Nagesh, J. | bioRxiv Science

bioRxiv2020

Double Domain Swapping in Human γC and γD Crystallin Drives Early Stages of Aggregation

Human{gamma} D (H{gamma}D) and{gamma} C (H{gamma}C) are double domained crystallin (Crys) proteins expressed in the nucleus of the eye lens. Structural perturbations in the protein often trigger aggregation, which eventually leads to cataract. To decipher the underlying molecular mechanism, it is important to characterize the partially unfolded conformations of Crys proteins. Using coarse grained protein models and molecular dynamics simulations, we studied the role of on-pathway folding intermediates in the early stages of aggregation. The multi-dimensional free energy surface revealed at least three different folding pathways with the population of partially structured intermediates. The two dominant pathways confirm sequential folding of the N-terminal [Ntd] and the C-terminal domains [Ctd], while the third, least favored pathway involves intermediates where both the domains are partially folded. A native like intermediate (I*), featuring the folded domains and disrupted inter domain contacts, gets populated in all the three pathways. I* forms domain swapped dimers by swapping the entire Ntds and Ctds with other monomers. Population of such oligomers can explain the increased resistance to unfolding resulting in hysteresis observed in the folding experiments of H{gamma}D Crys. An ensemble of double domain swapped dimers are also formed during refolding, where intermediates consisting of partially folded Ntds and Ctds swap secondary structures with other monomers. The double domain swapping model presented in our study provides structural insights into the early events of aggregation in Crys proteins and identifies the key secondary structural swapping elements, where introducing mutations will aid in regulating the overall aggregation propensity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/210443v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@fd37e6org.highwire.dtl.DTLVardef@60e7cdorg.highwire.dtl.DTLVardef@128386org.highwire.dtl.DTLVardef@178f240_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract figure C_FIG

biochemistry↗

bioRxiv2020

High resolution ensemble description of metamorphic and intrinsically disordered proteins using an efficient hybrid parallel tempering scheme

Determining the conformational ensemble for proteins with multi-funneled complex free-energy landscapes is often not possible with classical structure-biology methods that produce time and ensemble averaged data. With vastly improved force fields and advances in rare-event sampling methods, molecular dynamics (MD) simulations offer a complementary approach towards determining the collection of 3-dimensional structures that proteins can adopt. However, in general, MD simulations need to either impose restraints or reweigh the generated data to match experiments. The limitations extend beyond systems with high free-energy barriers as is the case with metamorphic proteins such as RFA-H. The predicted structures in even weakly-funneled intrinsically disordered proteins (IDPs) such as Histatin-5 (His-5) are too compact relative to experiments. Here, we employ a new computationally-efficient parallel-tempering based advanced-sampling method applicable across proteins with extremely diverse free-energy landscapes. And we show that the calculated ensemble averages match reasonably well with the NMR, SAXS and other biophysical experiments without the need to reweigh. We benchmark our method against standard model systems such as alanine di-peptide, TRP-cage and {beta}-hairpin and demonstrate significant enhancement in the sampling efficiency. The method successfully scales to large metamorphic proteins such as RFA-H and to highly disordered IDPs such as His-5 and produces experimentally-consistent ensemble. By allowing accurate sampling across diverse landscapes, the method enables for ensemble conformational sampling of deep multi-funneled metamorphic proteins as well as highly flexible IDPs with shallow multi-funneled free-energy landscape. Significance/Authors SummaryGenerating high-resolution ensemble of intrinsically disordered proteins, particularly the highly flexible ones with high-charge and low-hydrophobicity and with shallow multi-funneled free-energy landscape, is a daunting task and often not possible since information from biophysical experiments provide time and ensemble average data at low resolutions. At the other end of the spectrum are the metamorphic proteins with multiple deep funnels and elucidating the structures of the transition intermediates between the fold topologies is a non-trivial exercise. In this work, we propose a new parallel-tempering based advanced-sampling method where the Hamiltonian is designed to allow faster decay of water orientation dynamics, which in turn facilitates accurate and efficient sampling across a wide variety of free-energy landscapes.

biophysics↗