Hidden genetic variations harbor potential for the evolution of new traits. Molecular chaperones, that assist protein folding, may conceal genetic variations in protein coding regions. Here, we investigate if the chemical milieu of cells has the potential to alleviate intracellular protein folding; potentially implicating a role of osmolytes in concealing genetic variations. Using the model osmolyte TMAO, we uncover that it can buffer mutations that impose kinetic traps in the folding pathways of two model proteins. Using this information, we rationally designed TMAO-dependent mutants in vivo, starting from a TMAO-independent protein. Strikingly, we delineate different osmolytes to have a unique spectrum of buffered-mutations. Consequently, the chemical milieu of cells may alter the folding pathways of unique mutant variants in polymorphic populations and lead to unanticipated spectra of genetic buffering. KeywordsProtein folding; chaperone; chemical chaperone; Proteostasis; TMAO; genetic buffering; canalization Alterations in gene sequences are a major source of variation in protein structure and function leading to phenotypic variation 12 . Since native structures of proteins are only marginally stable, majority of non-conserved changes in the protein sequences lead to the formation of non-functional or metastable proteins 3,4 . Metastable proteins are functional under permissive conditions but are rendered inactive under special circumstances, which could be a major source of hidden genetic diversity, a phenomenon referred to as geneticcanalization 5 . Metastability, through canalization, may allow robust protein structures to adopt alternate conformations without losing in vivo activity; a significant step towards evolution of new protein function [6][7][8][9][10][11][12] . To establish this link we need to understand if protein folding modulators assist intracellular folding of metastable mutants.Since intracellular milieu is enriched with osmolytes that act as chemical chaperones, we asked if these could favor genetic canalization. Although there are preliminary reports of osmolyte dependent folding in vivo 13 ,14 ,15 , it is unknown if osmolytes affect canalization by assisting the folding of metastable proteins. Since osmolytes may also affect chaperone

The fundamental backbone dynamics of unfolded proteins arising due to intrinsic ϕ-ψ dihedral angle fluctuations dictate the course of protein folding, binding, assembly, and function. These internal fluctuations are also critical for protein misfolding associated with a range of human diseases. However, direct observation and unambiguous assignment of this inherent dynamics in chemically denatured proteins is extremely challenging due to various experimental limitations. To directly map the backbone torsional mobility in the ϕ-ψ dihedral angle space, we used a model intrinsically disordered protein, namely, α-synuclein, that adopts an expanded state under native conditions. We took advantage of nonoccurrence of tryptophan in α-synuclein and created a number of single-tryptophan variants encompassing the entire polypeptide chain. We then utilized highly sensitive picosecond time-resolved fluorescence depolarization measurements that allowed us to discern the site-specific torsional relaxation at a low protein concentration under physiological conditions. For all the locations, the depolarization kinetics exhibited two well-separated rotational-correlation-time components. The shorter, subnanosecond component arises due to the local mobility of the indole side chain, whereas the longer rotational-correlation-time component (1.37 ± 0.15 ns), independent of global tumbling, represents a characteristic timescale for short-range conformational exchange in the ϕ-ψ dihedral space. This correlation time represents an intrinsic timescale for torsional relaxation and is independent of position, which is expected for an extended polypeptide chain having little or no propensity to form persistent structures. We were also able to capture this intrinsic timescale at the N-terminal unstructured domain of the prion protein. Our estimated timescale of the segmental mobility is similar to that of unfolded proteins studied by nuclear magnetic resonance in conjunction with molecular dynamics simulations. Our results have broader implications for a diverse range of functionally and pathologically important intrinsically disordered proteins and disordered regions.

Various small molecules present in biological systems can assist protein folding in vitro and are known as chemical chaperones. De novo design of chemical chaperones with higher activity than currently known examples is desirable to ameliorate protein misfolding and aggregation in multiple contexts. However, this development has been hindered by limited knowledge of their activities. It is thought that chemical chaperones are typically poor solvents for a protein backbone and hence facilitate native structure formation. However, it is unknown if different chemical chaperones can act differently to modulate folding energy landscapes. Using a model slow folding protein, double-mutant Maltose-binding protein (DM-MBP), we show that a canonical chemical chaperone, trimethylamine-N-oxide (TMAO), accelerates refolding by decreasing the flexibility of the refolding intermediate (RI). Among a number of small molecules that chaperone DM-MBP folding, proline and serine stabilize the transition state (TS) enthalpically, while trehalose behaves like TMAO and increases the rate of barrier crossing through nonenthalpic processes. We propose a two-group classification of chemical chaperones based upon their thermodynamic effect on RI and TS, which is also supported by single molecule Förster resonance energy transfer (smFRET) studies. Interestingly, for a different test protein, the molecular mechanisms of the two groups of chaperones are not conserved. This provides a glimpse into the complexity of chemical chaperoning activity of osmolytes. Future work would allow us to engineer synergism between the two classes to design more efficient chemical chaperones to ameliorate protein misfolding and aggregation problems.

Conformational switching of the prion protein (PrP) from an α-helical normal cellular form (PrP(C)) to an aggregation-prone and self-propagating β-rich scrapie form (PrP(Sc)) underlies the molecular basis of pathogenesis in prion diseases. Anionic lipids play a critical role in the misfolding and conformational conversion of the membrane-anchored PrP into the amyloidogenic pathological form. In this work, we have used a diverse array of techniques to interrogate the early intermediates during amyloid formation from recombinant human PrP in the presence of a membrane mimetic anionic detergent such as sodium dodecyl sulfate. We have been able to detect and characterize two distinct types of interconvertible oligomers. Our results demonstrate that highly ordered large β-oligomers represent benign off-pathway intermediates that lack the ability to mature into amyloid fibrils. On the contrary, structurally labile small oligomers are capable of switching to an ordered amyloid-state that exhibits profound toxicity to mammalian cells. Our fluorescence resonance energy transfer measurements revealed that the partially disordered PrP serves as precursors to small amyloid-competent oligomers. These on-pathway oligomers are eventually sequestered into higher order supramolecular assemblies that conformationally mature into polymorphic amyloids possessing varied nanoscale morphology as evident by the atomic force microscopy imaging. The nanoscale diversity of fibril architecture is attributed to the heterogeneous ensemble of early obligatory oligomers and offers a plausible explanation for the existence of multiple prion strains in vivo.