Loop propensity of the sequence YKGQP from staphylococcal nuclease: implications for the folding of nuclease.

Recently we performed molecular dynamics (MD) simulations on the folding of the hairpin peptide DTVKLMYKGQPMTFR from staphylococcal nuclease in explicit water. We found that the peptide folds into a hairpin conformation with native and nonnative hydrogen-bonding patterns. In all the folding events observed in the folding of the hairpin peptide, loop formation involving the region YKGQP was an important event. In order to trace the origins of the loop propensity of the sequence YKGQP, we performed MD simulations on the sequence starting from extended, polyproline II and native type I' turn conformations for a total simulation length of 300 ns, using the GROMOS96 force field under constant volume and temperature (NVT) conditions. The free-energy landscape of the peptide YKGQP shows minima corresponding to loop conformation with Tyr and Pro side-chain association, turn and extended conformational forms, with modest free-energy barriers separating the minima. To elucidate the role of Gly in facilitating loop formation, we also performed MD simulations of the mutated peptide YKAQP (Gly --> Ala mutation) under similar conditions starting from polyproline II conformation for 100 ns. Two minima corresponding to bend/turn and extended conformations were observed in the free-energy landscape for the peptide YKAQP. The free-energy barrier between the minima in the free-energy landscape of the peptide YKAQP was also modest. Loop conformation is largely sampled by the YKGQP peptide, while extended conformation is largely sampled by the YKAQP peptide. We also explain why the YKGQP sequence samples type II turn conformation in these simulations, whereas the sequence as part of the hairpin peptide DTVKLMYKGQPMTFR samples type I' turn conformation both in the X-ray crystal structure and in our earlier simulations on the folding of the hairpin peptide. We discuss the implications of our results to the folding of the staphylococcal nuclease.     

Folding and stability of the three-stranded beta-sheet peptide Betanova: insights from molecular dynamics simulations

The dynamics of the three-stranded beta-sheet peptide Betanova has been studied at four different temperatures (280, 300, 350, and 450 K by molecular dynamics simulation techniques, in explicit water. Two 20-ns simulations at 280 K indicate that the peptide remains very flexible under "folding" conditions sampling a range of conformations that together satisfy the nuclear magnetic resonance (NMR)-derived experimental constraints. Two simulations at 300 K (above the experimental folding temperature) of 20 ns each show partial formation of "native"-like structure, which also satisfies most of the NOE constraints at 280 K. At higher temperature, the presence of compact states, in which a series of hydrophobic contacts remain present, are observed. This is consistent with experimental observations regarding the role of hydrophobic contacts in determining the peptide's stability and in initiating the formation of turns and loops. A set of different structures is shown to satisfy NMR-derived distance restraints and a possible mechanism for the folding of the peptide into the NMR-determined structure is proposed.     

β‐Hairpin folding and stability: molecular dynamics simulations of designed peptides in aqueous solution

The structural properties of a 10‐residue and a 15‐residue peptide in aqueous solution were investigated by molecular dynamics simulation. The two designed peptides, SYINSDGTWT and SESYINSDGTWTVTE, had been studied previously by NMR at 278 K and the resulting model structures were classified as 3:5 β‐hairpins with a type I + G1 β‐bulge turn. In simulations at 278 K, starting from the NMR model structure, the 3:5 β‐hairpin conformers proved to be stable over the time period evaluated (30 ns). Starting from an extended conformation, simulations of the decapeptide at 278 K, 323 K and 353 K were also performed to study folding. Over the relatively short time scales explored (30 ns at 278 K and 323 K, 56 ns at 353 K), folding to the 3:5 β‐hairpin could only be observed at 353 K. At this temperature, the collapse to β‐hairpin‐like conformations is very fast. The conformational space accessible to the peptide is entirely dominated by loop structures with different degrees of β‐hairpin character. The transitions between different types of ordered loops and β‐hairpins occur through two unstructured loop conformations stabilized by a single side‐chain interaction between Tyr2 and Trp9, which facilitates the changes of the hydrogen‐bond register. In agreement with previous experimental results, β‐hairpin formation is initially driven by the bending propensity of the turn segment. Nevertheless, the fine organization of the turn region appears to be a late event in the folding process. Copyright © 2004 European Peptide Society and John Wiley & Sons, Ltd.     

Protein flexibility: multiple molecular dynamics simulations of insulin chain B

Multiple molecular dynamics simulations totaling more than 100 ns were performed on chain B of insulin in explicit solvent at 300 K and 400 K. Despite some individual variations, a comparison of the protein dynamics of each simulation showed similar trends and most structures were consistent with NMR experimental values, even at the elevated temperature. The importance of packing interactions in determining the conformational transitions of the protein was observed, sometimes resulting in conformations induced by localized hydrophobic interactions. The high temperature simulation generated a more diverse range of structures with similar elements of secondary structure and populated conformations to the simulations at room temperature. A broad sampling of the conformational space of insulin chain B illustrated a wide range of conformational states with many transitions at room temperature in addition to the conformational states observed experimentally. The T-state conformation associated with insulin activity was consistently present and a possible mechanism of behavior was suggested.     

Molecular modeling of an antigenic complex between a viral peptide and a class I major histocompatibility glycoprotein.

Computer simulation of the conformations of short antigenic peptides (5-10 residues) either free or bound to their receptor, the major histocompatibility complex (MHC)-encoded glycoprotein H-2 Ld, was employed to explain experimentally determined differences in the antigenic activities within a set of related peptides. Starting for each sequence from the most probable conformations disclosed by a pattern-recognition technique, several energy-minimized structures were subjected to molecular dynamics simulations (MD) either in vacuo or solvated by water molecules. Notably, antigenic potencies were found to correlate to the peptides propensity to form and maintain an overall alpha-helical conformation through regular i,i + 4 hydrogen bonds. Accordingly, less active or inactive peptides showed a strong tendency to form i,i + 3 hydrogen bonds at their N-terminal end. Experimental data documented that the C-terminal residue is critical for interaction of the peptide with H-2 Ld. This finding could be satisfactorily explained by a 3-D Q.S.A.R. analysis postulating interactions between ligand and receptor by hydrophobic forces. A 3-D model is proposed for the complex between a high-affinity nonapeptide and the H-2 Ld receptor. First, the H-2 Ld molecule was built from X-ray coordinates of two homologous proteins: HLA-A2 and HLA-Aw68, energy-minimized and studied by MD simulations. With HLA-A2 as template, the only realistic simulation was achieved for a solvated model with minor deviations of the MD mean structure from the X-ray conformation. Water simulation of the H-2 Ld protein in complex with the antigenic nonapeptide was then achieved with the template-derived optimal parameters. The bound peptide retains mainly its alpha-helical conformation and binds to hydrophobic residues of H-2 Ld that correspond to highly polymorphic positions of MHC proteins. The orientation of the nonapeptide in the binding cleft is in accordance with the experimentally determined distribution of its MHC receptor-binding residues (agretope residues). Thus, computer simulation was successfully employed to explain functional data and predicts alpha-helical conformation for the bound peptide.     

Molecular dynamics simulations of synthetic peptide folding

Because the time scale of protein folding is much greater than that of the widely used simulations of native structures, a detailed report of molecular dynamics simulations of folding has not been available. In this study, we Included the average solvent effect in the potential functions to simplify the calculation of the solvent effect and carried out long molecular dynamics simulations of the alanine-based synthetic peptides at 274 K. From either an extended or a randomly generated conformation, the simulations approached a helix-coil equilibrium in about 3 ns. The multiple minima problem did not prevent helix folding. The calculated helical ratio of Ac-AAQAAAAQAAAAQAAY-NH₂ was 47%, in good agreement with the circular dichroism measurement (about 50%). A helical segment with frayed ends was the most stable conformation, but the hydrophobic interaction favored the compact, distorted helix-turn-helix conformations. The transition between the two types of conformations occurred in a much larger time scale than helix propagation. The transient hydrogen bonds between the glutamine side chain and the backbone carbonyl group could reduce the free energy barrier of helix folding and unfolding. The substitution of a single alanine residue in the middle of the peptide with valine or glycine decreased the average helical ratio significantly, in agreement with experimental observations. © 1996 Wiley-Liss, Inc.     

Induced‐fit or preexisting equilibrium dynamics? Lessons from protein crystallography and MD simulations on acetylcholinesterase and implications for structure‐based drug design

Crystal structures of acetylcholinesterase complexed with ligands are compared with side-chain conformations accessed by native acetylcholinesterase in molecular dynamics (MD) simulations. Several crystallographic conformations of a key residue in a specific binding site are accessed in a simulation of native acetylcholinesterase, although not seen in rotomer plots. Conformational changes upon ligand binding thus involve preexisting equilibrium dynamics. Consequently, rational drug design could benefit significantly from conformations monitored by MD simulations of native targets.     

A shorter peptide model from staphylococcal nuclease for the folding-unfolding equilibrium of a beta-hairpin shows that unfolded state has significant contribution from compact conformational states

It is important to understand the conformational features of the unfolded state in equilibrium with folded state under physiological conditions. In this paper, we consider a short peptide model LMYKGQPM from staphylococcal nuclease to model the conformational equilibrium between a hairpin conformation and its unfolded state using molecular dynamics simulation under NVT conditions at 300K using GROMOS96 force field. The free energy landscape has overall funnel-like shape with hairpin conformations sampling the minima. The "unfolded" state has a higher free energy of approximately 12kJ/mol with respect to native hairpin minimum and occupies a plateau region. We find that the unfolded state has significant contributions from compact conformations. Many of these conformations have hairpin-like topology. Further, these compact conformational forms are stabilized by hydrophobic interactions. Conversion between native and non-native hairpins occurs via unfolded states. Frequent conversions between folded and unfolded hairpins are observed with single exponential kinetics. We compare our results with the emerging picture of unfolded state from both experimental and theoretical studies.     

Folding of alpha(r)beta and epsilonbeta reverse turns; a nanosecond molecular dynamics simulation of the hexapeptide MSALNT and the octapeptide NMSALNTL in water.

Folding of the hexapeptide MSALNT and the octapeptide NMSALNTL were investigated using 2.8 ns molecular dynamics (MD) simulations in aqueous solution. In the simulation, the central sequence SALN of the hexapeptide folded rapidly within 200 ps into an alpha(r)beta turn conformation (type VIII conformation) and remained in this conformation for the rest of the trajectory. The sequence SALN of the octapeptide needed 2 ns to fold via epsilonbeta conformations into a similar conformation. The results join the sequences into a growing group of sequences which have a tendency to form secondary structures and thereby to direct protein folding. The structures of the reverse turn conformations were in accordance with the experimental results (Hakalehto et al., Eur J. Biochem. 250, 19-29 (1997)). The main driving force of folding seems to be the hydrophobic interaction between the side chains of Ala and Leu at the i+1 and i+2 positions of the beta-turn.     

Refolding simulations of an isolated fragment of barnase into a native-like β hairpin: Evidence for compactness and hydrogen bonding as concurrent stabilizing factors

Experimental evidence and theoretical models both suggest that protein folding is initiated within specific fragments intermittently adopting conformations close to that found in the protein native structure. These folding initiation sites encompassing short portions of the protein are ideally suited for study in isolation by computational methods aimed at peering into the very early events of folding. We have used Molecular Dynamics (MD) technique to investigate the behavior of an isolated protein fragment formed by residues 85 to 102 of barnase that folds into a β hairpin in the protein native structure. Three independent MD simulations of 1.3 to 1.8 ns starting from unfolded conformations of the peptide portrayed with an all-atom model in water were carried out at gradually decreasing temperature. A detailed analysis of the conformational preferences adopted by this peptide in the course of the simulations is presented. Two of the unfolded peptide conformations fold into a hairpin characterized by native and a larger bulk of nonnative interactions. Both refolding simulations substantiate the close relationship between interstrand compactness and hydrogen bonding network involving backbone atoms. Persistent compactness witnessed by side-chain interactions always occurs concomitantly with the formation of backbone hydrogen bonds. No highly populated conformations generated in a third simulation starting from the remotest unfolded conformer relative to the native structure are observed. However, nonnative long-range and medium-range contacts with the aromatic moiety of Trp94 are spotted, which are in fair agreement with a former nuclear magnetic resonance study of a denaturing solution of an isolated barnase fragment encompassing the β hairpin. All this lends reason to believe that the 85–102 barnase fragment is a strong initiation site for folding. Proteins 29:212–227, 1997. © 1997 Wiley-Liss, Inc.     

Beta-hairpin conformation of fibrillogenic peptides: structure and alpha-beta transition mechanism revealed by molecular dynamics simulations

Understanding the conformational transitions that trigger the aggregation and amyloidogenesis of otherwise soluble peptides at atomic resolution is of fundamental relevance for the design of effective therapeutic agents against amyloid-related disorders. In the present study the transition from ideal alpha-helical to beta-hairpin conformations is revealed by long timescale molecular dynamics simulations in explicit water solvent, for two well-known amyloidogenic peptides: the H1 peptide from prion protein and the Abeta(12-28) fragment from the Abeta(1-42) peptide responsible for Alzheimer's disease. The simulations highlight the unfolding of alpha-helices, followed by the formation of bent conformations and a final convergence to ordered in register beta-hairpin conformations. The beta-hairpins observed, despite different sequences, exhibit a common dynamic behavior and the presence of a peculiar pattern of the hydrophobic side-chains, in particular in the region of the turns. These observations hint at a possible common aggregation mechanism for the onset of different amyloid diseases and a common mechanism in the transition to the beta-hairpin structures. Furthermore the simulations presented herein evidence the stabilization of the alpha-helical conformations induced by the presence of an organic fluorinated cosolvent. The results of MD simulation in 2,2,2-trifluoroethanol (TFE)/water mixture provide further evidence that the peptide coating effect of TFE molecules is responsible for the stabilization of the soluble helical conformation.     

Exploring the energy landscape of protein folding using replica-exchange and conventional molecular dynamics simulations

Two independent replica-exchange molecular dynamics (REMD) simulations with an explicit water model were performed of the Trp-cage mini-protein. In the first REMD simulation, the replicas started from the native conformation, while in the second they started from a nonnative conformation. Initially, the first simulation yielded results qualitatively similar to those of two previously published REMD simulations: the protein appeared to be over-stabilized, with the predicted melting temperature 50-150K higher than the experimental value of 315K. However, as the first REMD simulation progressed, the protein unfolded at all temperatures. In our second REMD simulation, which starts from a nonnative conformation, there was no evidence of significant folding. Transitions from the unfolded to the folded state did not occur on the timescale of these simulations, despite the expected improvement in sampling of REMD over conventional molecular dynamics (MD) simulations. The combined 1.42 micros of simulation time was insufficient for REMD simulations with different starting structures to converge. Conventional MD simulations at a range of temperatures were also performed. In contrast to REMD, the conventional MD simulations provide an estimate of Tm in good agreement with experiment. Furthermore, the conventional MD is a fraction of the cost of REMD and continuous, realistic pathways of the unfolding process at atomic resolution are obtained.     

Structural and dynamic effects of alpha-helix deletion in Sso7d: implications for protein thermal stability

Sso7d is a 62-residue protein from the hyperthemophilic archaeon Sulfolobus solfataricus with a denaturation temperature close to 100 degrees C around neutral pH. An engineered form of Sso7d truncated at leucine 54 (L54Delta) is significantly less stable, with a denaturation temperature of 53 degrees C. Molecular dynamics (MD) studies of Sso7d and its truncated form at two different temperatures have been performed. The results of the MD simulations at 300 K indicate that: (1) the flexibility of Sso7d chain at 300 K agrees with that detected from X-ray and NMR structural studies; (2) L54Delta remains stable in the native folded conformation and possesses an overall dynamic behavior similar to that of the parent protein. MD simulations performed at 500 K, 10 ns long, indicate that, while Sso7d is in-silico resistant to high temperature, the truncated variant partially unfolds, revealing the early phases of the thermal unfolding pathway of the protein. Analysis of the trajectories of L54Delta suggests that the unzipping of the N-terminal and C-terminal beta-strands should be the first event of the unfolding pathway, and points out the regions more resistant to thermal unfolding. These findings allow one to understand the role played by specific interactions connecting the two ends of the chain for the high thermal stability of Sso7d, and support recent hypotheses on its folding mechanism emerged from site-directed mutagenesis studies.     

beta-hairpin stability and folding: molecular dynamics studies of the first beta-hairpin of tendamistat

The stability and (un)folding of the 19-residue peptide, SCVTLYQSWRYSQADNGCA, corresponding to the first beta-hairpin (residues 10 to 28) of the alpha-amylase inhibitor tendamistat (PDB entry 3AIT) has been studied by molecular dynamics simulations in explicit water under periodic boundary conditions at several temperatures (300 K, 360 K and 400 K), starting from various conformations for simulation lengths, ranging from 10 to 30 ns. Comparison of trajectories of the reduced and oxidized native peptides reveals the importance of the disulphide bridge closing the beta-hairpin in maintaining a proper turn conformation, thereby insuring a proper side-chain arrangement of the conserved turn residues. This allows rationalization of the conservation of those cysteine residues among the family of alpha-amylase inhibitors. High temperature simulations starting from widely different initial configurations (native beta-hairpin, alpha and left-handed helical and extended conformations) begin sampling similar regions of the conformational space within tens of nanoseconds, and both native and non-native beta-hairpin conformations are recovered. Transitions between conformational clusters are accompanied by an increase in energy fluctuations, which is consistent with the increase in heat capacity measured experimentally upon protein folding. The folding events observed in the various simulations support a model for beta-hairpin formation in which the turn is formed first, followed by hydrogen bond formation closing the hairpin, and subsequent stabilization by side-chain hydrophobic interactions.     

Dissecting the stability of a beta-hairpin peptide that folds in water: NMR and molecular dynamics analysis of the beta-turn and beta-strand contributions to folding.

NMR studies of the folding and conformational properties of a beta-hairpin peptide, several peptide fragments of the hairpin, and sequence-modified analogues, have enabled the various contributions to beta-hairpin stability in water to be dissected. Temperature and pH-induced unfolding studies indicate that the folding-unfolding equilibrium approximates to a two-state model. The hairpin is highly resistant to denaturation and is still significantly folded in 7 M urea at 298 K. Thermodynamic analysis shows the hairpin to fold in water with a significant change in heat capacity, however, DeltaCp degrees in 7 M urea is reduced. V/Y-->A mutations on one strand of the hairpin reduce folding to <10 %, consistent with a hydrophobic stabilisation model. We show that in a truncated peptide (residues 6-16) lacking the hydrophobic residues on one beta-strand, the type I' Asn-Gly turn in the sequence SINGKK is significantly populated in water in the absence of interstrand hydrophobic contacts. Unrestrained molecular dynamics simulations of unfolding, using an explicit solvation model, show that the conformation of the NG turn persists for longer than the AG analogue, which has a much lower propensity for type I' turn formation from a data base analysis of preferred turns. The origin of the high stability of the Asn-Gly turn is not entirely clear; data base analysis of 66 NG turns, together with molecular dynamics simulations, reveals no participation of the Asn side-chain in turn-stabilising interactions with the peptide backbone. However, hydration analysis of the molecular dynamics simulations reveals a pocket of "high density" water bridging between the Asn side-chain and peptide main-chain that suggests solvent-mediated interactions may play an important role in modulating phi,psi propensities in the NG turn region.     

Conformational dynamics of a short antigenic peptide in its free and antibody bound forms gives insight into the role of β‐turns in peptide immunogenicity

Earlier immunological experiments with a synthetic 36‐residue peptide (75‐110) from Influenza hemagglutinin have been shown to elicit anti‐peptide antibodies (Ab) which could cross‐react with the parent protein. In this article, we have studied the conformational features of a short antigenic (Ag) peptide (⁹⁸YPYDVPDYASLRS¹¹⁰) from Influenza hemagglutinin in its free and antibody (Ab) bound forms with molecular dynamics simulations using GROMACS package and OPLS‐AA/L all‐atom force field at two different temperatures (293 K and 310 K). Multiple simulations for the free Ag peptide show sampling of ordered conformations and suggest different conformational preferences of the peptide at the two temperatures. The free Ag samples a conformation crucial for Ab binding (β‐turn formed by “DYAS” sequence) with greater preference at 310 K while, it samples a native‐like conformation with relatively greater propensity at 293 K. The sequence “DYAS” samples β‐turn conformation with greater propensity at 310 K as part of the hemagglutinin protein also. The bound Ag too samples the β‐turn involving “DYAS” sequence and in addition it also samples a β‐turn formed by the sequence “YPYD” at its N‐terminus, which seems to be induced upon binding to the Ab. Further, the bound Ag displays conformational flexibility at both 293 K and 310 K, particularly at terminal residues. The implications of these results for peptide immunogenicity and Ag–Ab recognition are discussed. Proteins 2015; 83:1352–1367. © 2015 Wiley Periodicals, Inc.     

Folding simulations of Trp‐cage mini protein in explicit solvent using biasing potential replica‐exchange molecular dynamics simulations

Replica exchange molecular dynamics (RexMD) simulations are frequently used for studying structure formation and dynamics of peptides and proteins. A significant drawback of standard temperature RexMD is, however, the rapid increase of the replica number with increasing system size to cover a desired temperature range. A recently developed Hamiltonian RexMD method has been used to study folding of the Trp‐cage protein. It employs a biasing potential that lowers the backbone dihedral barriers and promotes peptide backbone transitions along the replica coordinate. In two independent applications of the biasing potential RexMD method including explicit solvent and starting from a completely unfolded structure the formation of near‐native conformations was observed after 30–40 ns simulation time. The conformation representing the most populated cluster at the final simulation stage had a backbone root mean square deviation of ～1.3 Å from the experimental structure. This was achieved with a very modest number of five replicas making it well suited for peptide and protein folding and refinement studies including explicit solvent. In contrast, during five independent continuous 70 ns molecular dynamics simulations formation of collapsed states but no near native structure formation was observed. The simulations predict a largely collapsed state with a significant helical propensity for the helical domain of the Trp‐cage protein already in the unfolded state. Hydrogen bonded bridging water molecules were identified that could play an active role by stabilizing the arrangement of the helical domain with respect to the rest of the chain already in intermediate states of the protein. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

Mechanism of fiber assembly: treatment of Aβ peptide aggregation with a coarse-grained united-residue force field

The growth mechanism of β-amyloid (Aβ) peptide fibrils was studied by a physics-based coarse-grained united-residue model and molecular dynamics (MD) simulations. To identify the mechanism of monomer addition to an Aβ_1-40 fibril, we placed an unstructured monomer at a distance of 20 Å from a fibril template and allowed it to interact freely with the latter. The monomer was not biased towards fibril conformation by either the force field or the MD algorithm. With the use of a coarse-grained model with replica-exchange molecular dynamics, a longer timescale was accessible, making it possible to observe how the monomers probe different binding modes during their search for the fibril conformation. Although different assembly pathways were seen, they all follow a dock-lock mechanism with two distinct locking stages, consistent with experimental data on fibril elongation. Whereas these experiments have not been able to characterize the conformations populating the different stages, we have been able to describe these different stages explicitly by following free monomers as they dock onto a fibril template and to adopt the fibril conformation (i.e., we describe fibril elongation step by step at the molecular level). During the first stage of the assembly (“docking”), the monomer tries different conformations. After docking, the monomer is locked into the fibril through two different locking stages. In the first stage, the monomer forms hydrogen bonds with the fibril template along one of the strands in a two-stranded β-hairpin; in the second stage, hydrogen bonds are formed along the second strand, locking the monomer into the fibril structure. The data reveal a free-energy barrier separating the two locking stages. The importance of hydrophobic interactions and hydrogen bonds in the stability of the Aβ fibril structure was examined by carrying out additional canonical MD simulations of oligomers with different numbers of chains (4-16 chains), with the fibril structure as the initial conformation. The data confirm that the structures are stabilized largely by hydrophobic interactions and show that intermolecular hydrogen bonds are highly stable and contribute to the stability of the oligomers as well.     

Modeling conformational changes in cyclosporin A.

NMR and X-ray structures for the immunosuppressant cyclosporin A (CsA) reveal a remarkable difference between the unbound (free) conformation in organic solvents and the conformation bound to cyclophilin. We have performed computer simulations of the molecular dynamics of CsA under a variety of conditions and confirmed the stability of these two conformations at room temperature in water and in vacuum. However, when the free conformation was modeled in vacuum at 600 K, a transition pathway leading to the bound conformation was observed. This involved a change in the cis MeLeu-9 peptide bond to a trans conformation and the movement of the side chains forming the dominant hydrophobic cluster (residues MeBmt-1, MeLeu-4, MeLeu-6, and MeLeu-10) to the opposite side of the plane formed by the backbone atoms in the molecular ring. The final conformation had a backbone RMS deviation from the bound conformation of 0.53 A and was as stable in dynamics simulations as the bound conformation. Our calculations allowed us to make a detailed analysis of a transition pathway between the free and the bound conformations of CsA and to identify two distinct regions of coordinated movement in CsA, both of which underwent transitions independently.