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.

Monte Carlo study of the formation and conformational properties of dimers of Aß42 variants.

Small soluble oligomers, and dimers in particular, of the amyloid ß-peptide (Aß) are believed to play an important pathological role in Alzheimer's disease. Here, we investigate the spontaneous dimerization of Aß42, 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 ß-sheets. Parallel, in-register ß-sheet structure, as in models for Aß 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 Aß fibrils, is found to increase upon dimerization and to correlate with experimentally measured rates of fibril formation for the different Aß42 variants. Our findings hint at reorganization of this part of the molecule as a potentially critical step in Aß aggregation.

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

The properties of the amyloid-beta peptide that lead to aggregation associated with Alzheimer's disease are not fully understood. This study aims at identifying conformational differences among four variants of full-length Abeta42 that are known to display very different aggregation properties. By extensive all-atom Monte Carlo simulations, we find that a variety of beta-sheet structures with distinct turns are readily accessible for full-length Abeta42. In the simulations, wild type (WT) Abeta42 preferentially populates two major classes of conformations, either extended with high beta-sheet content or more compact with lower beta-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 Abeta42 tends to form a turn. The aggregation-accelerating E22G mutation associated with early onset of Alzheimer's 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 Abeta42 variants, we identify specific conformational properties of residues 23-26 that might play a key role in aggregation.

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 beta-barrel formation: an all-atom Monte Carlo study of Abeta16-22 oligomerization.

Using all-atom Monte Carlo simulations with implicit water, combined with a cluster size analysis, we study the aggregation of Abeta(16) (-22), a peptide capable of forming amyloid fibrils. We consider a system of six initially randomly oriented Abeta(16) (-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 beta-sheet rich aggregates at low temperature. At the crossover between these two regimes, we find that clusters of all sizes occur, whereas the beta-strand content is low. In one of several runs, we observe the spontaneous formation of a beta-barrel with six antiparallel strands. The beta-barrel stands out as the by far most long-lived aggregate seen in our simulations.

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 beta-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.

Dissecting the mechanical unfolding of ubiquitin.

The unfolding behavior of ubiquitin under the influence of a stretching force recently was investigated experimentally by single-molecule constant-force methods. Many observed unfolding traces had a simple two-state character, whereas others showed clear evidence of intermediate states. Here, we use Monte Carlo simulations to investigate the force-induced unfolding of ubiquitin at the atomic level. In agreement with experimental data, we find that the unfolding process can occur either in a single step or through intermediate states. In addition to this randomness, we find that many quantities, such as the frequency of occurrence of intermediates, show a clear systematic dependence on the strength of the applied force. Despite this diversity, one common feature can be identified in the simulated unfolding events, which is the order in which the secondary-structure elements break. This order is the same in two- and three-state events and at the different forces studied. The observed order remains to be verified experimentally but appears physically reasonable.