Secondary Structure Analysis of the C-Terminus of Galpha-Interacting Vesicle Associated Protein Using Circular Dichroism Spectroscopy

• Main Author: Maddox, Adam L.
• Language: EN
• Published: California State University, Long Beach 2017
• Subjects: Biochemistry|Biophysics
• Online Access: http://pqdtopen.proquest.com/#viewpdf?dispub=10601759

<p>A vast majority of cell signaling is mediated through activation of hetero-trimeric G proteins. G-Interacting Vesicle associated protein (GIV) is a non-receptor Guanine nucleotide exchange factor (GEF), which activates Gi family of heterotrimeric G proteins downstream of activated receptor tyrosine kinases (RTKs). GIV?s GEF function is mediated by a stretch of highly conserved ~20 residues, which is followed by a putative SH2-like domain in the C-terminus of the protein. Previous studies have shown that the C-terminal 211 amino acids of GIV (referred to as ?GIV-CT? henceforth) are capable of functioning autonomously from its recruitment to the cell surface upon activation of RTKs promoting downstream signals by binding to and activating Gi proteins. However, despite all the functional information and computational predictions, the structural insights into how GIV-CT is able to perform all these functions is missing. Here, we have attempted to probe the structural aspects of GIV-CT using circular dichroism (CD) spectroscopy ? the most commonly used method for determining the secondary structure of peptides and proteins. N-terminally His6-tagged GIV-CT wild type (WT) and a phosphomimetic mutant (S1674D; binds and activates Gi better than the WT) were expressed and purified from E. coli BL21-DE3 cells using Co2+-NTA affinity chromatography followed by cation exchange chromatography. Our preliminary CD spectroscopy analyses showed a random coil profile for GIV-CT WT as well as S1674D mutant, both of which could be induced to adopt an -helical conformation by addition of trifluoroethanol (TFE). Further analysis of CD spectra to predict secondary structure characteristics was carried out using DichroWeb, an online deconvolution program, which compares CD data for known protein structure to that of unknown protein structure. Wild type GIV-CT secondary structure was predicted to contain 1% ?-helix, 19% ?-sheet and 47% random coil while our mutant contained 1% ?-helix, 19% ?-sheet and 43% random coil. -turn and `denatured? helix and sheet percentages make up the final total of 100% secondary structure. In the presence of TFE, GIV-CT WT and S1674D ?-helical content increased to 23% and 25%, respectively. Together, our data suggest that although recombinant GIV-CT may be predominantly in an unstructured state, it likely has a propensity to fold into a regular secondary structure, perhaps upon interacting with a binding partner.