FORCE PROFILE ANALYSIS TO STUDY THE COTRANSLATIONAL FOLDING OF A MEMBRANE PROTEASE

Protein folding is critical for cellular function, and any misfolding can cause diseases such as Alzheimer's and Parkinson's. In this study, we explored the co-translational insertion and folding pathways of GlpG, a multispanning inner membrane protein of Escherichia coli. GlpG, a member of the rhomboid protease family, consists of 276 residues, including a cytoplasmic N-terminal domain and six transmembrane helices. We used Force Profile Analysis (FPA) to examine the effects of specific residues on the insertion and folding of GlpG into the E. coli inner membrane. Our previous data identified regions with notable fluctuations in transmembrane helices 1 and 2 (TMH1, TMH2), with a significant deviation at residue 159. To investigate this further, we introduced point mutations at surrounding residues and also some farther residues, such as R137A and H145-H150 to AA. Our analysis identified mutations that impacted this unusual extreme minimum point and also another subsequent deep. Histidine mutations, in particular, showed pronounced effects on both the extreme minimum point and the subsequent deep. These findings advance our understanding of co-translational folding mechanisms and highlight key residues important for proper insertion and folding into the inner membrane.