Search for a Unified Mechanism for  Protein Misfolding and Aggregation


The WIDE-RANGING OBJECTIVE of the project is to significantly develop our insight into protein misfolding and aggregation process, reducing the difference among the diverse, still matching observations of disease-causing proteins. There are three main aspects that must be combined for the accomplishment of the project: (1) the key questions to be answered; (2) the techniques to be used; (3) the protein systems which are amenable to study by the techniques in (2) and which will address the problems in (1). This is an intricate matrix. Each of the aims below encompasses numerous components of this matrix, but overall intention is as follows. There are eight problems to be addressed: (a) detection of the region(s) of a sequence that forms and stabilizes the fibril core and/or plays a primary role in fibril formation; (b) kinetics of fibril formation; (c) characterization of the conformational landscapes of the misfolded monomers, oligomers, protofibrils, and fibrillar aggregates; (d) identification of the interactions that stabilize a particular form over others in the growth process; (e) free energy landscape of aggregation; (f) basis of protein complementarity, in which a protein chain can bind to itself; (g) binding free energy estimation of a filament to its fibril; (h) role of solvent models and force field parameters in the formation of oligomers, protofilaments, and amyloid fibrils. 


(a) Mechanical fingerprinting and malleability map (OBJECTIVE 1): A full atomistic description of both the protein and the solvent will be used for this approach. A substantial mechanical force will be applied to perturb the native configuration, and the resulting moderate deviations will be examined; however, the forces will not be too high to produce complete unfolding. Constant-velocity SMD simulations containing two tethering points will be applied on a protein where the centre of mass (COM) of the protein and the Cα atom of a specific amino acid will act as the two tethering points. This will generate a force-extension profile whose integration will give us a work-extension profile. Repetition of the pulling simulation for all the residues will generate a work profile of a protein sequence. This technique will identify – (1) variability of mechanical stability along a protein sequence, (2) effect of mutation(s) on local (region-specific) as well as global stability (cumulative distribution of mechanical work values) of a protein, and (3) positioning of misfolding-prone region(s) in a sequence.


(b) Coarse-grained unrestrained simulations (OBJECTIVE 2): To capture the aggregation kinetics, unconstrained MD simulations of millisecond timescale will be performed with multiple copies of the misfolding-prone sequences predicted by the procedure described in OBJECTIVE 1. The coarse-grained description of both peptides and solvent will be used for this purpose. Multiple copies of the same/different peptide(s) will be used for this part. To probe the aggregation propensity of the system, the number of clusters formed by the peptides will be analysed as a function of simulation time. Moreover, the relative orientation between pairs of interacting peptides will be estimated by computing the angles between their end-to-end vectors. The researcher will further count the number of conformations that will evolve during each simulation having at least two peptides within a specific cut-off distance. The fraction of such conformations will provide another measure of aggregation propensity and will help to compare the aggregation propensities between the wild-type sequence and the different mutated sequences.


(c) Enhanced conformational space sampling (OBJECTIVE 3): The replica exchange methodology will be used with the atomistic description for the peptide sequences predicted in OBJECTIVE 1. In this scheme, parallel trajectories with specific parameter sets will be initiated. Each trajectory will be given an opportunity to exchange conformations periodically with trajectories at immediately close temperatures following a Metropolis criterion which will guarantee the resulting thermodynamics is accurate. For each system, the total number of replicas will be determined by the number of atoms in the system, the temperature range, and the replica exchange ratio. The temperatures will be spaced exponentially between the minimum and the maximum values. To further improve the sampling method, additional exchange of conformations will also be allowed between two parallel trajectories with the identical temperature replicas. This method will give us the conformational landscapes of monomers and higher order multimers for an aggregation-prone peptide. Structural analysis (e.g. relative orientation of the peptides) will be performed following the technique(s) described in OBJECTIVE 2. These conformations with full atomistic description will be fed into the scheme of OBJECTIVE 2 to improve the precision of the identification of the species achieved with coarse-grained description. The process of continuous feedback between atomistic and coarse-grained simulations will give an accurate understanding of both kinetics and thermodynamics for the aggregation process.


(d) Binding free energy estimation (OBJECTIVE 4): For each system, a constant-velocity SMD simulation will be performed, in which the strands will be pulled apart using harmonic tethers applied to the COMs of each strand along the fibrillar axis. Snapshots from such SMD trajectories will then be used as the starting configurations for the umbrella sampling windows. In each of the resulting windows, a 100 ns MD simulation will be run. Analysis of the results will be performed using the WHAM, which will yield the potential of mean force (PMF) as a function of the inter-strand separation. The work to separate the strands (∆G) will then be calculated as the difference between the plateau value of the PMF at large separation, and its minimum value.


(e) Improved Force Fields and Solvent Models (OBJECTIVE 5): Existing force fields are optimized for globular folded proteins which may lead to erroneous conformational spaces of the aggregation process by over-stabilizing secondary structures in the unfolded states. The researcher will test the popular force fields that are generally used for protein simulations (CHARMM27, Amber03, OPLS-AA) to benchmark the simulations against available experimental data by comparing the conformational ensembles obtained from OBJECTIVE 2 and 3 and an optimized force field to study aggregation process will be developed by tweaking the existing force field parameters. Furthermore, the extent of the contribution of water in the process of protein aggregation is yet to be understood. Nearly all studies on amyloid association approaches have been principally analyzed exercising a perspective where the protein is at the centre of attention. The researcher will deliver an evaluation on the impact of water during protein aggregation: (1) how the energy landscape of the aggregation process is dictated by the water-mediated interactions, and (2) what is the contribution of water in the advanced stages of fibril formation. The researcher will specifically test the robustness of the results by comparing several popularly used water models such as SPE, SPC-E, TIP3P, TIP4P, TIP4P-D, etc.