The project has addressed the following areas in the past year: 1. Interpreting all atom folding simulations. It has recently become possible to fold proteins on a computer, but this in itself is insufficient to guarantee that the mechanism of folding is correct. However, there are very few experiments that can be used to test the mechanism. One which can is phi-value analysis. We have developed a method to compute phi-values from simulations and used to to interpret recent all-atom simulations of protein folding. We find that in most cases the computed phi-values are close to the experimental ones, but there are some discrepancies (Ref. 1). We show how heterogeneity in folding mechanism can be revealed by the sensitivity of the phi-value to the type of mutation made. A second line of investigation has been testing the assumptions made by folding theories. We had previously shown that the approximation that native contacts determine folding mechanism was valid in the context of the all-atom simulations. This year, we also showed that the dynamics can be simplified to one-dimensional diffusion along an appropriately chosen coordinate (Ref. 2). 2. Misfolding in multidomain proteins. We have developed a model for the susceptibility of different protein folds to intramolecular domain-swapped misfolding using coarse-grained simulations. The model can identify which proteins are known to misfold and which are known not to do so, the distinction between them being based mainly on the topology of the folded state (Ref 3). In collaboration with Ben Schuler (University of Zurich), we have also worked to interpret new time-resolved FRET experiments on misfolding in titin domains. Our coarse-grained simulations provided a plausible molecular mechanism for the intermediate states identified by FRET (Ref. 4). 3. Folding of membrane proteins. We are focussing our efforts on membrane protein folding using both coarse-grained and atomistic simulations, which is very challenging due to the high viscosity of lipid membranes. We find, using two-helix dimers as a testbed, that while current coarse-grained models yield reasonable values for dissociation constants, they often predict incorrect structures for the folded state. We have just completed a manuscript on this topic. We are also looking at all atom simulations, where the sampling problem is even more severe, but we have overcome this to a large extent by using a solute tempering replica exchange approach which we are now using to study the same problems at an all-atom resolution, with promising results. 4. Structure of unfolded and intrinsically disordered proteins in chemical denaturant. One of the outstanding controversies in protein folding concerns the effects of chemical denaturants on the structure of unfolded or disordered proteins, i.e. do chemical denaturants cause the unfolded chain to expand, or not? FRET and SAXS experiments had given different answers to this question. We have addressed the problem from two directions: first, a bottom-up approach in which a molecular simulation force field for the denaturant was carefully optimized (Ref 5). Simulations with this force field showed that increased denaturant concentration caused the protein to expand, but were also consistent with FRET and SAXS data from our collaborators Ben Schuler (University of Zurich) and Alex Grishaev (NIST) respectively (Ref 6). Thus, the two types of experiment are not necessarily in contradiction. We then addressed the inverse problem, determining structural data directly from the experiments, and showed that the apparent differences were essentially due to the models used in interpreting the data (Ref. 7). 5. A key unresolved aspect of protein folding dynamics is the contribution of interactions within the chain to slowing folding, known as 'internal friction'. Building on our previous results where we showed that a common origin for internal friction in all proteins is crossing of local torsional barriers in the energy landscape, we have developed a simple model to explore whether there is any contribution from the height of the global free energy barrier to the internal friction. We find that the internal friction is explained essentially by the local torsion barriers only (Ref. 8). We are now working on interpreting internal friction in unfolded and disordered chains. 6. In collaboration with David de Sancho (San Sebastian), and Jochen Blumberger (University College London) we have been investigating the diffusion of gas molecules to the active sites of hydrogenase enzymes. We have now applied the methodology to an FeFe hydrogenase, and combined the results with estimates of the rate for the chemical step from ab initio calculations to obtain a description of the overall kinetics. The results have been used to make mutants to alter the oxygen sensitivity of the enzyme, which have been tested by our experimental collaborator, Christophe Leger. (Ref. 9) 7. We are working in collaboration with Tuomas Knowles in Cambridge in order to describe the formation of amyloid fibers, and in particular the molecular mechanism of secondary nucleation. We are taking two-approaches. The first is to build a simple coarse-grained model based on the known structure of the fiber. The second is to characterise the affinity, association rate and binding mode of the peptides with the surface of an existing fiber, which can be checked against experimental data from the Knowles and Buell labs. 8. Force field development. Accurate energy functions, or force fields, are essential in order to obtain useful results from molecular simulations. In work published last year, we showed the critical importance of balancing protein-water interactions in force fields to obtain reliable results on the equilibrium properties of unfolded states. In follow-up work, we have shown that this balance is also critical for reproducing dynamics in the unfolded state (Ref. 10). In work with collaborators, we have looked at the suitability of current RNA force fields to fold small RNA tetraloops. We found that although the latest generation of RNA force fields is promising, there are significant shortcomings, most likely in the strength of hydrogen bonds between the base pairs in the stem (Ref. 11).
| Best, Robert B; Lindorff-Larsen, Kresten (2018) Editorial overview: Theory and simulation: Interpreting experimental data at the molecular level. Curr Opin Struct Biol 49:iv-v |
| Doma?ski, Jan; Sansom, Mark S P; Stansfeld, Phillip J et al. (2018) Balancing Force Field Protein-Lipid Interactions To Capture Transmembrane Helix-Helix Association. J Chem Theory Comput 14:1706-1715 |
| Zheng, Wenwei; Best, Robert B (2018) An Extended Guinier Analysis for Intrinsically Disordered Proteins. J Mol Biol 430:2540-2553 |
| Zheng, Wenwei; Zerze, Gül H; Borgia, Alessandro et al. (2018) Inferring properties of disordered chains from FRET transfer efficiencies. J Chem Phys 148:123329 |
| Tian, Pengfei; Louis, John M; Baber, James L et al. (2018) Co-Evolutionary Fitness Landscapes for Sequence Design. Angew Chem Int Ed Engl 57:5674-5678 |
| Borgia, Alessandro; Borgia, Madeleine B; Bugge, Katrine et al. (2018) Extreme disorder in an ultrahigh-affinity protein complex. Nature 555:61-66 |
| Meng, Fanjie; Bellaiche, Mathias M J; Kim, Jae-Yeol et al. (2018) Highly Disordered Amyloid-? Monomer Probed by Single-Molecule FRET and MD Simulation. Biophys J 114:870-884 |
| Best, Robert B (2018) Race to the native state. Proc Natl Acad Sci U S A 115:2267-2269 |
| De Sancho, David; Schönfelder, Jörg; Best, Robert B et al. (2018) Instrumental Effects in the Dynamics of an Ultrafast Folding Protein under Mechanical Force. J Phys Chem B : |
| Dignon, Gregory L; Zheng, Wenwei; Kim, Young C et al. (2018) Sequence determinants of protein phase behavior from a coarse-grained model. PLoS Comput Biol 14:e1005941 |
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