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Hydrogen Bond Shaping of Membrane Protein Structure

  • Author(s): Cao, Zheng
  • Advisor(s): Bowie, James U
  • et al.
Abstract

The intricate functions of membrane proteins would not be possible without bends or breaks that are remarkably common in transmembrane helices. The frequent distortions are nevertheless surprising because backbone hydrogen bonds should be strong in an apolar environment, potentially rigidifying helices. It is therefore mysterious how distortions can be generated by evolutions. Through my studies on bacteriorhodopsin and Ca2+-ATPase, I found that helix distortions are facilitated by shifting hydrogen bonding partners. My results explained how evolution has been able to liberally exploit transmembrane helix bending by for optimizing membrane protein structure, function and dynamics.

It has recently been found that the stability of bacteriorhodopsin assessed through unfolding did not achieve equilibrium. Here I made a new equilibrium test by measuring the transition of folded bacteriorhodopsin to unfolded bacterioopsin. My result suggests that the energetic effects of most mutations that have been studied before may be focused on the folded state.

To further investigate how hydrogen bonds may shape transmembrane helices, I sought a non-perturbing method to measure strengths of both backbone and side-chain hydrogen bond strengths, because backbone hydrogen bonds cannot be probed by mutation and mutagenesis to remove side-chains may not mimic the breaking of side-chain hydrogen bonds. I therefore decided to employ the equilibrium hydrogen / deuterium fractionation factors (phi-value) for hydrogen bonds. I used model compounds to study the relationship between phi-value and the hydrogen bond free energy. By applying this relationship, I found that hydrogen bonding stabilizes enzyme intermediate state by up to 4 kcal/mol more than the resting state and marginally favor soluble-protein unfolding.

To measure hydrogen bond strengths in a membrane protein, I focused on the isolated voltage-sensor domain of the voltage-dependent potassium-selective channel. By utilizing its 2D NMR spectrum, I obtained phi-values and thus free energies for 70 % of the backbone hydrogen bonds in this membrane protein. I found that it has similar backbone hydrogen bond strength to water-soluble proteins on average. Moreover, I found that the flexible (dynamic) point of a transmembrane helix in this protein can be predicted from the backbone hydrogen bond strengths in that helix.

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