Hydrogen Bond Shaping of Membrane Protein Structure

Seminar series
Physical Chemistry Seminar
Thu, May 2 12:00pm
2033 Young Hall
Speaker Zheng Cao
University of California, Los Angeles
Dept. of Chemistry & Biochemistry

Abstract: The intricate functions of membrane proteins would not be possible without bends or breaks that are remarkably common in transmembrane helices. The frequent helix distortions are nevertheless surprising because backbone hydrogen bonds should be strong in an apolar membrane, potentially rigidifying helices. It is therefore mysterious how distortions can be generated by the evolutionary currency of random point mutations. To investigate the energetic cost of bending transmembrane helices, I engineered a transition between distinct helix conformations in bacteriorhodopsin.  Through protein stability measurements of the mutant proteins, I estimate the bending cost to be smaller than 1 kcal/mol.  Why is it so easy to bend transmembrane helices?  Inspection of the mutant crystal structures suggests that flexibility is allowed by the facile shifting of backbone hydrogen bond patterns. To further investigate how hydrogen bonds may shape transmembrane helices, I sought a method to measure backbone hydrogen bond strengths.  As backbone hydrogen bonds cannot be probed by mutation, I needed a non-perturbing method for measuring their strengths.  I therefore decided to employ protium/deuterium (H/D) isotope exchange equilibrium fractionation factors: φ = ([D]/[H])protein/([D]/[H])solvent . As φ values depend on hydrogen bond strength, they provide ready access to energetic information.  I first used model compounds to study the relationship between φ value and the hydrogen bond free energy.  To measure hydrogen bond strengths in a membrane protein, I am focusing on the KvAP voltage sensor domain, because its 15N-1H HSQC NMR spectrum has been previously assigned.  Thus, by measuring changes in 15N-1H HSQC peak volume as a function of the D2O/H2O ratio, I can measure the hydrogen bond free energy for nearly all the backbone hydrogen bonds in the protein.  These data will provide a comprehensive view of hydrogen bonding in a membrane protein for the first time and provide a pathway to investigating how differing hydrogen bond strengths may shape membrane protein structure.