Identification and Characterization of an Inside-Out Folding Intermediate of T4 Phage Sliding Clamp

Abstract

Protein folding process involves formation of transiently occurring intermediates that are difficult to isolate and characterize. It is both necessary and interesting to characterize the structural conformations adopted by these intermediates, also called molten globules (MG), to understand protein folding. Here, we investigated the equilibrium (un)folding intermediate state of T4 phage gene product 45 (gp45, also known as DNA polymerase processivity factor or sliding clamp) obtained during chemical denaturation. We show that gp45 undergoes substantial conformational rearrangement during unfolding and forms an expanded dry-MG. By monitoring the fluorescence of tryptophans that were strategically introduced at various sites, we demonstrate that the urea-treated molecule has its surface residues flip inside the core, and closely placed residues move farther. We were also able to isolate and purify the MG form of gp45 in native condition (i.e., nondenaturing buffer, at physiological pH and temperature); characteristics of this purified molecule substantially match with urea-treated wild-type gp45. To the best of our knowledge, this is one of the few reports that demonstrate the isolation and purification of a protein folding intermediate in native condition. We believe that our work not only allows us to dissect the process of protein folding, but will also help in the designing of folding inhibitors against sliding clamps to treat a wide variety of diseases from bacterial infection to cancer, due to the vast presence of clamps in all the domains of life.

An intramolecular cross-talk in D29 mycobacteriophage endolysin governs the lytic cycle and phage-host population dynamics

“…SDM was carried out to generate all the mutants using transfer-PCR (polymerase chain reaction) ( 48 , 55 ). PCR was carried out using mutagenic primers and plasmids harboring respective WT genes as templates.…”

CcdB at pH 4 Forms a Partially Unfolded State with a Dry Core

“…Although NMR can provide structural information at amino acid resolution, limited insights have been obtained about transient nonnative states using NMR because of the longer time associated with NMR measurements. Nevertheless, a few studies have reported the 2D NMR characterization of the DMG state because it populates under equilibrium under high pressure (31,58,59). The CcdB_4 and high-pressure ubiquitin state are compact as judged by the dispersion of crosspeaks.…”