Limited proteolysis in the investigation of beta2-microglobulin amyloidogenic and fibrillar states

Amyloid fibrils of patients treated with regular haemodialysis essentially consists of beta2-microglobulin (beta2-m) and its truncated species DeltaN6beta2-m lacking six residues at the amino terminus. The truncated fragment shows a higher propensity to self-aggregate and constitutes an excellent candidate for the analysis of a protein in the amyloidogenic conformation. The surface topology and the conformational analysis of native beta2-m and the truncated DeltaN6beta2-m species both in the soluble and in the fibrillar forms were investigated by the limited proteolysis/mass spectrometry strategy. The conformation in solution of a further truncated mutant DeltaN3beta2-m lacking three residues at the N-terminus was also examined. This approach appeared particularly suited to investigate the regions that are solvent-exposed, or flexible enough to be accessible to protein-protein interactions and to describe the conformation of transient intermediates. Moreover, proteolysis experiments can also be tailored to investigate amyloid fibrils by discriminating the protein regions constituting the unaccessible core of the fibrils and those still flexible and exposed to the solvent. Although native beta2-m and DeltaN3beta2-m shared essentially the same conformation, significative structural differences exist between the native and the DeltaN6beta2-m proteins in solution with major differences located at the end moiety of strand V and subsequent loop with strand VI and at both the N- and C-termini of the proteins. On the contrary, an identical distribution of preferential proteolytic sites was observed in both proteins in the fibrillar state, which was nearly superimposible to that observed for the soluble form of DeltaN6beta2-m. These data revealed that synthetic fibrils essentially consists of an unaccessible core comprising residues 20-87 of the beta2-m protein with exposed and flexible N- and C-terminal ends. Moreover, proteolytic cleavages observed in vitro at Lys 6 and Lys 19 reproduce specific cleavages that have to take place in vivo to generate the truncated forms of beta2-m occurring in natural fibrils. On the basis of these results, a molecular mechanism for fibril formation has been proposed.     

Properties of some variants of human beta2-microglobulin and amyloidogenesis

Three variants of human beta(2)-microglobulin (beta(2)-m) were compared with wild-type protein. For two variants, namely the mutant R3Abeta(2)-m and the form devoid of the N-terminal tripeptide (DeltaN3beta(2)-m), a reduced unfolding free energy was measured compared with wild-type beta(2)-m, whereas an increased stability was observed for the mutant H31Ybeta(2)-m. The solution structure could be determined by (1)H NMR spectroscopy and restrained modeling only for R3Abeta(2)-m that showed the same conformation as the parent species, except for deviations at the interstrand loops. Analogous conclusions were reached for H31Ybeta(2)-m and DeltaN3beta(2)-m. Precipitation and unfolding were observed over time periods shorter than 4-6 weeks with all the variants and, sometimes, with wild-type protein. The rate of structured protein loss from solution as a result of precipitation and unfolding always showed pseudo-zeroth order kinetics. This and the failure to observe an unfolded species without precipitation suggest that a nucleated conformational conversion scheme should apply for beta(2)-m fibrillogenesis. The mechanism is consistent with the previous and present results on beta(2)-m amyloid transition, provided a nucleated oligomeric species be considered the stable intermediate of fibrillogenesis, the monomeric intermediate being the necessary transition step along the pathway from the native protein to the nucleated oligomer.     

Topological investigation of amyloid fibrils obtained from beta2-microglobulin

Amyloid fibrils of patients treated with regular hemodialysis essentially consists of beta2-microglobulin (beta2-m) and its truncated species DeltaN6beta2-m lacking six residues at the amino terminus. The truncated fragment has a more flexible three-dimensional structure and constitutes an excellent candidate for the analysis of a protein in the amyloidogenic conformation. The surface topology of synthetic fibrils obtained from intact beta2-m and truncated DeltaN6beta2-m was investigated by the limited proteolysis/mass spectrometry approach that appeared particularly suited to gain insights into the structure of beta2-m within the fibrillar polymer. The distribution of prefential proteolytic sites observed in both fibrils revealed that the central region of the protein, which had been easily cleaved in the full-length globular beta2-m, was fully protected in the fibrillar form. In addition, the amino- and carboxy-terminal regions of beta2-m became exposed to the solvent in the fibrils, whereas they were masked completely in the native protein. These data indicate that beta2-m molecules in the fibrils consist of an unaccessible core comprising residues 20-87 with the strands I and VIII being not constrained in the fibrillar polymer and exposed to the proteases. Moreover, proteolytic cleavages observed in vitro at Lys 6 and Lys 19 reproduce specific cleavages that have to occur in vivo to generate the truncated forms of beta2-m occurring in natural fibrils. On the basis of these data, a possible mechanism for fibril formation from native beta2-m is discussed and an explanation for the occurrence of truncated protein species in natural fibrils is given.     

CD4 binding partially locks the bridging sheet in gp120 but leaves the beta2/3 strands flexible

The structure of the free form HIV gp120, critical for therapeutic agent development, is unavailable due to its high flexibility. Previous thermodynamic data, structural analysis and simulation results have suggested a large conformational change in the core domain upon CD4 binding. The bridging sheet, which consists of four beta-strands with beta20/21 nestling against the inner/outer domains and beta2/3 facing outward, more exposed to the solvent, was proposed to be unfolded in the native state. In order to test this proposition and to characterize the native conformations, we performed potential mean force (PMF) molecular dynamics (MD) simulations on the CD4-bound crystal structure. We pushed the bridging sheet away from the inner and outer domain to explore the accessible conformational space for the bridging sheet. In addition, we performed conventional MD simulations on structures with the bridging sheet partially unfolded to investigate the stability of the association between the inner and outer domains. Based on the free energy profiles, we find that the whole bridging sheet is unlikely to unfold without other concurrent conformational changes. On the other hand, the partial bridging sheet, beta strands 2/3, can switch its conformation from the folded to the unfolded state. Furthermore, relaxation of conformation with partially unfolded bridging sheet through MD simulations leads to a conformation with beta strands 20/21 quickly re-anchoring against the inner and outer domains. Such a conformation, although lacking some of the hydrophobic interactions present in the CD4-bound structure, displayed high stability as further indicated by other restrained MD simulations. The relevance of this conformation to the free form structure and the pathway for conformational change from the free form to the CD4-bound structure is discussed in detail in light of the available unliganded SIV gp120 crystal structure.     

Computer simulations of a tumor surface octapeptide epitope

Molecular dynamics using CHARMM and GEMM programs with the Star Technologies ST 100 array processor functioning at the speed of super computers was used as a searching algorithm for conformational exploration of the octapeptide Gly-Asn-Thr-Ile-Val-Ala-Glu. This poorly soluble octapeptide is the N-terminal epitope of an 11 KD glycoprotein antigen residing on human ductal carcinoma (breast) cells. Very long (nanoseconds) simulations were required. Both an alpha-helix and the N-acetyl-N1-methylamide derived minimized starting structures gave the same lowest potential energy conformation with simulations at 600 K. The same conformation was found only when using the latter starting conformation with simulations at 300 K. The lowest potential energy conformation was stabilized by 4 hydrophobic contacts and 13 H bonds completing one turn of a left-handed helix.     

Effects of force fields on the conformational and dynamic properties of amyloid β(1‐40) dimer explored by replica exchange molecular dynamics simulations

The conformational space and structural ensembles of amyloid beta (Aβ) peptides and their oligomers in solution are inherently disordered and proven to be challenging to study. Optimum force field selection for molecular dynamics (MD) simulations and the biophysical relevance of results are still unknown. We compared the conformational space of the Aβ(1‐40) dimers by 300 ns replica exchange MD simulations at physiological temperature (310 K) using: the AMBER‐ff99sb‐ILDN, AMBER‐ff99sb*‐ILDN, AMBER‐ff99sb‐NMR, and CHARMM22* force fields. Statistical comparisons of simulation results to experimental data and previously published simulations utilizing the CHARMM22* and CHARMM36 force fields were performed. All force fields yield sampled ensembles of conformations with collision cross sectional areas for the dimer that are statistically significantly larger than experimental results. All force fields, with the exception of AMBER‐ff99sb‐ILDN (8.8 ± 6.4%) and CHARMM36 (2.7 ± 4.2%), tend to overestimate the α‐helical content compared to experimental CD (5.3 ± 5.2%). Using the AMBER‐ff99sb‐NMR force field resulted in the greatest degree of variance (41.3 ± 12.9%). Except for the AMBER‐ff99sb‐NMR force field, the others tended to under estimate the expected amount of β‐sheet and over estimate the amount of turn/bend/random coil conformations. All force fields, with the exception AMBER‐ff99sb‐NMR, reproduce a theoretically expected β‐sheet‐turn‐β‐sheet conformational motif, however, only the CHARMM22* and CHARMM36 force fields yield results compatible with collapse of the central and C‐terminal hydrophobic cores from residues 17‐21 and 30‐36. Although analyses of essential subspace sampling showed only minor variations between force fields, secondary structures of lowest energy conformers are different.     

The actin binding site of thymosin beta 4 mapped by mutational analysis.

We characterized in detail the actin binding site of the small actin-sequestering protein thymosin beta 4 (T beta 4) using chemically synthesized full-length T beta 4 variants. The N-terminal part (residues 1-16) and a hexapeptide motif (residues 17-22) form separate structural entities. In both, we identified charged and hydrophobic residues that participate in the actin interaction using chemical cross-linking, complex formation in native gels and actin-sequestering experiments. Quantitative data on the activity of the variants and circular dichroism experiments allow to present a model in which the N-terminal part needs to adopt an alpha-helix for actin binding and interacts through a patch of hydrophobic residues (6M-I-F12) on one side of this helix. Also, electrostatic contacts between actin and lysine residues 18, in the motif, and 14, in the N-terminal alpha-helix, appear important for binding. The residues critical for contacting actin are conserved throughout the beta-thymosin family and in addition to this we identify a similar pattern in the C-terminal headpiece of villin and dematin.     

Mass-weighted molecular dynamics simulation of cyclic polypeptides.

A modified molecular dynamics (MD) method in which atomic masses are weighted was developed previously for studying the conformational flexibility of neuroregulating tetrapeptide Phe-Met-Arg-Phe-amide (FMRF-amide). The method has now been applied to longer and constrained molecules, namely a disulfide-linked cyclic hexapeptide, c[CYFQNC], and its linear and "pseudo-cyclic" analogues. The sampling of dehedral conformational space of teh linear hexapeptide in mass-weighted MD simulations was found to be improved significantly over conventional MD simulations, as in the case of the shorter FMRF-amide molecule studied previously. In the cyclic hexapeptide, the internal constraint of the molecule due to the intramolecular disulfide bond (hence the absence of free terminals in the molecule) does not adversely affect the significant improvement of conformational sampling in mass-weighted MD simulations over normal MD simulations. The pseudo-cyclic polypeptide is identical to the linear CYFQNC molecule in amino acid sequence (i.e., side chains of the cysteine residues are reduced), but the positions of its two terminal heavy atoms were held fixed in space such that the molecule has a nearly cyclic conformation. For this molecule, the mass-weighted MD simulation generated a wide range of polypeptide backbone conformations covering the internal dihedral degrees of freedom; moreover, the physical space of the pseudo-cyclic structure was also sampled in a complete revolution of the entire molecular fragment about the two fixed termini during the simulation. These characteristics suggest that mass-weighted MD can also be an extremely useful method for conformational analyses of constrained molecules and, in particular, for modeling loops on protein surfaces.     

Conformational transitions of flanking purines in HIV-1 RNA dimerization initiation site kissing complexes studied by CHARMM explicit solvent molecular dynamics

Dimerization of HIV-1 genomic RNA is initiated by kissing loop interactions at the Dimerization Initiation Site (DIS). Dynamics of purines that flank the 5' ends of the loop-loop helix in HIV-1 DIS kissing complex were explored using explicit solvent molecular dynamics (MD) simulations with the CHARMM force field. Multiple MD simulations (200 ns in total) of X-ray structures for HIV-1 DIS Subtypes A, B, and F revealed conformational variability of flanking purines. In particular, the flanking purines, which in the starting X-ray structures are bulged-out and stack in pairs, formed a consecutive stack of four bulged-out adenines at the beginning of several simulations. This conformation is seen in the crystal structure of DIS Subtype F with no interference from crystal packing, and was frequently reported in our preceding MD studies performed with the AMBER force field. However, as CHARMM simulations progressed, the four continuously stacked adenines showed conformational transitions from the bulged-out into the bulged-in geometries. Although such an arrangement has not been seen in any X-ray structure, it has been suggested by a recent NMR investigation. In CHARMM simulations, in the longer time scale, the flanking purines display the tendency to move to bulged-in conformations. This is in contrast with the AMBER simulations, which indicate a modest prevalence for bulged-out flanking base positions in line with the X-ray data. The simulations also suggest that the intermolecular stacking between purines from the opposite hairpins can additionally stabilize the kissing complex.     

The N-terminal alpha-helix of pancreatic phospholipase A2 determines productive-mode orientation of the enzyme at the membrane surface

Phospholipase A(2) (PLA(2)) hydrolyzes glycerophospholipids to free fatty acid and lyso-phospholipid, which serve as precursors for the biosynthesis of eicosanoids and other lipid-derived mediators of inflammation and allergy. PLA(2) activity strongly increases upon binding to the surface of aggregated phospholipid. The N-terminal approximately ten residue alpha-helix of certain PLA(2) isoforms plays important roles in the interfacial activation of the enzyme by providing residues for membrane binding of PLA(2) and by contributing to the formation of the substrate-binding pocket. However, the relative contributions of the N-terminal alpha-helix and the rest of the protein in membrane binding of PLA(2) and its productive-mode orientation at the membrane surface are not well understood. Here we use a variety of biophysical approaches to determine the role of the N-terminal helix in membrane binding strength, orientation, and activity of human pancreatic PLA(2). While the full-length PLA(2) binds to membranes with a defined orientation, an engineered PLA(2) fragment DeltaN10 that lacks the N-terminal ten residues binds to membranes with weaker affinity and at random orientation, and exhibits approximately 100-fold lower enzymatic activity compared to the full-length PLA(2), indicating the key role of the N terminus in PLA(2) function. The results of polarized infrared spectroscopic experiments permit determination of the orientation of membrane-bound PLA(2) and identification of its interfacial binding surface. Moreover, the full-length PLA(2) demonstrates increased conformational flexibility in solution and is stabilized upon membrane binding, while the DeltaN10 fragment is more rigid than the full-length PLA(2) both in free and membrane-bound states. Our results suggest that the N-terminal alpha-helix supports the activation of PLA(2) by (a) enhancing the membrane binding strength, (b) facilitating a productive-mode orientation of PLA(2) at the membrane surface, and (c) conferring conformational integrity and plasticity to the enzyme.     

Effective energy function for proteins in solution

A Gaussian solvent-exclusion model for the solvation free energy is developed. It is based on theoretical considerations and parametrized with experimental data. When combined with the CHARMM 19 polar hydrogen energy function, it provides an effective energy function (EEF1) for proteins in solution. The solvation model assumes that the solvation free energy of a protein molecule is a sum of group contributions, which are determined from values for small model compounds. For charged groups, the self-energy contribution is accounted for primarily by the exclusion model. Ionic side-chains are neutralized, and a distance-dependent dielectric constant is used to approximate the charge-charge interactions in solution. The resulting EEF1 is subjected to a number of tests. Molecular dynamics simulations at room temperature of several proteins in their native conformation are performed, and stable trajectories are obtained. The deviations from the experimental structures are similar to those observed in explicit water simulations. The calculated enthalpy of unfolding of a polyalanine helix is found to be in good agreement with experimental data. Results reported elsewhere show that EEF1 clearly distinguishes correctly from incorrectly folded proteins, both in static energy evaluations and in molecular dynamics simulations and that unfolding pathways obtained by high-temperature molecular dynamics simulations agree with those obtained by explicit water simulations. Thus, this energy function appears to provide a realistic first approximation to the effective energy hypersurface of proteins.     

Conformational dynamics of beta(2)-microglobulin analyzed by reduction and reoxidation of the disulfide bond

Although native beta(2)-microglobulin (beta2-m), the light chain of the major histocompatibility complex class I antigen, assumes an immunoglobulin domain fold, it is also found as a major component of dialysis-related amyloid fibrils. In the amyloid fibrils, the conformation of beta2-m is considered to be largely different from that of the native state, and a monomeric denatured form is likely to be a precursor to the amyloid fibril. To obtain insight into the conformational dynamics of beta2-m leading to the formation of amyloid fibrils, we studied the reduction and reoxidation of the disulfide bond by reduced and oxidized dithiothreitol, respectively, and the effects on the reduction of the chaperonin GroEL, a model protein that might destabilize the native state of beta2-m. We show that beta2-m occasionally unfolds into a denatured form even under physiological conditions and that this transition is promoted upon interaction with GroEL. The results imply that in vivo interactions of beta2-m with other proteins or membrane components could destabilize its native structure, thus stabilizing the amyloid precursor.     

Beta-hairpin folding mechanism of a nine-residue peptide revealed from molecular dynamics simulations in explicit water

The beta-hairpin fold mechanism of a nine-residue peptide, which is modified from the beta-hairpin of alpha-amylase inhibitor tendamistat (residues 15-23), is studied through direct folding simulations in explicit water at native folding conditions. Three 300-nanosecond self-guided molecular dynamics (SGMD) simulations have revealed a series of beta-hairpin folding events. During these simulations, the peptide folds repeatedly into a major cluster of beta-hairpin structures, which agree well with nuclear magnetic resonance experimental observations. This major cluster is found to have the minimum conformational free energy among all sampled conformations. This peptide also folds into many other beta-hairpin structures, which represent some local free energy minimum states. In the unfolded state, the N-terminal residues of the peptide, Tyr-1, Gln-2, and Asn-3, have a confined conformational distribution. This confinement makes beta-hairpin the only energetically favored structure to fold. The unfolded state of this peptide is populated with conformations with non-native intrapeptide interactions. This peptide goes through fully hydrated conformations to eliminate non-native interactions before folding into a beta-hairpin. The folding of a beta-hairpin starts with side-chain interactions, which bring two strands together to form interstrand hydrogen bonds. The unfolding of the beta-hairpin is not simply the reverse of the folding process. Comparing unfolding simulations using MD and SGMD methods demonstrate that SGMD simulations can qualitatively reproduce the kinetics of the peptide system.     

Effects of different force fields on the structural character of α synuclein β-hairpin peptide (35–56) in aqueous environment

The hallmark of Parkinson’s disease (PD) is the intracellular protein aggregation forming Lewy Bodies (LB) and Lewy neuritis which comprise mostly of a protein, alpha synuclein (α-syn). Molecular dynamics (MD) simulation methods can augment experimental techniques to understand misfolding and aggregation pathways with atomistic resolution. The quality of MD simulations for proteins and peptides depends greatly on the accuracy of empirical force fields. The aim of this work is to investigate the effects of different force fields on the structural character of β hairpin fragment of α-syn (residues 35–56) peptide in aqueous solution. Six independent MD simulations are done in explicit solvent using, AMBER03, AMBER99SB, GROMOS96 43A1, GROMOS96 53A6, OPLS-AA, and CHARMM27 force fields with CMAP corrections. The performance of each force field is assessed from several structural parameters such as root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), solvent accessible surface area (SASA), formation of β-turn, the stability of folded β-hairpin structure, and the favourable conformations obtained for different force fields. In this study, CMAP correction of CHARMM27 force field is found to overestimate the helical conformation, while GROMOS96 53A6 is found to most successfully capture the conformational dynamics of α-syn β-hairpin fragment as elicited from NMR.     

Increase in the conformational flexibility of beta 2-microglobulin upon copper binding: a possible role for copper in dialysis-related amyloidosis

A key pathological event in dialysis-related amyloidosis is the fibril formation of beta(2)-microglobulin (beta 2-m). Because beta 2-m does not form fibrils in vitro, except under acidic conditions, predisposing factors that may drive fibril formation at physiological pH have been the focus of much attention. One factor that may be implicated is Cu(2+) binding, which destabilizes the native state of beta 2-m and thus stabilizes the amyloid precursor. To address the Cu(2+)-induced destabilization of beta 2-m at the atomic level, we studied changes in the conformational dynamics of beta 2-m upon Cu(2+) binding. Titration of beta 2-m with Cu(2+) monitored by heteronuclear NMR showed that three out of four histidines (His13, His31, and His51) are involved in the binding at pH 7.0. (1)H-(15)N heteronuclear NOE suggested increased backbone dynamics for the residues Val49 to Ser55, implying that the Cu(2+) binding at His51 increased the local dynamics of beta-strand D. Hydrogen/deuterium exchange of amide protons showed increased flexibility of the core residues upon Cu(2+) binding. Taken together, it is likely that Cu(2+) binding increases the pico- to nanosecond fluctuation of the beta-strand D on which His51 exists, which is propagated to the core of the molecule, thus promoting the global and slow fluctuations. This may contribute to the overall destabilization of the molecule, increasing the equilibrium population of the amyloidogenic intermediate.     

Conformational space exploration of Met- and Leu-enkephalin using the MOLS method, molecular dynamics, and Monte Carlo simulation--a comparative study

We report here a comparative study of the molecular conformational energy landscape generated using the mutually orthogonal Latin squares (MOLS) method, molecular dynamics (MD), and Monte Carlo (MC) simulation. The MOLS method, as described earlier from our laboratory, uses an experimental design technique to rapidly and exhaustively sample the low energy conformations of a molecule. MD and MC simulations have been used to perform similar tasks. In the comparison reported here, the three methods were applied to a pair of neuropeptides, namely Met- and Leu-enkephalin. A set of 1500 conformations of these enkephalins were generated using these methods with CHARMM22 force field, and the resulting samples were analyzed to determine the extent and nature of coverage of the conformational space. The results indicate that the MOLS method samples a larger number of possible conformations and identifies conformations closer to the experimental structures than the MD and MC simulations.     

Effects of helix and fingertip mutations on the thermostability of xyn11A investigated by molecular dynamics simulations and enzyme activity assays

Local conformational changes and global unfolding pathways of wildtype xyn11A recombinant and its mutated structures were studied through a series of atomistic molecular dynamics (MD) simulations, along with enzyme activity assays at three incubation temperatures to investigate the effects of mutations at three different sites to the thermostability. The first mutation was to replace an unstable negatively charged residue at a surface beta turn near the active site (D32G) by a hydrophobic residue. The second mutation was to create a disulphide bond (S100C/N147C) establishing a strong connection between an alpha helix and a distal beta hairpin associated with the thermally sensitive Thumb loop, and the third mutation add an extra hydrogen bond (A155S) to the same alpha helix. From the MD simulations performed, MM/PBSA energy calculations of the unfolding energy were in a good agreement with the enzyme activities measured from the experiment, as all mutated structures demonstrated the improved thermostability, especially the S100C/N147C proved to be the most stable mutant both by the simulations and the experiment. Local conformational analysis at the catalytic sites and the xylan access region also suggested that mutated xyn11A structures could accommodate xylan binding. However, the analysis of global unfolding pathways showed that structural disruptions at the beta sheet regions near the N-terminal were still imminent. These findings could provide the insight on the molecular mechanisms underlying the enhanced thermostability due to mutagenesis and changes in the protein unfolding pathways for further protein engineering of the GH11 family xylanase enzymes.     

Domain motions of the Mip protein from Legionella pneumophila

The homodimeric 45.6 kDa (total mass) Mip protein, a virulence factor from Legionella pneumophila, was investigated with solution NMR spectroscopy and molecular dynamics (MD) simulations. Two Mip monomers are dimerized via an N-terminal helix bundle that is connected via a long alpha-helix to a C-terminal FKBP domain in each subunit. More than 85% of the amino acids were identified in triple-resonance NMR spectra. (15)N relaxation analysis showed a bimodal distribution of R(1)/R(2) values, with the lower ratio in the N-terminal domain. Relaxation dispersion measurements confirmed that these reduced ratios did not originate from conformational exchange. Thus, two different correlation times (tau(c)) can be deduced, reflecting partly uncoupled motions of both domains. Relaxation data of a Mip(77)(-)(213) monomer mutant were similar to those observed in the dimer, corroborating that the FKBP domain, including part of the connecting helix, behaves as one dynamic entity. MD simulations (18 ns) of the Mip dimer also yielded two different correlation times for the two domains and thus confirm the independence of the domain motions. Principal component analysis of the dihedral space covariance matrix calculated from the MD trajectory suggests a flexible region in the long connecting helix that acts as a hinge between the two domains. Such motion provides a possible explanation of how Mip can bind to complex molecular components of the extracellular matrix and mediate alveolar damage and bacterial spread in the lung.     

Unusual thionation of a cyclic hexapeptide. Conformational changes and dynamics.

One carbonyl oxygen of the cyclic hexapeptide cyclo(-Gly1-Pro2-Phe3-Val4-Phe5-Phe6-) (A) can be selectively exchanged with sulphur using Yokoyama's reagent. Surprisingly it was not the C=] of Gly1 but that of Phe5 which was substituted and cyclo(-Gly1-Pro2-Phe3-Val4-Phe5 psi [CS-NH]Phe6-) (B) was obtained. Thionation results in a conformational change of the peptide backbone although the C=O of Phe5 and the corresponding C=S are not involved in internal hydrogen bonds. Two isomers in slow exchange, containing a cis Gly1-Pro2 bond in a beta VIa-turn (minor) and a trans Gly-Pro bond in a beta II'-turn (major), were analyzed by restrained molecular dynamics in vacuo and in DMSO as well as using time dependent distance constraints. It is impossible to fit all experimental data to a static structure of each isomer. Interpreting the conflicting NOEs, local segment flexibility is found. MD simulations lead to a dynamic model for each structure with evidence of an equilibrium between a beta I- and beta II-turn about the Val4-Phe5 amide bond in both the cis and trans isomers. Additionally proton relaxation rates in the rotating frame (R1 rho) were measured to verify the assumption of this fast beta I/beta II equilibrium within each isomer. Significant contributions to R1 rho-rates from intramolecular motions were found for both isomers. Therefore it is possible to distinguish between at least four conformers interconverting on different time scales based on NMR data and MD refinement. This work shows that thionation is a useful modification of peptides for conformation-activity investigations.