Solution conformation of the Pseudomonas syringae MSU 16H phytotoxic lipodepsipeptide Pseudomycin A determined by computer simulations using distance geometry and molecular dynamics from NMR data

Pseudomycin A is a cyclic lipodepsinonapeptide phytotoxin produced by a strain of the plant pathogenic bacterium Pseudomonas syringae. Like other members of this family of bacterial metabolites, it is characterised by a fatty acylated cyclic peptide with mixed chirality and lactonic closure. Several biological activities of Pseudomycin A are lower than those found for some of its congeners, a difference which might depend on the diverse number and distribution of charged residues in the peptide moiety. Hence, it was of interest to investigate its conformation in solution. After the complete interpretation of the two-dimensional NMR spectra, NOE data were obtained and the structure was determined by computer simulations, applying distance geometry and molecular dynamics procedures. The conformation of the large ring of Pseudomycin A in solution includes three rigid structural regions interrupted by three short flexible regions that act as hinges. The overall three-dimensional structure of the cyclic moiety is similar to that of previously studied bioactive lipodepsinonapeptides produced by other pseudomonads.     

Internal motions and hydration of sucrose in a diluted water solution

This paper represents a synthesis of our most recent work on the hydration, internal and overall dynamics of sucrose in a diluted water solution. The studies were carried out as a total ensemble of 1.2 nanosecond condensed phase molecular dynamics trajectories. In this study the focus is on a 500 ps trajectory starting with the solute in the crystalline conformation. The presence of water was found to significantly alter the accessible conformational space of the solute. All potential intra-molecular hydrogen bonds were found to be exchanged to surrounding water molecules and the simulations suggest that the sucrose conformation is stabilized by the dynamic presence of two interring bridging water molecules: O-2g...Ow...O-3f and O-2g...Ow...O-lf. The overall shift in conformation of the solute induced by the presence of water was found to improve the theoretical models of experimental traits. It is demonstrated that the hydration structure and the internal and overall motions of sucrose compare extremely well with NMR data such as glycosidic heteronuclear coupling constants and the molecular tumbling time, with X-ray data of two partially hydrated sucrose structures in a protein complex and with translational diffusion coefficients and hydration numbers established from experimental studies.     

A model of water structure inside the HLA-A2 peptide binding groove

Based on molecular dynamics simulations, it is proposed that water within the binding groove of the human MHC class I molecule HLA-A2 plays a role in the formation of its complex with the influenza matrix protein (residues 58-66; GILGFVFTL) peptide. In these simulations, a loosely structured network of water molecules is present in the binding groove between the peptide and the MHC molecule, and may be important in completing the peptide-MHC interface. In two independent 400 ps simulations where groove-based water molecules were included, the peptide remained essentially in the conformation observed in the crystal structure. In contrast, in a 400 ps simulation in which no water molecules were placed between the peptide and the MHC molecule, the crystal structure conformation was rapidly lost. The basis for this behavior appears to be that the groove-based water molecules help to maintain the appropriate orientation of the Arg-97 side chain of HLA-A2 and, in turn, the conformation of the central part of the peptide.     

Design, synthesis and conformational analysis of hGM-CSF(13-31)-Gly-Pro-Gly-(103-116)

On the basis of the X-ray structure and results from structure-activity relationship studies, the following GM-CSF analogue was designed and synthesized by solid-phase methodology: hGM-CSF[13-31]-Gly-Pro-Gly-[103-116]-NH2. This analogue was constructed to comprise helices A and D of the native hGM-CSF, covalently linked in an antiparallel orientation by the tripeptide spacer Gly-Pro-Gly, which is known as a turn-inducing sequence. The conformational analysis of the analogue by CD spectroscopy revealed an essentially random structure in water, while alpha-helix formation was observed upon addition of TFE. In 40% TFE the helix content was approximately 45%. By two-dimensional NMR experiments in 1:1 water/trifluoroethanol mixture two helical sequences were identified comprising the segments corresponding to helix A and helix D. In addition to medium-range NOESY connectivities, a long-range cross-peak was found involving the leucine residues at positions 13 and 35. Based on the experimentally derived data (54 NOEs), the structure was refined by restrained molecular dynamics simulations over 120 ps at various temperatures. A representative conformation derived from the computer simulation is mainly characterized by two helical segments connected by a loop region. The overall three-dimensional structure of the analogue is comparable to the X-ray structure of hGM-CSF in that helices A and D are oriented in an antiparallel fashion, forming a two alpha-helix bundle. Nevertheless, there are small differences in the topology of the helices between the solution structure of the designed analogue and the X-ray structure of hGM-CSF. The possible implications of these conformational features at the effects of biological activity are discussed.     

Molecular dynamics investigations of DNA triple helical models: unique features of the Watson-Crick duplex

We have built computer models of triple helical structures with a third poly(dT) strand Hoogsteen base paired to the major groove of a poly(dA).poly(dT) Watson-Crick (WC) base-paired duplex in the canonical A-DNA as well as B-DNA. For the A-DNA form, the sugar-phosphate backbone of the third strand intertwines and clashes with the poly(dA) strand requiring a radical alteration of the duplex to access the hydrogen bonding sites in the major groove. In contrast, when the duplex was in the canonical B-DNA form, the third strand was readily accommodated in the major groove without perturbing the duplex. The triple helical model, with the duplex in the B-DNA form, was equilibrated for 400ps using molecular dynamics simulations including water molecules and counter-ions. During the entire simulations, the deoxyriboses of the adenine strand oscillate between the S-type and E-type conformations. However, 30% of the sugars of the thymine strands-II & III switch to the N-type conformation early in the simulations but return to the S-type conformation after 200ps. In the equilibrium structure, the WC duplex portion of the triplex is unique and its geometry differs from both the A- or B-DNA. the deoxyriboses of the three strands predominantly exhibit S-type conformation. Besides the sugar pucker, the major groove width and the base-tilt are analogous to B-DNA, while the X-displacement and helical twist resemble A-DNA, giving a unique structure to the triplex and the Watson & Crick and Hoogsteen duplexes.     

Kinetics of peptide folding: computer simulations of SYPFDV and peptide variants in water

The folding of Ser-Tyr-Pro-Phe-Asp-Val (SYPFDV), and sequence variants of this peptide (SYPYD and SYPFD) are studied computationally in an explicit water environment. An atomically detailed model of the peptide is embedded in a sphere of TIP3P water molecules and its optimal structure is computed by simulated annealing. At distances from the peptide that are beyond a few solvation shells, a continuum solvent model is employed. The simulations are performed using a mean field approach that enhances the efficiency of sampling peptide conformations. The computations predict a small number of conformations as plausible folded structures. All have a type VI turn conformation for the peptide backbone, similar to that found using NMR. However, some of the structures differ from the experimentally proposed ones in the packing of the proline ring with the aromatic residues. The second most populated structure has, in addition to a correctly folded backbone, the same hydrophobic packing as the conformation measured by NMR. Our simulations suggest a kinetic mechanism that consists of three separate stages. The time-scales associated with these stages are distinct and depend differently on temperature. Electrostatic interactions play an initial role in guiding the peptide chain to a roughly correct structure as measured by the end-to-end distance. At the same time or later the backbone torsions rearrange due to local tendency of the proline ring to form a turn: this step depends on solvation forces and is helped by loose hydrophobic interactions. In the final step, hydrophobic residues pack against each other. We also show the existence of an off the pathway intermediate, suggesting that even in the folding of a small peptide "misfolded" structures can form. The simulations clearly show that parallel folding paths are involved. Our findings suggest that the process of peptide folding shares many of the features expected for the significantly larger protein molecules.     

Conformation of desmopressin, an analogue of the peptide hormone vasopressin, in aqueous solution as determined by NMR spectroscopy

Desmopressin (1-desamino-[DArg8]vasopressin, is a synthetic analogue of the neurohypophyseal peptide hormone vasopressin which has high antidiuretic and antibleeding potency. The structure of desmopressin has been determined in aqueous solution by two-dimensional NMR techniques and molecular dynamics simulations. Both standard and time-averaged distance restraints were used in structure calculations because of the inherent flexibility in small peptides. 21 models calculated with standard restraints were compared with structures refined with time-averaged distance restraints and were found to be good representatives of the conformational ensemble of desmopressin. The macrocyclic ring forms an inverse gamma-turn centered around Gln4. Residues 1 and 2, the disulphide bridge and the three-residue acyclic tail were found to be flexible in solution. Residues 4-6 in the ensemble of calculated structures contain essentially the same backbone conformation as in the crystal structure of pressinoic acid, the cyclic moiety of vasopressin, whereas residues 2-6 superimpose on the NMR-derived conformation of oxytocin bound to neurophysin. The results presented in this work suggest that, in addition to the differences in sequence between desmopressin and vasopressin, differences in conformational and dynamic properties between the two compounds explain their pharmacological differences.     

Modeling the interaction between FK506 and FKBP12: a mechanism for formation of the calcineurin inhibitory complex

FK506 is a naturally occurring immunosuppressant whose mode of action involves formation of an initial complex with the cytosolic protein FKBP12. The composite surface of this complex then binds to and inhibits the protein phosphatase calcineurin (PP2B). To investigate why FK506 does not inhibit calcineurin directly we have conducted molecular modeling and conformational studies on published structures of FK506 both alone and in complex with FKBP12. From studies of the structure of FK506 in CDCl3 and Z-Arg32-ascomycin in water (a water soluble analogue of FK506) we suggest that the FK506 molecule can be viewed as consisting of three separate regions. The pipecolate region which extends from C24 to C10 including the pipecolate ring shows strongly conserved conformation in both solvents. The loop region which extends from C25 to C16 shows general conservation of the loop structure and the pyranose region made up of the pyranose ring and C15-C17 which shows highly variable conformation depending on solvent. Comparison of the structure of Z-Arg32-ascomycin in water with structures of FK506 bound to FKBP12 indicate that the conformation of the pipecolate region is conserved during the binding process. The conformation of the loop region was generally conserved but a significant reduction (approximately 1.7 A) in the diameter of the loop in the bound structure was observed. The conformation of the pyranose ring and C15-C17 region was found to be significantly altered in the bound structure resulting in displacements of the C13 and C15 methoxyl groups of 2.8 and 3.5 A, respectively. From computer models and molecular dynamics simulations of interactions between FK506 and FKBP12 we suggest that the conformational changes observed in bound FK506 are induced by the interaction between the 80's loop of FKBP12 and the pyranose ring of FKBP12. These interactions result in the formation of a complex with the both correct shape and surface polarity for interaction with calcineurin.     

The solution structure of the histidine-containing protein (HPr) from Staphylococcus aureus as determined by two-dimensional 1H-NMR spectroscopy

The three-dimensional solution structure of the heat-stable phosphocarrier protein HPr from Staphylococcus aureus was determined from two-dimensional NMR data by restrained molecular dynamics. It consists of a large twisted antiparallel beta-pleated sheet with four strands A, B, C, and D of amino acids 2-7, 34-37, 40-42 and 60-65. Three right-handed helices A, B, C (amino acids 18-27, 47-53 and 71-85) are positioned on top of this sheet. The aromatic ring of His15 is located in a cleft formed by amino acids 12-17 and 55-58, only the nitrogen (N delta 1) atom which can be phosphorylated by enzyme I is exposed to the water. The side chains of Thr12 and Arg17 are located close to the histidine ring. The regulatory serine residue (Ser46) is located in a hydrophobic patch, its hydroxyl group is water-accessible but forms hydrogen bonds with the amide groups of the backbone. The general features of the three-dimensional structure are similar to those found in HPr proteins from different microorganisms such as Escherichia coli, Bacillus subtilis and Streptococcus faecalis.     

Solution structure of a DNA duplex containing the exocyclic lesion 3,N4-etheno-2'-deoxycytidine opposite 2'-deoxyguanosine

Vinyl chloride reacts with cellular DNA producing 3,N4-etheno-2'-deoxycytidine (epsilonC) along with other exocyclic adducts. The solution structure of an oligodeoxynucleotide duplex containing an epsilonC.dG base pair was determined by high-resolution NMR spectroscopy and molecular dynamics simulations. NMR data indicated that the duplex adopts a right-handed helical structure having all residues in anti orientation around the glycosylic torsion angle. The epsilonC adduct has a sugar pucker in the C3'-endo/C4'-exo region while the rest of the residues are in the C2'-endo/C3'-exo range. NOE interactions established Watson-Crick alignments for canonical base pairs of the duplex. The imino proton of the lesion-containing base pair resonated as a sharp signal that was resistant to water exchange, suggesting hydrogen bonding. Restrained molecular dynamics simulations generated three-dimensional models in excellent agreement with the spectroscopic data. The refined structures are slightly bent at the lesion site without major perturbations of the sugar-phosphate backbone. The adduct is displaced and shifted toward the major groove of the helix while its partner on the complementary strand remains stacked. The epsilonC(anti).dG(anti) base pair alignment is sheared and stabilized by the formation of hydrogen bonds. The biological implications of structures of epsilonC-containing DNA duplexes are discussed.     

Molecular dynamics of amphotericin B. II. Dimer in water

Molecular dynamics simulations were performed for a dimer of the antifungal antibiotic, amphotericin B, in water. In the first step of the work three appropriately selected versions of the dimer structure were taken into consideration. In each version antibiotic molecules were placed antiparallel with polar and ionizable groups outside the hydrophobic core formed by polyene chromophores. During short dynamic simulations versions of the dimer structure were compared in respect of the energy of dimerization. The highest energy was observed for the structure in which polyene chromophores superimposed each other as much as possible and this version was subjected to the main simulation. The analysis of 66 snapshot geometries stored during 33 ps dynamic trajectory allowed us to draw three main conclusions: (i) the relative orientation of the amino-sugar moiety and chromophore as well as conformation of the antibiotic macrolide ring were different in both molecules and could exhibit dynamic changes, (ii) the dimer structure exhibited intrinsic asymmetry which could be responsible for characteristic circular dichroism spectra of the aggregated form of the antibiotic, (iii) relatively high stability of the dimer structure resulted not only from hydrophobic interactions between chromophores but also from hydrogen bonds networks that were observed around polar terminals of antibiotic molecules. Implications of these features of the dimer structure for its susceptibility on the ionic state of carboxyl and/or amino groups are also discussed.     

Interactions of hydrated metal ions with nucleotides: the crystal structure of barium adenosine 5'-monophosphate heptahydrate

The crystal and molecular structure of barium adenosine 5'-monophosphate heptahydrate was determined from x-ray diffraction data. Crystals of barium adenosine 5'-monophosphate heptahydrate are monoclinic, space group C2, with a = 32.559(3), b = 6.969(3), c = 9.597(1) A, and beta = 100.31(1) degrees. Intensity data were collected with an automated diffractometer. The structure was solved by the heavy-atom method and refined by least-squares to R = 0.034. This structure provides an example of an outer-sphere metal-nucleotide complex, in which a completely hydrated metal ion interacts with the nucleotide only through water bridges. The barium ion is coordinated to eight water molecules, which form a slightly distorted square antiprism. Seven of the eight water molecules from the barium hydration shell are hydrogen bonded to phosphate groups; three of these water molecules are also hydrogen bonded to other suitable acceptor sites on the base and ribose moieties. The conformation about the glycosidic bond is anti, with chiCN = 69 degrees, and, as in most nucleotide structures, the conformation about the C(4')-C(5') bond is gauche-gauche. However, the ribose displays an unusual conformation (best described as C(4')-exo) not previously observed in crystal structures of nucleosides or nucleotides, other than 3',5'-cyclic nucleotides. It is possible that this unusual conformation is a consequence of the metal-water-nucleotide bridging interactions.     

The solution conformations of amino acids from molecular dynamics simulations of Gly-X-Gly peptides: comparison with NMR parameters

The conformations that amino acids can adopt in the random coil state are of fundamental interest in the context of protein folding research and studies of protein-peptide interactions. To date, no detailed quantitative data from experimental studies have been reported; only nuclear magnetic resonance parameters such as chemical shifts and J coupling constants have been reported. These experimental nuclear magnetic resonance data represent averages over multiple conformations, and hence they do not provide unique structural information. I have performed relatively long (2.5 ns) molecular dynamics simulations of Gly-X-Gly tripeptides, surrounded by explicit water molecules, where X represents eight different amino acids with long side chains. From the trajectories one can calculate time averaged backbone chemical shifts and 3J(NH alpha) coupling constants and compare these with experimental data. These calculated quantities are quite close to the experimental values for most amino acids, suggesting that these simulations are a good model for the random coil state of the tripeptides. On the basis of my simulations I predict 3J(alphabeta) coupling constants and present dihedral distributions for the phi, psi, as well as chi1 and chi2 angles. Finally, I present correlation plots for these dihedral angles.     

Dual recognition of double-stranded DNA by 2'-aminoethoxy-modified oligonucleotides: the solution structure of an intramolecular triplex obtained by NMR spectroscopy

The solution structure of an intramolecular triple helical oligonucleotide has been solved by NMR. The third strand of the pyrimidine x purine x pyrimidine triplex is composed of 2'-aminoethoxy-modified riboses, whereas the remaining part of the nucleic acid is DNA. The structure around the aminoethoxy modification was obtained with the help of selective isotope labeling in conjunction with isotope-editing experiments. Dinucleotide steps and interstrand connectivities, as well as the complete backbone conformation of the triplex, were derived from J-couplings, NOEs, and 31P chemical shifts. The structure of this triplex, solved by distance geometry, explains the extraordinary stability and increase in rate of triplex formation induced by 2'-aminoethoxy-modified oligonucleotides: apart from the formation of seven base triples, a well-defined hydrogen-bonding network is formed across the Crick-Hoogsteen groove involving the amino protons of the aminoethoxy moieties and the phosphates of the purine strand of the DNA. The modified strand adopts a conformation which is close to an A-type helix, whereas the DNA duplex conformation is best described as an unwound B-type helix. The groove dimensions and helical parameters of the 2'-aminoethoxy-modified rY x dRdY triplex are surprisingly well conserved in comparison with DNA triplexes.     

High-resolution solution NMR structure of the minimal active domain of the human immunodeficiency virus type-2 nucleocapsid protein

The retroviral nucleocapsid (NC) protein is a multifunctional protein essential for RNA genome packaging and viral infectivity. The NC protein, NCp8, of the human immunodeficiency virus type-II (HIV-2) is a 49 amino acid peptide containing two zinc fingers, of the type C-X2-C-X4-H-X4-C, connected by seven amino acid residues, called the "basic amino acid cluster." It has been shown that the N-terminal zinc finger flanked by the basic amino acid cluster is the minimal active domain for the specific binding to viral RNA and other functions. However, the structure-activity relationships of NCp8 have not been investigated in detail. In the present study, the three-dimensional structure of a 29 amino acid peptide, including the minimal active domain (NCp8-fl), was determined by two-dimensional 1H NMR spectroscopy with simulated annealing calculations. A total of 15 converged structures of NCp8-fl were obtained on the basis of 355 experimental constraints, including 343 distance constraints obtained from nuclear Overhauser effect connectivities, 12 torsion angle (phi, chi1) constraints, and four constraints for zinc binding. The root-mean-square deviation of the 15 converged structures was 0.29 +/- 0.04 A for the backbone atoms (N, C(alpha), C) and 1.27 +/- 0.13 A for all heavy atoms. Interestingly, the basic amino acid cluster itself was defined well, with a loop-like conformation in which three arginine residues in the cluster and one arginine residue in the zinc finger are located approximately in the same plane of the molecule and are exposed to the solvent. The structure-activity relationships are discussed on the basis of the comparison of this well-defined structure with those of other NC proteins.     

Solution structure of Lqh-8/6, a toxin-like peptide from a scorpion venom--structural heterogeneity induced by proline cis/trans isomerization

Lqh-8/6 is a minor fraction isolated from the venom of the scorpion Leiurus quinquestriatus hebraeus. Here we describe the purification, amino acid sequencing and solution structure determination by NMR and molecular modeling of this peptide. Lqh-8/6 is a small polypeptide (38 residues) which contains 8 half-cystines and is highly similar to another venom component, chlorotoxin. Standard homonuclear methods were used to sequentially assign the proton NMR spectra and to collect spatial restraints for structure determination. Two populations, identified early in the assignment step, are in slow interconversion on the NMR timescale. The two conformers were shown to originate from a cis/trans peptidyl-prolyl isomerization. Using a distance geometry program and simulated annealing protocol under the NMR restraints we obtained 10 final structures for the major conformation (trans isomer). None of the structures showed NOE violations larger than 0.05 nm, and the rmsd value relative to the mean structure (considering the main chain atoms in well-defined secondary structure) is 0.07 nm. The three-dimensional structure contains a short alpha-helix strapped on a small antiparallel beta-strand and an N-terminal extended fragment. The sequence/structure and structure/function relationships of the new scorpion toxin-like peptide are discussed in the context of the present structure determination. This toxin shows a stable, highly populated cis conformer of a peptidyl-prolyl peptide bond.     

Intrinsic nature of the three-dimensional structure of proteins as determined by distance geometry with good sampling properties

A protocol for distance geometry calculation is shown to have excellent sampling properties in the determination of three-dimensional structures of proteins from nuclear magnetic resonance (NMR) data. This protocol uses a simulated annealing optimization employing mass-weighted molecular dynamics in four-dimensional space (Havel, T.F. (1991) Prog. Biophys. Mol. Biol., 56, 43-78). It attains an extremely large radius of convergence, allowing a random coil conformation to be used as the initial estimate for the succeeding optimization process. Computations are performed with four systems of simulated distance data as tests of the protocol, using an unconstrained L-alanine 30mer and three different types of proteins, bovine pancreatic trypsin inhibitor, the alpha-amylase inhibitor Tendamistat, and the N-terminal domain of the 434-repressor. The test of the unconstrained polypeptide confirms that the sampled conformational space is that of the statistical random coil. In the larger and more complicated systems of the three proteins, the protocol gives complete convergence of the optimization without any trace of initial structure dependence. As a result of an exhaustive conformational sampling by the protocol, the intrinsic nature of the structures generated with distance restraints derived from NMR data has been revealed. When the sampled structures are compared with the corresponding X-ray structures, we find that the averages of the sampled structures always show a certain pattern of discrepancy from the X-ray structure. This discrepancy is due to the short distance nature of the distance restraints, and correlates with the characteristic shape of the protein molecule.     

Chronic neurologic effects of pesticide overexposure

To investigate the molecular mechanisms involved in paramyxovirus-induced cell fusion, the function and structure of a peptide with a 20-amino-acid sequence from the leucine zipper region (heptad repeat region 2) of the Newcastle disease virus fusion protein (F) were characterized. A peptide with the sequence ALDKLEESNSKLDKVNVKLT (amino acids 478-497 of the F protein) was found to inhibit syncytia formation after virus infection and after transfection of Cos cells with the HN (hemagglutinin-neuraminidase) and F protein cDNAs. Using an F protein gene that requires addition of exogenous trypsin for cleavage, it was shown that the peptide exerted its inhibitory effect prior to cleavage. The three-dimensional conformation of the peptide in aqueous solution was determined through the use of NMR and molecular modeling. Results showed that the peptide formed a helix with properties between an alpha-helix and a 3(10)-helix and that leucine residues aligned along one face of the helix. Side chain salt bridges and hydrogen bonds likely contributed to the stability of the peptide secondary structure. Analysis of the aqueous solution conformation of the peptide suggested mechanisms for specificity of interaction with the intact F protein.     

The crystal structure of the sevenfold mutant of barley beta-amylase with increased thermostability at 2.5 A resolution

The three-dimensional structure of the sevenfold mutant of barley beta-amylase (BBA-7s) with increased thermostability was determined by X-ray crystallography. The enzyme was purified as a single component and crystallized by a hanging drop method in the presence of 14 % PEG 6000. The crystals belong to space group P43212 with cell dimensions a=b=72.11 A, c=250.51 A. The diffraction data up to 2.5 A were collected after soaking the crystal in 100 mM maltose with Rsym of 8.6 %. The structure was determined by a molecular replacement method using soybean beta-amylase (SBA) as a search model and refined to an R-factor of 18.7 %. The final model included 500 amino acid residues, 141 water molecules and three glucose residues, which were located at subsites 1-2 and 4 in the active site. The r.m.s. distance of 485 Calpha atoms between BBA-7s and SBA was 0.62 A. Out of the seven mutated amino acids, four (Ser295Ala, Ile297Val, Ser351Pro and Ala376Ser) were substitutions from the common residues with SBA to the thermostable forms. A comparison of the structures of BBA-7s and SBA indicated that the side-chain of Ser376 makes new hydrogen bonds to the main-chain of an adjacent beta-strand, and that the side-chains of Val297 reduce an unfavorable interaction between the side-chains of Ala314. The mutation of Ser295Ala breaks the hydrogen bond between Ser295 OG and Tyr195 OH, which seems to be the reason for the unoccupied glucose residue at subsite 3. The tandem mutations at 350-352 including substitutions to two Pro residues suggested the reduction of main-chain entropy in the unfolded structure of this solvent-exposed protruded loop.