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Underline Mechanisms of Remodeling Diverse Topological Substrate Proteins through Bacterial Clp ATPase using Computer Simulations

Fonseka, Hewafonsekage Yasan Yures
http://orcid.org/0000-0001-6591-3626
http://rave.ohiolink.edu/etdc/view?acc_num=ucin1637061123711353

Abstract Details

2021, PhD, University of Cincinnati, Arts and Sciences: Chemistry.
Protein quality control is one of the key cellular activities in any living cell, as it provides folding assistance or degradation mechanisms to prevent undesirable off-pathway reactions such as misfolding or aggregates using molecular chaperones. Bacterial Caseinolytic proteases (Clp) promote the degradation pathways by unfolding and translocating tagged substrate proteins (SPs) using powerful ring-shaped AAA+ (ATPases Associated with diverse cellular Activities) motor component with a narrow central pore and later deliver the unfolded polypeptide chain into the peptidase chamber for ultimate destruction into small peptide fragments. ATP fueled conformational transitions of subunits generate a repetitive mechanical unfolding force at the central pore loops and apply this force onto transient residues of SP that reside at the central pore and promote the unfolding and translocation mechanisms. Some aspects of these processes are addressed in experimental and computational studies. Nevertheless, the effects of mechanical anisotropies, non-native contacts, and topological features of SPs on allosteric cycle coupled Clp-mediated unfolding and translocation of SPs into peptidase chamber still remain unclear. To answer these questions, I presented the following three studies using an implicit atomistic model and performed Langevin dynamics simulations coupled with targeted molecular dynamics (TMD): (1) non-conserved allosteric cycles featured Clp nanomachine mediated remodeling mechanisms of three knotted SPs with 3.1, 5.2 and 6.1-knot types. These simulations reveal knot sliding along the contour of the knotted protein traversed through a rugged conformational landscape. Translocation hindrance of knotted SPs results from the synergetic coordination between knotted topologies and non-native contacts. In contrast to homopolymers, transmission of tension along the peptide chain occurs very differently. Disruption or formation of divergent type contacts along with backbone-backbone contacts can create multiple unfolding pathways with distinct energy barriers. (2) elucidate the effect of mechanical anisotropy on unfolding and translocation of stable substrate proteins with similar global stability but distinct local environments by Clp ATPases. Here, I consider stable DHFR and its two types of circular permutations (CPs), i.e., CP P25 and CP K38, as the SP. The resulting unfolding pathways reveal the unfolding energy barrier is largely determined by the force directionality. Clp nanomachine permits different unfolding mechanisms to regulate the unfolding energy barriers and features different unfolding pathways. Detailed analysis reveals the translocation hindrances arise from a combined effect of formation or disruption of non-native contacts, force directionality, and the arrangement of the primary sequence upon the unfolding. (3) This study focuses on the remodeling mechanism of bulky HaloTag protein using Clp Nanomachine with restrained or unrestrained geometries, which mimics the single-molecule experiments and in-vivo setups respectively. Regardless of unfolding direction or geometry, all unfolding pathways produce significant amounts of non-native contacts that later hinder the translocation mechanisms of HaloTag. In C-N directional unfolding process of HaloTag produces one stable intermediate state. In unrestrained geometry, nanomachine is not able to destabilize this intermediate state, but in restrained geometry, this stable intermediate unravels with the assistance of constant opposing force. Overall, these simulations shed light on mechanistic details of protein unfolding and translocation mediated by AAA+ ATPase nanomachines.
George Stan, Ph.D. (Committee Chair)
In-Kwon Kim (Committee Member)
Andrew Herr, Ph.D. (Committee Member)
92 p.
Biophysics
Protein unfolding; protein translocation; knotted proteins; non-native contacts; force directionality; Protein degradation

Recommended Citations

Citations

  • Fonseka, H. Y. Y. (2021). Underline Mechanisms of Remodeling Diverse Topological Substrate Proteins through Bacterial Clp ATPase using Computer Simulations [Doctoral dissertation, University of Cincinnati]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1637061123711353

    APA Style (7th edition)

  • Fonseka, Hewafonsekage Yasan Yures. Underline Mechanisms of Remodeling Diverse Topological Substrate Proteins through Bacterial Clp ATPase using Computer Simulations. 2021. University of Cincinnati, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ucin1637061123711353.

    MLA Style (8th edition)

  • Fonseka, Hewafonsekage Yasan Yures. "Underline Mechanisms of Remodeling Diverse Topological Substrate Proteins through Bacterial Clp ATPase using Computer Simulations." Doctoral dissertation, University of Cincinnati, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1637061123711353

    Chicago Manual of Style (17th edition)

ucin1637061123711353
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© 2021, some rights reserved.Creative Commons License Underline Mechanisms of Remodeling Diverse Topological Substrate Proteins through Bacterial Clp ATPase using Computer Simulations by Hewafonsekage Yasan Yures Fonseka is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. Based on a work at etd.ohiolink.edu.
This open access ETD is published by University of Cincinnati and OhioLINK.
Release 3.2.12