Detailed Stability and Unfolding Study of Mycobacterium Global Transcription Regulator Protein

Abstract

Background and objectives:The cyclic adenosine monophosphate Receptor Protein of Mycobacterium tuberculosis (CRP Mt ) (Rv3676), is a global transcriptional regulator and plays a pivotal role in the survival and infection of Mycobacterium. This signaling protein (CRP Mt ) shares several common structural and functional features with the CRP from Escherichia coli. Structurally, CRP Mt is a homodimer that undergoes allosteric changes upon cyclic AMP binding. This binding also triggers the activation of several genes responsible for various physiological processes in this bacterium. Despite the importance of CRP for mycobacterial survival, limited information is available regarding the stability and unfolding properties of the protein. The main objective of this study is to study stability, unfolding and dynamics of CRP Mt in terms of its structure. Methods:In this study, we monitored the stability and unfolding of CRP Mt using various biophysical and computational techniques. Results:We experimentally studied protein unfolding in the presence of chemical denaturants [urea and guanidine hydrochloride (GdnHCl)]. The results from these chemical-induced unfolding studies suggest that CRP Mt follows a two-state transition and that chemical-induced protein denaturation is reversible. According to circular dichroism and activity data, CRP Mt structure and function were restored upon refolding. We also studied the stability and unfolding of the CRP Mt protein against temperature variations and protease action (trypsin). Limited proteolysis experiments provide insights into the minimum domain structure requirement for CRP Mt activity. Interestingly, temperature-induced CRP Mt unfolding was completely different compared to chemical-induced unfolding. The thermal unfolding of CRP Mt was found to be irreversible, leading to the formation of insoluble aggregates at elevated temperatures. To understand why the thermal unfolding of the protein differed from chemically induced unfolding, we carried out a detailed molecular dynamics simulation analysis of the protein at three different temperatures. The results from these molecular dynamics simulations mechanistically validate the significant differences between chemical and temperature-induced CRP Mt unfolding. Conclusion:Our study provides detailed insights into the stability and folding/unfolding properties of CRP Mt , which could be useful in developing new anti-mycobacterial medicines.