pH-Triggered Membrane Insertion of Proteins

Abstract

Several classes of membrane proteins refold from their original soluble conformations in response to acidification and insert into lipid bilayers. Despite the recent advances in unraveling the molecular mechanisms of pH-triggered insertion of membrane proteins, many aspects of this process remain unknown. In this study we used two proteins that share structural similarities as model systems: the diphtheria toxin translocation domain (T domain) and the anti-apoptotic regulator Bcl-xL. We addressed the following specific questions: (1) Do structurally similar T domain and Bcl-xL share common mechanisms of membrane insertion? (2) What is the role of titratable histidine residues on the late stages of transmembrane insertion of the T domain? (3) How do the physicochemical properties of the lipid bilayer modulate the pH-triggered membrane insertion of Bcl-xL? First, we characterized the main features of the pH-triggered membrane insertion pathway of Bcl-xL using circular dichroism (CD) and a battery of fluorescence-based methods and compared them to those previously determined for the T domain. We demonstrate that both proteins follow distinct membrane insertion pathways despite of the structural similarities of the initial solution and putative final transmembrane folds. Second, we used site-directed mutagenesis and functional and spectroscopic assays to characterize the membrane interactions of the C-terminal histidines of the T domain. We determine that H322 is critical for proper refolding of the N-terminal helices within the membrane, which is critical for the formation of the Open-Channel State of the T domain within the lipid bilayer. Finally, we determined the role of various lipids on the membrane interactions of Bcl-xL using several fluorescence-based techniques. We demonstrate that the initial membrane association of Bcl-xL is modulated by the membrane surface potential, while the transmembrane insertion is regulated by additional properties, e.g. mechanical stress. We conclude that the pH-triggered membrane insertion of proteins can be modulated at multiple levels, including protonation of specific titratable residues and changes in the membrane lipid composition. These differences in the mechanisms of regulation are relevant to the physiological function of the corresponding membrane proteins (e.g., T domain and Bcl-xL).