Stereochemical studies on peptide and protein structures: Implications for validation, flexibility, and dynamics

Abstract

Accuracy of 3-D structures of proteins is crucial, both in terms of its agreement with the experimental data used to determine the structure and stereochemistry, especially when they are utilized for applications like drug design. Hence, stereochemical validation of 3-D structures is of fundamental importance. In this thesis, we have performed stereochemical studies on various systems, starting from peptide structures to high-resolution protein structures and large multi-protein assemblies. In peptide structures, we noted that subtle deviations in backbone bond lengths and angles from ideal bond geometry significantly alters the allowed Ramachandran (ϕ,ψ) space, which is visualized using bond geometry-specific steric maps. We utilized these bond geometry-specific steric maps for stereochemical validation of residues in protein structures where we distinguish between genuine (ϕ,ψ) angle outliers and outliers due to modeling errors. This was done by analyzing if the observed (ϕ,ψ) value of a residue occurs in disallowed or allowed regions of their own bond geometry-specific steric map. We showed that these maps are significantly affected through variations in backbone bond geometry during atomic vibrations. Thus, disallowed (ϕ,ψ) regions at one timepoint can become allowed at another timepoint. We also suggested how high energy barriers in the (ϕ,ψ) space are potentially crossed during conformational transitions. Our analysis of the relationship between conformational strain in protein structures due to unfavorable (ϕ,ψ) angles and flexibility in local regions of proteins showed that they are only weakly related. This is likely due to the variable nature of allowed (ϕ,ψ) space itself, where adjustments to bond lengths and angles could lower the supposedly high-energy associated with an unfavorable (ϕ,ψ) conformation. With the emergence of an increasing number of cryoEM structures, we assessed their stereochemical quality and highlighted areas requiring improvement. We also showed that global resolution of cryo-EM structures is not a robust indicator of their quality. Our comparison of atomic packing in cryo-EM and crystal structures revealed that cryo-EM structures are less tightly packed than crystal structures, which is likely due to the nature of samples used in structure determination. We also suggest that the level of atomic packing seen in cryo-EM structures resembles the native state better. Overall, in this thesis, our studies on stereochemistry of peptides and proteins have generated a new framework for Ramachandran angle validation. We have also explored the implications of these studies on the flexibility and dynamics of proteins. Stereochemical studies on cryo-EM structures, which are predominantly multi-protein assemblies, have highlighted the red flags in these structures that their users should be aware of.