Mechanism of substrate protein remodeling by molecular chaperones

Protein quality control regulates the natural load of proteins by providing folding assistance or degradation mechanism to prevent misfolding or aggregation. It is maintained by the complex regulatory network of molecular chaperons. The study of such fundamental biological system helps in designing different biological applications like targeted therapeutic treatments. The goal of this work is to elucidate protein quality control mechanisms associated with chaperonin folding assistance and protein degradation. Chaperonins are large double ring assemblies that assist folding of substrate proteins (SPs) under non-permissive conditions. Spectacular ATP driven conformational changes take place within each chaperonin ring. Distinct allosteric mechanisms have been described for the two chaperonin classes. Bacterial (group I) chaperonins, such as GroEL undergo concerted subunit motions within each ring, while archaeal and eukaryotic chaperonins (group II) undergo sequential subunit motions. In the protein degradation pathway, nanomachines such as ClpP cleave the unwanted protein. Here we study four aspects regarding this problem: (1) Allosteric mechanisms of group II chaperonin, (2) Kinetics of multi-domain protein folding confined to cylindrical nanopores (3). Structural and bioinformatics analyses to understand substrate recognition mechanisms in group II chaperonin and (4) Allosteric mechanisms of ClpP. We use normal mode analysis to understand how distinct allosteric mechanisms arise in two groups of chaperonins. Our results show that the lower frequency modes are important for the thermosome monomers whereas distinct higher frequency modes are attributed to functional specialization of these subunit types. Our results also indicate weaker long-range inter-subunit correlation of amino acid pairs in archaeal chaperonin compared to GroEL. These results support distinct allosteric mechanisms of the two chaperonin types. In order to understand the effect of encapsulation inside the chaperonin cavity, we studied the confinement of protein inside nanopores. We perform coarse-grained Langevin dynamics simulations of actin confined to single-walled carbon nanotubes. Actin is a multi-domain protein which comprises two non-contiguous subdomains and its folding is strictly dependent on the eukaryotic chaperonin CCT. Our bulk simulations show that long-distance native contacts are difficult to form, restricting actin to fold spontaneously. By contrast, weak confinement promotes folding and enhances the overall stability of the native state. To probe conditions that mimic the chaperonin action, we also perform folding simulations of actin in quasi-native conformations. In this case, we find that folding yield is optimized in a moderate confinement regime. To understand the substrate recognition mechanism, we used structural and bioinformatics analyses. Based on these studies we propose substrate binding sites in helical protrusion and two helices. The conservation of hydrophobic and charged amino acids at these sites indicates universal and specific aspects of the substrate recognition mechanisms. We further elucidate substrate specific mechanisms by identifying conservation of negative or positive character of the charged amino acid within the subsets of eukaryotic subunits. To understand the allosteric motion of ClpP, we use normal mode analysis. Our results indicate that multiple higher frequency modes contribute in functional role of ClpP transitions.