Simon Mitternacht

An effective all-atom potential for proteins

We describe and test an implicit solvent all-atom potential for simulations of protein folding and aggregation. The potential is developed through studies of structural and thermodynamic properties of 17 peptides with diverse secondary structure. Results obtained using the final form of the potential are presented for all these peptides. The same model, with unchanged parameters, is furthermore applied to a heterodimeric coiled-coil system, a mixed α/β protein and a three-helix-bundle protein, with very good results. The computational efficiency of the potential makes it possible to investigate the free-energy landscape of these 49–67-residue systems with high statistical accuracy, using only modest computational resources by todays standards. PACS Codes: 87.14.E-, 87.15.A-, 87.15.Cc

Comparing the folding free-energy landscapes of Abeta42 variants with different aggregation properties.

The properties of the amyloid‐β peptide that lead to aggregation associated with Alzheimers disease are not fully understood. This study aims at identifying conformational differences among four variants of full‐length Aβ42 that are known to display very different aggregation properties. By extensive all‐atom Monte Carlo simulations, we find that a variety of β‐sheet structures with distinct turns are readily accessible for full‐length Aβ42. In the simulations, wild type (WT) Aβ42 preferentially populates two major classes of conformations, either extended with high β‐sheet content or more compact with lower β‐sheet content. The three mutations studied alter the balance between these classes. Strong mutational effects are observed in a region centered at residues 23–26, where WT Aβ42 tends to form a turn. The aggregation‐accelerating E22G mutation associated with early onset of Alzheimers disease makes this turn region conformationally more diverse, whereas the aggregation‐decelerating F20E mutation has the reverse effect, and the E22G/I31E mutation reduces the turn population. Comparing results for the four Aβ42 variants, we identify specific conformational properties of residues 23–26 that might play a key role in aggregation. Proteins 2010.

Monte Carlo Study of the Formation and Conformational Properties of Dimers of Aβ42 Variants.

Small soluble oligomers, and dimers in particular, of the amyloid β-peptide (Aβ) are believed to play an important pathological role in Alzheimers disease. Here, we investigate the spontaneous dimerization of Aβ42, with 42 residues, by implicit solvent all-atom Monte Carlo simulations, for the wild-type peptide and the mutants F20E, E22G and E22G/I31E. The observed dimers of these variants share many overall conformational characteristics but differ in several aspects at a detailed level. In all four cases, the most common type of secondary structure is intramolecular antiparallel β-sheets. Parallel, in-register β-sheet structure, as in models for Aβ fibrils, is rare. The primary force driving the formation of dimers is hydrophobic attraction. The conformational differences that we do see involve turns centered in the 20-30 region. The probability of finding turns centered in the 25-30 region, where there is a loop in Aβ fibrils, is found to increase upon dimerization and to correlate with experimentally measured rates of fibril formation for the different Aβ42 variants. Our findings hint at reorganization of this part of the molecule as a potentially critical step in Aβ aggregation.

Binding Leverage as a Molecular Basis for Allosteric Regulation

Allosteric regulation involves conformational transitions or fluctuations between a few closely related states, caused by the binding of effector molecules. We introduce a quantity called binding leverage that measures the ability of a binding site to couple to the intrinsic motions of a protein. We use Monte Carlo simulations to generate potential binding sites and either normal modes or pairs of crystal structures to describe relevant motions. We analyze single catalytic domains and multimeric allosteric enzymes with complex regulation. For the majority of the analyzed proteins, we find that both catalytic and allosteric sites have high binding leverage. Furthermore, our analysis of the catabolite activator protein, which is allosteric without conformational change, shows that its regulation involves other types of motion than those modulated at sites with high binding leverage. Our results point to the importance of incorporating dynamic information when predicting functional sites. Because it is possible to calculate binding leverage from a single crystal structure it can be used for characterizing proteins of unknown function and predicting latent allosteric sites in any protein, with implications for drug design.

SPACER: server for predicting allosteric communication and effects of regulation

The SPACER server provides an interactive framework for exploring allosteric communication in proteins with different sizes, degrees of oligomerization and function. SPACER uses recently developed theoretical concepts based on the thermodynamic view of allostery. It proposes easily tractable and meaningful measures that allow users to analyze the effect of ligand binding on the intrinsic protein dynamics. The server shows potential allosteric sites and allows users to explore communication between the regulatory and functional sites. It is possible to explore, for instance, potential effector binding sites in a given structure as targets for allosteric drugs. As input, the server only requires a single structure. The server is freely available at http://allostery.bii.a-star.edu.sg/.

Unraveling hidden regulatory sites in structurally homologous metalloproteases

Monitoring enzymatic activity in vivo of individual homologous enzymes such as the matrix metalloproteinases (MMPs) by antagonist molecules is highly desired for defining physiological and pathophysiological pathways. However, the rational design of antagonists targeting enzyme catalytic moieties specific to one of the homologous enzymes often appears to be an extremely difficult task. This is mainly due to the high structural homology at the enzyme active sites shared by members of the protein family. Accordingly, controlling enzymatic activity via alternative allosteric sites has become an attractive proposition for drug design targeting individual homologous enzymes. Yet, the challenge remains to identify such regulatory alternative sites that are often hidden and scattered over different locations on the proteins surface. We have designed branched amphiphilic molecules exhibiting specific inhibitory activity towards individual members of the MMP family. These amphiphilic isomers share the same chemical nature, providing versatile nonspecific binding reactivity that allows to probe hidden regulatory residues on a given protein surface. Using the advantage provided by amphiphilic ligands, here we explore a new approach for determining hidden regulatory sites. This approach includes diverse experimental analysis, such as structural spectroscopic analyses, NMR, and protein crystallography combined with computational prediction of effector binding sites. We demonstrate how our approach works by analyzing members of the MMP family that possess a unique set of such sites. Our work provides a proof of principle for using ligand effectors to unravel hidden regulatory sites specific to members of the structurally homologous MMP family. This approach may be exploited for the design of novel molecular effectors and therapeutic agents affecting protein catalytic function via interactions with structure-specific regulatory sites.

Changing the Mechanical Unfolding Pathway of FnIII10 by Tuning the Pulling Strength

We investigate the mechanical unfolding of the tenth type III domain from fibronectin (FnIII(10)) both at constant force and at constant pulling velocity, by all-atom Monte Carlo simulations. We observe both apparent two-state unfolding and several unfolding pathways involving one of three major, mutually exclusive intermediate states. All three major intermediates lack two of seven native beta-strands, and share a quite similar extension. The unfolding behavior is found to depend strongly on the pulling conditions. In particular, we observe large variations in the relative frequencies of occurrence for the intermediates. At low constant force or low constant velocity, all three major intermediates occur with a significant frequency. At high constant force or high constant velocity, one of them, with the N- and C-terminal beta-strands detached, dominates over the other two. Using the extended Jarzynski equality, we also estimate the equilibrium free-energy landscape, calculated as a function of chain extension. The application of a constant pulling force leads to a free-energy profile with three major local minima. Two of these correspond to the native and fully unfolded states, respectively, whereas the third one can be associated with the major unfolding intermediates.

Spontaneous β‐barrel formation: An all‐atom Monte Carlo study of Aβ16–22 oligomerization

Using all‐atom Monte Carlo simulations with implicit water, combined with a cluster size analysis, we study the aggregation of Aβ16–22, a peptide capable of forming amyloid fibrils. We consider a system of six initially randomly oriented Aβ16–22 peptides, and investigate the thermodynamics and structural properties of aggregates formed by this system. The system is unaggregated without ordered secondary structure at high temperature, and forms β‐sheet rich aggregates at low temperature. At the crossover between these two regimes, we find that clusters of all sizes occur, whereas the β‐strand content is low. In one of several runs, we observe the spontaneous formation of a β‐barrel with six antiparallel strands. The β‐barrel stands out as the by far most long‐lived aggregate seen in our simulations. Proteins 2008.

Coherent conformational degrees of freedom as a structural basis for allosteric communication.

Conformational changes in allosteric regulation can to a large extent be described as motion along one or a few coherent degrees of freedom. The states involved are inherent to the protein, in the sense that they are visited by the protein also in the absence of effector ligands. Previously, we developed the measure binding leverage to find sites where ligand binding can shift the conformational equilibrium of a protein. Binding leverage is calculated for a set of motion vectors representing independent conformational degrees of freedom. In this paper, to analyze allosteric communication between binding sites, we introduce the concept of leverage coupling, based on the assumption that only pairs of sites that couple to the same conformational degrees of freedom can be allosterically connected. We demonstrate how leverage coupling can be used to analyze allosteric communication in a range of enzymes (regulated by both ligand binding and post-translational modifications) and huge molecular machines such as chaperones. Leverage coupling can be calculated for any protein structure to analyze both biological and latent catalytic and regulatory sites.

Thermal versus mechanical unfolding of ubiquitin

The authors studied the temperature‐induced unfolding of ubiquitin by all‐atom Monte Carlo simulations. The unfolding behavior is compared with that seen in previous simulations of the mechanical unfolding of this protein, based on the same model. In mechanical unfolding, secondary‐structure elements were found to break in a quite well‐defined order. In thermal unfolding, the authors saw somewhat larger event‐to‐event fluctuations, but the unfolding pathway was still far from random. Two long‐lived secondary‐structure elements could be identified in the simulations. These two elements have been found experimentally to be the thermally most stable ones. Interestingly, one of these long‐lived elements, the first β‐hairpin, was found to break early in the mechanical unfolding simulations. Their combined simulation results thus enable the authors to predict in detail important differences between the thermal and mechanical unfolding behaviors of ubiquitin. Proteins 2006.