PhD Thesis Defence: Towards Understanding Membrane Protein Folding and Stability with Solid-State NMR and Hydrogen Deuterium Exchange
Date and Time
A comprehensive understanding of membrane protein folding in the native-like lipid environment is one of the most formidable challenges in protein biophysics. The folding of alpha-helical membrane proteins has been described by a model featuring two energetically distinct stages. A potential third stage had been proposed involving interactions such as folding of loops, binding of ligands, and oligomerization. It may play a crucial role in the formation of a functional and stable protein; however, the nature of the stage is yet to be elucidated. In our research, we aim to probe the late stages of the membrane protein folding and interactions contributing to protein stability within the lipid bilayer, and to provide key evidence for the third stage of the folding.
We have developed a methodology that combines hydrogen-deuterium exchange (HDX) and solid-state NMR (ssNMR) detection to site-specifically follow the thermally induced unfolding of membrane proteins in their lipid environment. We first employed this methodology to obtain an atomistic description of the thermally induced unfolding pathway of a retinal-binding seven-helical photoreceptor Anabaena Sensory Rhodopsin (ASR). The pathway is visualized through ssNMR-detected snapshots of HDX patterns as a function of temperature, revealing the unfolding intermediate and its stabilizing factors involving interactions within the retinal binding pocket and at the intermonomer interface.
This methodology is then implemented for investigating the unfolding energy landscape of human aquaporin-1 (hAQP1), a homo-tetrameric transmembrane water channel. The site-specific exchange rates were probed at four different temperatures and the activation energies were extracted, from which we constructed an energy landscape corresponding to the third and the second stage. We described the specific unfolding events correlated to the unfolding transitions, and structurally characterize the unfolding intermediates. Our results suggest that the folding of this extracellular loop in hAQP1 plays a critical role in stabilizing the fold and the correct folding for the entire protein, which is further supported by sitedirected mutagenesis experiments.
- Dr. Hermann Eberl, Chair
- Dr. Vladimir Ladizhansky, Advisor
- Dr. Leonid Brown, Co-Advisor
- Dr. Siavash Vahidi, Graduate Faculty
- Dr. Giuseppe Melacini, External Examiner (McMaster University)