Confinement and Crowding Effects on Folding of a Multidomain Y-Family DNA Polymerase

Abstract

Proteins in vivo endure highly various interactions from the luxuriant surrounding macromolecular cosolutes. Confinement and macromolecular crowding are the two major effects that should be considered while comparing the results of protein dynamics from in vitro to in vivo. However, efforts have been largely focused on single domain protein folding up to now, and the quantifications of the in vivo effects in terms of confinements and crowders on modulating the structure and dynamics as well as the physical understanding of the underlying mechanisms on multidomain protein folding are still challenging. Here we developed a topology-based model to investigate folding of a multidomain Y-family DNA polymerase (DPO4) within spherical confined space and in the presence of repulsive and attractive crowders. We uncovered that the entropic component of the thermodynamic driving force led by confinements and repulsive crowders increases the stability of folded states relative to the folding intermediates and unfolded states, while the enthalpic component of the thermodynamic driving force led by attractive crowders gives rise to the opposite effects with less stability. We found that the shapes of DPO4 conformations influenced by the confinements and the crowders are quite different even when only the entropic component of the thermodynamic driving force is considered. We uncovered that under all in vivo conditions, the folding cooperativity of DPO4 decreases compared to that in bulk. We showed that the loss of folding cooperativity can promote the sequential domain-wise folding, which was widely found in cotranslational multidomain protein folding, and effectively prohibit the backtracking led by topological frustrations during multidomain protein folding processes.

Investigating the trade-off between folding and function in a multidomain Y-family DNA polymerase

“…Stopped-flow Förster resonance energy transfer (FRET) studies monitoring intradomain ( Raper and Suo, 2016 ) and interdomain ( Xu et al, 2009 ; Maxwell et al, 2014 ) conformational dynamics of DPO4 during DNA binding as well as nucleotide binding and incorporation revealed weak and strong interactions between and within each domain of DPO4, respectively. Using only topological information, an SBM with homogeneously weighted native contacts predicted a ‘divide-and-conquer’ domain-wise folding scenario for DPO4 folding ( Wang et al, 2012a ; Chu et al, 2020 ). These results suggested that domains in DPO4 can fold without any aid from other domains.…”

Combined Effects of Confinement and Macromolecular Crowding on Protein Stability

“…While there is enough evidence to confirm that confinement and crowding can significantly impact protein properties, it is not yet clear if they function similarly or differently to replicate in vivo cell environments [ 14 , 19 , 41 ]. Furthermore, only a few studies have explored the combined effects of confinement and crowding, an additional complexity that may arise due to the heterogeneous mixture of differently sized biomacromolecules, typical of native cellular environments [ 17 , 24 , 25 , 26 ].…”

“…We hope this study will provide a foundation to create more relevant models for the in vitro investigation of protein behavior and fates in vivo. Such models may enable exploration of the mechanisms of multidomain protein folding and protein association and interactions, or to develop systems for drug delivery or implantable biosensors [ 26 , 43 , 44 , 45 ].…”

“…In this study, we describe our attempts to build on previous research and develop conditions to study the combined effects of confinement and crowding on protein stability [ 17 , 24 , 25 , 26 ]. These studies are relevant as recent studies have shown that investigations of protein fates in the combined presence of confinement and crowding can shed light on the underlying mechanisms of both single- and multi-domain protein folding and stability in vivo [ 17 , 26 ]. We investigated the combined effects of confinement and crowding on the functional and structural stability of two model proteins, horseradish peroxidase (HRP) and β-galactosidase (β-gal), using agarose scaffolds incorporating various amounts of dextran, an inert polysaccharide.…”

“…In this study, we describe our attempts to build on previous research and develop conditions to study the combined effects of confinement and crowding on protein stability [17,[24][25][26]. These studies are relevant as recent studies have shown that investigations of protein fates in the combined presence of confinement and crowding can shed light on the underlying mechanisms of both single-and multi-domain protein folding and stability in vivo [17,26].…”