Zrinka Gattin

A Combined Solid‐State NMR and MD Characterization of the Stability and Dynamics of the HET‐s(218‐289) Prion in its Amyloid Conformation

Dynamic and rigid: The prion HET‐s(218–289) consists, in its amyloid form as shown here, of highly ordered and rigid parts and a very dynamic loop, which could be of great importance for fibril formation. Indeed, MD simulations explain the experimental NMR results and describe the dynamics of the salt‐bridge network that stabilizes the amyloid fibril, a feature not easily accessible by experiment.

Temperature and urea induced denaturation of the TRP-cage mini protein TC5b: A simulation study consistent with experimental observations

The effects of temperature and urea denaturation (6M urea) on the dominant structures of the 20‐residue Trp‐cage mini‐protein TC5b are investigated by molecular dynamics simulations of the protein at different temperatures in aqueous and in 6M urea solution using explicit solvent degrees of freedom and the GROMOS force‐field parameter set 45A3. In aqueous solution at 278 K, TC5b is stable throughout the 20 ns of MD simulation and the trajectory structures largely agree with the NMR‐NOE atom–atom distance data available. Raising the temperature to 360 K and to 400 K, the protein denatures within 22 ns and 3 ns, showing that the denaturation temperature is well below 360 K using the GROMOS force field. This is 40–90 K lower than the denaturation temperatures observed in simulations using other much used protein force fields. As the experimental denaturation temperature is about 315 K, the GROMOS force field appears not to overstabilize TC5b, as other force fields and the use of continuum solvation models seem to do. This feature may directly stem from the GROMOS force‐field parameter calibration protocol, which primarily involves reproduction of condensed phase thermodynamic quantities such as energies, densities, and solvation free energies of small compounds representative for protein fragments. By adding 6M urea to the solution, the onset of denaturation is observed in the simulation, but is too slow to observe a particular side‐chain side‐chain contact (Trp6‐Ile4) that was experimentally observed to be characteristic for the denatured state. Interestingly, using temperature denaturation, the process is accelerated and the experimental data are reproduced.

Atomic Models of De Novo Designed ccβ-Met Amyloid-Like Fibrils

The common characteristics of amyloid and amyloid-like fibrils from disease- and non-disease-associated proteins offer the prospect that well-defined model systems can be used to systematically dissect the driving forces of amyloid formation. We recently reported the de novo designed cc beta peptide model system that forms a native-like coiled-coil structure at low temperatures and which can be switched to amyloid-like fibrils by increasing the temperature. Here, we report a detailed molecular description of the system in its fibrillar state by characterizing the cc beta-Met variant using several microscopic techniques, circular dichroism spectroscopy, X-ray fiber diffraction, solid-state nuclear magnetic resonance, and molecular dynamics calculations. We show that cc beta-Met forms amyloid-like fibrils of different morphologies on both the macroscopic and atomic levels, which can be controlled by variations of assembly conditions. Interestingly, heterogeneity is also observed along single fibrils. We propose atomic models of the cc beta-Met amyloid-like fibril, which are in good agreement with all experimental data. The models provide a rational explanation why oxidation of methionine residues completely abolishes cc beta-Met amyloid fibril formation, indicating that a small number of site-specific hydrophobic interactions can play a major role in the packing of polypeptide-chain segments within amyloid fibrils. The detailed structural information available for the cc beta model system provides a strong molecular basis for understanding the influence and relative contribution of hydrophobic interactions on native-state stability, kinetics of fibril formation, fibril packing, and polymorphism.

Structure and dynamics of two β-peptides in solution from molecular dynamics simulations validated against experiment

We have studied two different β-peptides in methanol using explicit solvent molecular dynamics simulations and the GROMOS 53A6 force field: a heptapeptide (peptide 1) expected to form a left-handed 314-helix, and a hexapeptide (peptide 2) expected to form a β-hairpin in solution. Our analysis has focused on identifying and analyzing the stability of the dominant secondary structure conformations adopted by the peptides, as well as on comparing the experimental NOE distance upper bounds and 3J-coupling values with their counterparts calculated on the basis of the simulated ensembles. Moreover, we have critically compared the present results with the analogous results obtained with the GROMOS 45A3 (peptide 1) and 43A1 (peptide 2) force fields. We conclude that within the limits of conformational sampling employed here, the GROMOS 53A6 force field satisfactorily reproduces experimental findings regarding the behavior of short β-peptides, with accuracy that is comparable to but not exceeding that of the previous versions of the force field.GCE legendConformational clustering analysis of the simulated ensemble of a ß-hexapeptide with two different simulation setups (a and b). The central members of all of the clusters populating more than 5% of all of the structures are shown, together with the most dominant hydrogen bonds and the corresponding percentages of cluster members containing them

Interpreting Experimental Data by Using Molecular Simulation Instead of Model Building

A proper description of the conformational equilibrium of polypeptides or proteins is essential for a correct description of their function. The conformational ensembles from 16 molecular dynamic simulations of two beta- heptapeptides were used to interpret the primary NMR data, which were also compared to a set of NMR model structures (see graphic).One of the most used spectroscopic techniques for resolving the structure of a biomolecule, such as a protein or peptide, is NMR spectroscopy. Because only NMR signal intensities and frequencies are measured in the experiment, a conformational interpretation of the primary data, that is, measured data, is not straightforward, especially for flexible molecules. It is hampered by the occurrence of conformational and/or time-averaging, by insufficient number of experimental data and by insufficient accuracy of experimental data. All three problematic aspects of structure refinement based on NMR nuclear Overhauser effect (NOE) intensities and (3)J coupling data are illustrated by using two beta-heptapeptides in methanol as an example. We have performed 16 molecular dynamics (MD) simulations between 20 to 100 ns in length of unrestrained and NOE distance-restrained cases (instantaneous and time-averaged) of two beta-heptapeptides with a central beta-HAla(alpha-OH) amino acid in methanol at two different temperatures using two different GROMOS force-field parameter sets, 45 A3 and 53 A6. The created conformational ensembles were used to interpret the primary NMR data on these molecules. They also were compared to a set of NMR model structures derived by single-structure refinement in vacuo by using standard techniques. It is shown that the conformational interpretation of measured experimental data can be significantly improved by using unrestrained, instantaneous and time-averaged restrained MD simulations of the peptides by using a thermodynamically calibrated force field and by explicitly including solvent degrees of freedom.

Structure Determination of a Flexible Cyclic Peptide Based on NMR and MD Simulation 3J‐Coupling

Molecular dynamics (MD) simulations, in which experimental values such as nuclear Overhauser effects (NOEs), dipolar couplings, (3)J-coupling constants or crystallographic structure factors are used to bias the values of specific molecular properties towards experimental ones, are often carried out to study the structure refinement of peptides and proteins. However, (3)J-coupling constants are usually not employed because of the multiplicity of torsional angle values (phi) corresponding to each (3)J-coupling constant value. Here, we apply the method of adaptively enforced restraining using a local-elevation (LE) biasing potential energy function in which a memory function penalizes conformations in case both the average <(3)J> and the current (3)J-values deviate from the experimental target value. Then, the molecule is forced to sample other parts of the conformational space, thereby being able to cross high energy barriers and to bring the simulated average <(3)J> close to the measured <(3)J> value. Herein, we show the applicability of this method in structure refinement of a cyclo-beta-tetrapeptide by enforcing the (3)J-value restraining with LE on twelve backbone torsional angles. The resulting structural ensemble satisfies the experimental (3)J-coupling data better than the NMR model structure derived using conventional single-structure refinement based on these data. Thus, application of local-elevation search MD simulation in combination with biasing towards (3)J-coupling makes it possible to use (3)J-couplings quantitatively in structure determination of peptides.

Influence of Backbone Fluorine Substitution upon the Folding Equilibrium of a β-Heptapeptide

Explicit solvent molecular dynamics (MD) simulations of three beta-heptapeptides with a central beta- HAla(alpha-F) amino acid (Figure 1) in methanol are reported. They aim at an analysis of the conformational consequences of C(alpha) carbon atom bound fluoro atoms, and the particular configuration of the central fluoro-beta-amino acid: peptide 3 with an S configuration of the C(alpha) bound fluor atom, peptide 4 with an R configuration of the C(alpha) bound fluor atom, and peptide 5 with a difluoro substitution at the C(alpha) atom of residue 4. The NMR and CD spectra of these three beta-peptides were earlier (Mathad et al. Helv. Chim. Acta 2005, 88, 266-280) interpreted to indicate a decrease in propensity of 3(14)-helical structure from peptide 4 to peptide 5 to peptide 3. This result was at odds with previous experimental data for beta-heptapeptides with a central beta-HAla(alpha-Me) amino acid which showed that the beta-heptapeptide with the S,S configuration of the central beta-HAla(alpha-Me) was the most 3(14)-helical, whereas the S,R configuration did not lead to any detected helicity. The reported MD simulations resolve this paradox. The MD trajectories of all three peptides do agree with the primary, measured data: NMR nuclear Overhauser effect (NOE) atom-atom distance bounds and (3)J-coupling constants. A conformational analysis of the MD trajectory conformations shows, however, a decrease in 3(14)-helical character from peptide 3 to peptide 5 to peptide 4, which is in line with the results for the nonfluorinated peptides. It is shown that interpretation of NMR NOE data using single-structure refinement in vacuo based on local (along the sequence) and limited atom-atom distance data as in ref 1 (Mathad et al. Helv. Chim. Acta 2005, 88, 266-280) may lead to molecular structures that are not representative for the ensemble of molecular conformations.

A Molecular Dynamics Study of the ASC and NALP1 Pyrin Domains at Neutral and Low pH

The pyrin domain is one of four subfamilies of the death domain superfamily of proteins, all members of which share a similar three‐dimensional fold with a structure comprising five or six antiparallel α‐helices. The pyrin domain of the ASC (six‐helical fold) and of the NALP1 (five‐helical fold) proteins were simulated at two different pH values, 3.7 and 6.5, with two different force‐field parameter sets, and the molecular dynamics simulation trajectories were compared to NMR experimental data. The two force fields that were used did not show very different results. The simulations of NALP1 at pH 6.5 largely satisfied the experimental NOE atom–atom distance bounds that were measured at pH 6.5, and preserved its tertiary structure. The simulations at pH 3.7 showed a denaturation of the protein. The simulations of ASC at pH 3.7 only satisfied the experimental NOE atom–atom distance bounds that were measured at pH 3.7 if either three acidic side chains (Asp48, Glu64 and Asp75) or only two (Glu64 and Asp75) were not protonated. This indicates that the ASC tertiary structure is stabilized by salt bridges at low pH. A corresponding analysis for NALP1 at pH 3.7 only yielded one possible salt bridge, but this did not stabilize the tertiary structure at low pH. The results show that the particular protonation states of acidic side chains in the protein interior might be crucial to properly modeling these proteins at low pH.