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GAINING INSIGHTS INTO THE CONFORMATIONAL DYNAMICS OF PHOSPHOLIPASE C-BETA

<p>Phospholipase Cs (PLCs) are a family of enzymes that
hydrolyze membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP2) to
generate inositol triphosphate (IP3) and diacylglycerol (DAG). These second
messengers activate a variety of intracellular responses, including
inflammation, vascular smooth muscle contraction, and cardiac hypertrophy.
While much is known about how Gaq-mediated activation of PLCb occurs, the same
cannot be said for Gbg-mediated activation. Residues within the PLCb-Gbg
binding interface were previously identified in interior regions of the
protein, suggesting the PH domain must undergo a conformational change to allow
for Gbg-mediated activation. However, the role of PH domain conformational
dynamics in Gbg-mediated activation of PLCb has yet to be determined. In this
work, I discuss efforts to characterize conformational dynamics of the PLCb PH
domain and its role in interactions of the enzyme with liposomes and Gbg.
First, I generated a disulfide crosslink between the PH domain and EF hands1/2
of PLCb3, purified under oxidizing or reducing conditions, and conducted
biochemical and structural tests to determine any differences in structure
and/or function of the protein as compared to wild-type. Results of these
studies provided the first direct structural evidence of PLCb PH domain
dynamics in solution. Then, I discuss the rationale behind the generation of a
surface cysteine-less PLCb for use in solvatochromic fluorescence assays in the
presence and absence of liposomes and Gbg. Initial results of these studies
suggest the PLCb PH domain favors a buried conformation alone and in the
presence of Gbg or liposomes, and likely exists at an equilibrium between open
and closed states.</p>

  1. 10.25394/pgs.15025668.v1
Identiferoai:union.ndltd.org:purdue.edu/oai:figshare.com:article/15025668
Date21 July 2021
CreatorsMichelle M Van Camp (11161194)
Source SetsPurdue University
Detected LanguageEnglish
TypeText, Thesis
RightsCC BY 4.0
Relationhttps://figshare.com/articles/thesis/GAINING_INSIGHTS_INTO_THE_CONFORMATIONAL_DYNAMICS_OF_PHOSPHOLIPASE_C-BETA/15025668

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