ligand. In order to gain a better insight into the nature of these changes, two ligands, namely, P-wt and P-18r,werestudiedby Isothermal titration calorimetry (ITC). These were the only ligands for which ITC experiments were possible, both because of insufficient solubility of ligands (free ligands in aqueous buffer at near physiological pH start to precipitate at 0.5–0.8 mM concentrations) and a large amount of protein required. VEGF (10 lM) was titrated with 170 lM P-wt and 247 lM P-18r at 378C. Analysis of the entropy/enthalpy balance of VEGF-ligand interactions showed that although Gibbs FIGURE 5 Dissociation plots of VEGF in complex with each of 5 energies (DG) of the two ligands differed by only 3.2 kJ/mol, +10 ligands built for [PL2] ions. Increase in collisional voltage corre- differences in entropic (TDS)andenthalpic(DH) effects were sponds to the increase in gas-phase binding energy and is opposite much stronger: 20.2 kJ/mol and 17 kJ/mol respectively (see to the increase of solution binding affinity. Figure 6). The KD values, calculated with ITC, were in good DISCUSSION agreement with ones from NMR CSP. However, it remains unclear, why the driving force of interactions in both cases is Protein-Ligand Complex in Solution enthalpy, whereas in case of hydrophobic interactions the Table II summarizes results obtained for solution and gas- main binding force should come from the entropic part.48,49 phase stabilities. In solution, when the protein functions To address this question, the structure of the VEGF:::P-wt under native conditions, the strongest ligand is P-wt, i.e. the complex was examined with Molecular Operating Environ- ‘‘wild type’’ cyclopeptide, optimized for the interaction with ment (MOE) software.50 This program allows the prediction the same construct of VEGF.23 Although free P-wt is poorly of noncovalent interactions that may take place between the ordered, when bound to VEGF it adopts a conformation protein and the ligand, on the basis of their relative position. with a type I b-turn and a C0-terminal a-helix. In particular, For the VEGF:::P-wt binding interface in addition to the the a-helix forms part of the binding interface, pointing numerous hydrophobic interactions it predicted four hydro- hydrophobic residues Trp13, Phe16, Leu19 towards the pro- gen bonds: Phe16P-wt-Gln89VEGF1,Trp11P-wt-Tyr21VEGF2,Ile7P- tein hydrophobic binding pocket. Together with other hydrophobic residues, buried in the binding interface, (solvent accessible surface (SAS) < 60, calculated with GETAREA web service47), namely, Ile7, Ala8, Met10, and Trp11, they form a highly hydrophobic interface. Inversion of configuration of any of these residues would affect the hydrophobicity of this interface, thereby decreasing its affinity to VEGF. However, the correlation between the SAS of a given residue and the binding affinity of respective mutant ligand is not straightforward because of the effect of the change of chirality on the secondary structure of the ligand. For example, the Phe16 residue showed the lowest SAS value (<1%). However, P-16f exhibited a smaller decrease in binding affin- FIGURE 6 Entropy/enthalpy balance chart for interactions of ity than P-10m (SAS 50%). This observation indicates that VEGF with P-wt and P-18r peptides and their dissociation constants obtained with ITC.

Biopolymers (Peptide Science) Protein Surface Recognition in Solution and Gas Phase 697

wt-Asn62VEGF2, and Arg9P-wt-Asn62VEGF2 (lower indexes P-wt, VEGF1 and VEGF2 indicate, that the amino acid belongs to P- wt, 1st or 2nd monomer of VEGF dimer respectively). Thus, VEGF:::P-wt interactions are probably not purely hydrophobic, but also have an electrostatic component. This would explain the strong ‘‘favorable’’ DH component of DG. The affinities of the two ligands were very close, although their components differed substantially. This is an example of the well-known entropy-enthalpy compensation effect.51,52 +10 One interpretation of the underlying processes could be the FIGURE 7 Collision cross-sections of PL2 (bottom) and +11 following. The perturbation of hydrophobic surface of the PL2 (top) ions of complexes of VEGF with five ligands. The order from left to right corresponds to the increase in dissociation energy ligand, caused by substitution of L-Arg18 with D-Arg18, in gas phase. decreases the efficiency of hydrophobic interactions. The entropic component of DG includes the penalty for the loss phase, is to compare the collision cross-sections of the com- of conformational freedom and ‘‘favorable’’ component that plexes. Given that all the ligands have exactly the same mass, comes from the release of water molecules upon burial of the the reliability of this experiment is increased. hydrophobic part of the ligand in the hydrophobic pocket. In For this experiment, an Ion Mobility Spectrometer (IMS), P-18r, the latter is reduced, thereby increasing the total embedded in the Waters SYNAPT HDMS instrument, was ‘‘unfavorable’’ entropic component of DG. On the other used. The collision cross-section (O) of each complex was hand, perturbation of hydrophobic surface, may give more measured following the procedure described in details by flexibility and freedom for the ligand to adopt a conforma- Ruotulo et al.53 As standard proteins for building the calibration, more favorable for hydrogen bonding. This would tion curve we used equine myoglobin, equine cytochrome c, increase the enthalpic component of DG, which compensates bovine ubiquitin and bradykinin. In total, 27 points were the ‘‘unfavorable’’ entropic component. plotted for each set of conditions. The O values were meas- Although the ITC was available for only two ligands, MS ured for two sets of ions, PL +10 (m/z ¼ 2821) and PL +11 experiments (described in the next section) suggest that these 2 2 (m/z ¼ 2564), in nitrogen in low vacuum conditions. explanations can cover the five ligands studied. Although argon was used for the CID experiments, the theo-

retical difference between O in nitrogen (ON2) and O in ar- O Protein-Ligand Complex in Gas Phase gon ( Ar), given by Eq. (3) is less than 1%, which does not exceed the experimental error.54 Thus, the values obtained The stabilities of complexes of VEGF with ﬁve ligands in gas can also be used for the calculation of DU (see Materials and phase, measured by CID MS, are listed in Table II. They are Methods). ranked almost exactly in the opposite way to solution values. The only exception is the P-7i/P-18r pair, but their gas-phase R þ r 2 X ¼ X Ar ð3Þ Ar N2 þ stabilities are relatively close. Thus, in this system, ranking of R rN2 afﬁnities in the gas phase can be considered the reverse of that in solution. The question to be addressed in this context where rAr is the radius of argon atom, and rN2 is the average is the meaning of the values, listed in Table II. Both Vc and ‘‘radius’’ of N2 molecule. O values for the said complexes are DU are related to the total energy acquired by the ion in the plotted in Figure 7. The differences between the O values of collision cell before dissociation and transferred to its inter- the complexes are in the same order of magnitude as the ex- nal energy. However, since this energy is distributed among perimental error, thereby making it possible to consider all degrees of freedom, it is unclear which part is devoted to them similar in size. This allows us to assume, that all the breaking the bonds that keep the ligand attached. ligands occupied a similar surface area on the protein, and This question is to be addressed quantitatively in the thus, DU values, obtained in CID MSMS experiments, can be future studies. However, from the qualitative point of view, used to rank the ligands in terms of their capacity to remain the fraction of internal energy that can be used to disrupt the attached to VEGF in the gas phase. protein-ligand interaction should be related to the area occu- Why does this change in chirality of one amino acid cause pied by the ligand on the protein surface. A relatively the opposite effects on solution and gas-phase stabilities of straightforward way to check whether all the ligands bind in non-covalent interactions? The main difference between the the same way and, thus occupy the same area in the gas system surrounded by the solvent and one transferred to the

Biopolymers (Peptide Science) 698 Dyachenko et al. gas phase is that in the gas phase the effects caused by the sol- Germany). Other reagents, if not otherwise stated, were purchased vent are removed. In particular, hydrophobic interactions from Carlo Erba Reagenti SpA, Strada Rivoltana, I-20090, that stabilize complexes in solution are either drastically Rodano. Dimethylformamide (DMF) and dichloromethane (DCM) 55 solvents were used; piperidine/DMF 20%/80% (v/v) was used as weakened or completely eliminated in the gas phase. In a deprotection agent, TBTU (Iris Biotech) as a coupling activator, contrast, electrostatics-based interactions, including hydro- and N,N-diisopropylethylamine (DIEA, Fluka Chemie GmbH, CH- gen bonds,56 are strengthened by the removal of solvent 9471, Buchs, Switzerland) as an activator base. Fmoc-Rink-Amide because of the decrease in the dielectric permittivity of the MBHA (Iris Biotech) resin was used as a support. Cleavage from the media.36 Applying these considerations to the present case, resin was performed with trifluoroacetic acid/water/1,2-ethanedi- thiol/triisopropylsilane 94%/2.5%/2.5%/1%, resulting in peptides we propose the following explanation. Perturbation of having a carboxamide group at the C-termini. hydrophobic surface, caused by the change of chirality of one For the automatic synthesis of ligand CEM Liberty Automated amino acid, decreases the efficiency of hydrophobic interac- Microwave peptide Synthesizer (CEM Corporation, 3100 Smith Farm tions. Thus, these interactions do not compensate the Rd, Matthews, NC) was used, following standard procedures. As a sup- entropic penalty that the system pays for the loss of confor- port, Aminomethyl ChemMatrix resin (Matrix Innovation, 1450 City mational freedom of the ligand, and the affinity of ligand in Councillors Suite 230, Montreal (Quebec), Canada), previously func- tionalized by Fmoc-Rink-Amide Linker (Iris Biotech) was used. As a solution decreases. However, in the gas phase electrostatics decoupling agent, piperidine/DMF 20%/80% (v/v) with 1 mM of N- plays the major role and perturbation of the hydrophobic hydroxybenzotriazole (HOBt) was used, in order to prevent the racemi- surface may open up more possibilities for the ligand to zation of Cys.57 All other reagents were the same as in the manual SSPS. make electrostatic contact with the VEGF binding site. Thus, Oxidation of Cys and formation of the Cys5-Cys15 disulfide the stronger the perturbation of the hydrophobic surface of bond were performed using Potassium Ferricyanide K3Fe(CN)6 (Sigma-Aldrich Co Ltd, Gillingham, Dorset, SP8 4XT, UK). Each the ligand, the weaker it binds to VEGF in solution, but the peptide was dissolved in 5 mM NH4HCO3 aqueous buffer in a con- stronger the binding in the gas phase. This mechanism is centration of *20 mg/ml, resulting in 2 l of solution. Totally, 1 ml confirmed by the ITC study, where the enthalpic part, com- of 20 mM K3Fe(CN)6 was added every 30 min for 2 h. The reaction ing from electrostatic interactions and hydrogen bonds, was monitored by Matrix-Assisted Laser Desorption Ionization increases after inversion of chirality of Arg18. (MALDI) TOF MS. After 2 h, excess solvent was removed with a ro- tary evaporator and the sample was freeze-dried. The present example shows, how the CID MSMS technique MALDI TOF MS experiments were performed on an Applied provides a deeper insight into the nature of hydrophobic inter- Biosystems 4700 Proteomics Analyzer instrument (AB, 850 Lincoln actions by allowing examination of the system, unaffected by Centre Drive Foster City, CA 94404). For the data analysis Data the solvent. However, we show that the assumption of similar- Explorer 4.5 was used. Each ligand was dissolved in 25/75 CH3CN/ ity in behavior of non-covalent interactions in solutions and a water, purified using Waters HPLC semi preparative instrument, l 3 gas phase can be made only for limited range of cases. Gener- equipped with Waters Symmetry C18 5 M 30 100 mm preparative column and freeze dried. ally, it can be concluded, that when noncovalent interactions contain a significant hydrophobic component, gas phase affinities cannot be taken as an estimation of solution behavior ei- VEGF Expression 11-109 ther from quantitative, or qualitative point of view. VEGF121 was produced in a similar way to that described by Fairbrother et al.29 Isotopic labeling of (methyl 13C)-Met and In summary, a comparison of binding in gas phase and 15 solution can pave the way towards a better understanding of all- N was performed by using E. coli strain B834 (DE3) (auxo- thropic for Met, purchased from Cambridge Isotope Laboratories, molecular recognition at protein surfaces. Peptide ligands are Inc.). Expression was carried out in M9 minimal media supple- 13 15 highly suited for these types of studies. The comparison of mented with 80 mg/l Met( C)and 1 g/l NH4Cl. the relative affinities of collections of ligands with the same molecular weight facilitates the interpretation of data from MS because it is not necessary to make corrections that are CID MS The CID MS experiments were carried out using a Waters SYNAPT not very reliable with the present state of the technique. HDMSTOFmassspectrometerwithanembeddedIonMobilitySpec- trometry (IMS) cell. It was equipped with TriVersa NanoMate ion source from Advion (19 Brown Road, Ithaca, NY, 14850). The instru- MATERIALS AND METHODS ment was operated in TOF mode. Conditions were optimized in order to maintain the noncovalent interactions in the gas phase intact.42 Since the mass of ligands is below the extraction limit of most of Ligand Synthesis commercially available desalting columns, VEGF and a ligand for Manual ligand synthesis was performed using a standard solid state each sample were prepared separately. For the buffer exchange/ peptide synthesis (SSPS) protocol, with Fmoc-protected amino desalting of the protein BioRad BioSpin-6 columns were used two acids (Iris Biotech GmbH, Gaurstrasse, d-95615, Marktredwitz, times. Each ligand, previously purified using preparative HPLC with

Biopolymers (Peptide Science) Protein Surface Recognition in Solution and Gas Phase 699

C18 reversed phase column and freeze-dried, was dissolved in 200 curves were built, using three wave height values: 8.5 V, 9.5 V, and mM aqueous ammonium acetate (AmAc) buffer. In order to remove 10 V. A wave velocity of 300 m/s was used for all the cases. Data traces of K+, Millipore Microcon YM-3 centrifugal ultrafiltration were analyzed with MassLynx 4.1 and DriftScope 2.0 software. Drift +10 +11 devices were used. The complex solutions were prepared, with a times of analyzed [PL2] and [PL2] ions were fitted to the final concentration of VEGF being 15 lM, and a ligand—60 lM for resulting curves. The error of measurements was calculated, accord- P-wt and P-18r,90lM for P-7i and P-16f, and 150 lM for P-10m. ing to.53 A greater concentration of weaker ligands in solution was needed to provide the sufficient abundance of PL2 species in the sample. K Totally, 10 ll of each sample was analyzed. The pressure in the D Determination by NMR collision cell was kept equal to 3.77 3 10 2 mbar. Vc was varied The NMR CSP experiments were carried out on a Bruker Digital from 10 V to 60 V, with 2 V increment. Hundred scans were Avance 600MHz apparatus equipped with a cryoprobe. (Bruker recorded for each voltage. Spectra were processed and integrated Daltonics Inc. 40 Manning Road, Manning Park, Billerica, MA using MassLynx v.4.1 software. 01821) at the High Field NMR Unit of the University of Barcelona. Samples were prepared in a 25 mM PB buffer containing 50 mM NaCl and 0.02% NaN3 (H2O:D2O 9:1). The protein concentration Internal Energy Calculation in all cases was 100 lM. Titration with the ligands was performed +10 Thedissociationcurve,i.e.therelativeintensityof[PL2] ion signal, by dissolving previously defined amounts of freeze-dried peptide in plotted against Vc, was built for each complex. According to the ICT the sample and adding 0.5% of DMSO-d6 to ensure solubility. theory, introduced in45,46 and applied in,58 the increase in internal energy 15N-1H HSQC spectra were acquired at 318 K using the fhsqcf3gpph of the ion after single collision with a buffer gas molecule DUS is given by pulse sequence with 2048 3 256 complex points, with a total of 8 transients per increment. The f2 domain of the dataset was 2 2m ma increased to 512 by linear prediction and then zero-filled to yield a D ¼ ÀÁÀÁg cos2 u ð Þ US E0 2 4 2048 3 1024 data set. A qsine function was used for line broaden- M þ mg ma þ mg ing. All spectra were processed using Topspin 2.0. For the fitting the resulting Dd vs [L0] curves it was assumed, where E0 is the initial kinetic energy of the ion, mg is a mass of buffer that change in chemical shift is proportional to the fraction of gas molecule, ma is a mass of colliding atom in the surface of the pro- ligand, bound to the protein: tein, M is the mass of the protein and u is the angle between the tra- jectory of the ion and a straight line, drawn through the centers of ½L ½½L L Dd ¼ k b ¼ k 0 ð7Þ the masses of the ion and a buffer gas molecule. After averaging, and ½P ½P taking into account that an ion undertakes many collisions before dis- 0 0 sociation, total internal energy, acquired by the ion, DU,isgivenby where [Lb] is the concentration of ligand bound to the protein. !! 2 N Combining Eqs. (2) and (7) gives the final equation for the fitting: 2mgma DU ¼ E0 1 1 ÀÁÀÁ ð5Þ M þ m m þ m 2 Dd ¼ g a g qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2 KD þ ½þL0 2½P0 ðÞKD þ ½L0 þ 4½P0 ðÞKD ½þL0 ½P0 where N is the number of collisions, which can be calculated from k the pressure in cell p and collision cross-section r using 2

rnl For curve fitting, a nonlinear regression procedure, built into the N ¼ ð6Þ 60 kT Wolfram Mathematica software package, was used. where l is the path of the ion before dissociation, k is a Boltzmann Isothermal Titration Calorimetry constant and T is temperature in Kelvin. For the ITC experiments, a VP-ITC Isothermal Titration Calorime- The E was calculated from Vc , the collision voltage, at which 0 0 ter was used (Microcal, 800, Centennial Avenue, Piscataway, NJ the derivative of dissociation curve has a minimum. At this point, 08554) Both the ligand and the protein were dissolved in 25 mM PB the length of the collisional cell, 0.1 m, could be taken as an l value. buffer containing 50 mM NaCl and 0.02% NaN . Protein was The calculated error is a sum of errors of cross-section measurement 3 titrated with ligand at 378C. Volume of one injection was 10 ll, ref- and Vc determination. 0 erence power, 15 kcal/s. Results were analyzed with Origin 7 script, provided by Microcal. Cross-Section Determination +10 +11 Cross-sections of [PL2] and [PL2] complex ions for five complexes were determined as described in,53 using a Waters SYNAPT REFERENCES HDMS TOF mass spectrometer, working in the ion mobility mode. 1. Gordo, S.; Giralt, E. Protein Sci 2009, 18, 481–493. N2 buffer gas was used. For building the calibration curves, standard 2. Uetz, P.; Giot, L.; Cagney, G.; Mansfield, T. A.; Judson, R. S.; proteins with previously published collision cross-sections,59 Brady- Knight, J. R.; Lockshon, D.; Narayan, V.; Srinivasan, M.; kinin, Cytochrome c, Equine myoglobine, Bovine ubiquitine (Sigma Pochart, P.; Qureshi-Emili, A.; Li, Y.; Godwin, B.; Conover, D.; Aldrich, 3050 Spruce Street, St. Louis, MO 63103) were used. Three Kalbfleisch, T.; Vijayadamodar, G.; Yang, M.; Johnston, M.;

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&OOTLINE !UTHOR 0.!3 )SSUE $ATE 6OLUME )SSUE .UMBER -ATERIALS AND -ETHODS ! -* -INI THERMAL CYCLER "IO 2AD WAS USED TO ENSURE ACCURATE TEMPERATURE 'RO%, EXPRESSION AND PURIlCATION CONTROL !LL REAGENTS WERE PURCHASED FROM 3IGMA !LDRICH %XPRESSION AND PURIlCATION OF 'RO%, WERE CARRIED OUT AS DESCRIBED -ASS SPECTROMETRY MEASUREMENTS .ANOmOW ELECTROSPRAY IONIZATION %3) -3 EXPERIMENTS WERE CONDUCTED FOLLOWED BY PRECIPITATION IN VV ACETONE !LIQUOTS OF PURIlED 'RO%, WERE STORED AT O# IN - AMMONIUM ACETATE BUFFER P( USING A HIGH MASS 1 4/& TYPE INSTRUMENT ADAPTED FOR A 134!2 8, PLATFORM CONTAINING M- $44 #ONDITIONS WERE CAREFULLY CHOSEN TO ALLOW THE IONIZATION AND DETECTION OF 'RO%, ASSEMBLIES WITHOUT DISRUPTING NON COVALENT INTERACTIONS !LIQUOTS !40 HYDROLYSIS ASSAYS OF , WERE ELECTROSPRAYED FROM GOLD COATED BOROSILICATE CAPILLARIES PRE )NITIAL RATES OF !40 HYDROLYSIS BY 'RO%, WERE MEASURED USING THE PHOS PARED IN HOUSE AS DESCRIBED 4HE FOLLOWING EXPERIMENTAL PARAMETERS PHATE BINDING PROTEIN ASSAY IN M- %$!! 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WWWPNASORG &OOTLINE !UTHOR

Allosteric mechanisms can be distinguished using structural mass

spectrometry

Supplementary Information

Andrey Dyachenko1,2†‡, Ranit Gruber2†, Liat Shimon1, Amnon Horovitz2* and Michal Sharon1*

Affiliations: 1Departments of Biological Chemistry and 2Structural Biology, Weizmann Institute of Science, Rehovot Israel

‡Current address: Institute for Research in Biomedicine, Barcelona, Spain

†These authors contributed equally to this work

*Correspondence to: Michal Sharon Amnon Horovitz [email protected] [email protected] Tel: 972-8-9343947 Tel: 972-8-9343399

Data analysis and correction for non-specific binding For each mass spectrum, the areas of the peaks corresponding to apo GroEL and its ATP-bound states were calculated using a deconvolution software (peakﬁt v4, Jandel Scientiﬁc, San Rafael, CA). The reliability of the data deconvolution results was validated by comparison of the expected peak position calculated from the mass to charge ratio with the generated peakfit value. After averaging the relative intensities for at least three charge-states, the data were corrected in order to remove the contribution of non-specific binding, as described (1):

ఈିே ܥே ܫே െܫேିଵܭ௡ሾܵሿ ௝ିଵ ൌቆ ቇቌ෍ሺܭ௡ሾܵሿሻ ቍሾͳሿ ሾܧሿ ܫ଴ ௝ୀଵ

where CN is the total concentration of the GroEL species with N ATP molecules bound at speciﬁc sites, IN is the intensity corresponding to a population with N ATP molecules bound, [S] is the free ATP concentration, [E] is the concentration of apo GroEL, Kn is the nonspecific binding constant and designates the largest number of bound ATP molecules that is visible in the spectrum (this number includes the apo state so that if, for example, the highest number of GroEL-bound ATP molecules seen in the spectrum is 5, then α = 6). In cases where the peak corresponding to apo GroEL was not present, the following equation was used:

ே ேି௝ ఈିே ܫ ܥே ܰ ேିଵ ௝ିଵ ௝ିଵ ൌ൬ െܭ௡ሾܵሿ൰ቌ෍ሾܵሿ ܭ௡ ෑܭ௜ቍቌ෍ሺܭ௡ሾܵሿሻ ቍሾʹሿ ሾܧሿ ܫேିଵ ௝ୀଵ ௜ୀଵ ௝ୀଵ

Analysis of cooperativity

The data corrected for non-specific binding were used to calculate the Hill coefficient as a function of ATP concentration or degree of saturation. The Hill equation is given by:

K[S]nH Y = [S3] 1+ K[S]nH

where Y is the fractional saturation, [S] is the ATP concentration, K is the apparent binding constant of ATP to all N sites of the protein and nH is the Hill coefficient which is assumed to be ATP concentration-independent. This equation was used to fit the data in Fig. S3. Eq. S3 can be rearranged, as follows:

Y log( ) [S] Y n = 1 Y = [S4] H log[S] Y(1 Y) [S]

where nH is no longer assumed to be a constant and is a function of [S]. The degree of saturation is given by:

N i iKi[S] = i=0 Y N [S5] i NK i[S] i=0 where N is the total number of specific ATP binding sites and i is the number of ATP-bound sites. Combining Eqs. S4 and S5 yields:

i2 (i)2 (i) n = = observed [S6] H i (i)binomial i(1 ) N In this equation, the Hill coefficient is expressed as the ratio between the observed and binomial standard deviations of the binding numbers. Eq. S6 was used to calculate the values of the ATP concentration-dependent Hill coefficients from the MS data shown in Fig. 3A. The 14 apparent ATP binding constants of GroEL were calculated from the corrected MS intensities, as follows: I K = i(corr) i I [S] i1(corr) [S7]

where Ki is the apparent binding constant of the ith molecule of ATP (when i-1 molecules are already bound), Ii(corr) and Ii-1(corr) are the respective MS intensities at a given ATP concentration that correspond to GroEL with i and i-1 bound ATP molecules (after correction for non-specific binding) and [S] is the ATP concentration. Each Ki was calculated at different ATP concentrations and then averaged and the standard deviation determined. The apparent binding constants were converted into intrinsic binding constants by applying a statistical correction that accounts for the number of ways the ith ATP molecule can bind or dissociate from GroEL when i-1 molecules are already bound, as follows:

+ app = N i 1 int Ki Ki i [S8] where N is the total number of binding sites. Given the scheme in Fig. 3C, it is possible to express the intrinsic binding constant for the ith site, as follows:

Ki L L + Ki L + Ki Kint = T 1 2 R 2 R' [S9] i i1 + i1 + i1 KT L1L2 KR L2 KR'

where KT, KR and KR’ are the ATP binding constants of the TT, TR and RR states and L1 and L2 are the allosteric equilibrium constants for the TT TR and TR RR transitions, respectively (L1

= [TT]/[TR] and L2 = [TR]/[RR]). In the derivation of Eq. S9, we assumed, for simplicity, that the affinities for ATP of the two different rings in the TR state can be represented by a single binding constant. The values of the parameters obtained from the fit to Eq. S9 were used to calculate the relative populations in the scheme in Fig. 3C.

Reference

1. Shimon L, Sharon M, & Horovitz A (2010) A method for removing effects of nonspecific binding on the distribution of binding stoichiometries: application to mass spectroscopy data. Biophys J 99(5):1645-1649.

Table S1. Measured and calculated masses of ATP bound forms of GroEL

Chaperonin complex Theoretical mass (Da) Measured mass (Da)a

ATP 507

ATP·Mg2+ 531 533 ± 17b

GroEL 57,198c 57,198 ± 2

(GroEL)14 800,766 801,047 ± 10

(GroEL)14(ATP)1 801,298 801,579 ± 16

(GroEL)14(ATP)2 801,829 802,109 ± 29

(GroEL)14(ATP)3 802,361 802,647 ± 47

(GroEL)14(ATP)4 802,892 803,211 ± 14

(GroEL)14(ATP)5 803,424 803,725 ± 21

(GroEL)14(ATP)6 803,955 804,292 ± 31

(GroEL)14(ATP)7 804,487 804,793 ± 26

(GroEL)14(ATP)8 805,018 805,333 ± 40

(GroEL)14(ATP)9 805,550 805,865 ± 36

(GroEL)14(ATP)10 806,081 806,403 ± 32

(GroEL)14(ATP)11 806,613 806,922 ± 29

(GroEL)14(ATP)12 807,144 807,452 ± 34

(GroEL)14(ATP)13 807,676 807,986 ± 44

(GroEL)14(ATP)14 808,207 808,518 ± 37

aMass errors represent the standard deviation of 4 charge states in 4 different ATP concentrations. bCalculated from the average mass difference between two successive ATP-bound states of GroEL. cThe first Met residue is removed.

Figure S1. Screening buffer conditions for gaining optimal resolution. Nano-ESI mass spectra of intact GroEL acquired in the presence of either 1M ammonium acetate, 1M ammonium acetate supplemented with 10 mM imidazole, 200 mM EDAA or triethylammonium acetate (TEAA). To ensure cooperative ATP binding 1M MgAc was added to all samples. The presence of 10 mM imidazole lowers the average charge state distribution of GroEL, however, a more pronounced effect that significantly increases the resolution, is observed when EDAA and TEAA were used. While TEAA gave the highest charge state reduction, the peaks were considerably broadened due to its adherence to GroEL’s outer surface. Therefore, we used in our analysis the EDAA buffer.

Figure S2. Charge states series give rise to a set of reliable replicas with low standard deviation. The diagram shows the relative populations of GroEL with different numbers of bound ATP molecules determined from the data corresponding to different charge states. The relative populations are remarkably similar among the different charge states with a standard deviation of up to 0.024. The different charge states are color-labeled and the standard deviation values are indicated above the bars corresponding to each population.

Figure S3. Plot of the fractional saturation determined from the MS data as a function of ATP concentration. The data were fitted to the Hill equation. For further details, see Supplementary Eqs.

Figure S4. ATP binding and hydrolysis by GroEL, in the presence of the EDAA buffer, displays a mono-phasic dependence on ATP concentration. (A) Initial rates of ATP hydrolysis by GroEL, in the presence of the EDAA buffer, at different ATP concentrations. The data were fitted to the Hill equation. For further details, see Supplementary Equations. (B) Simulation of the fractional saturation of the ATP binding sites in GroEL by different -1 -1 -1 concentrations of ATP using the values of KTT = 0.03 M , KTR = 0.11 M , KRR = 1.8 M , 3 15 LTT RR = 3x10 and LTR RR = 3x10 obtained from the fit of the data in Fig. 5 to Eq. S9.

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