Membrane Proteins Have Distinct Fast Internal Motion and Residual Conformational Entropy.

Evan S O'Brien,Kushol Gupta,Brian Fuglestad,Henry J Lessen,Karen G Fleming,A Joshua Wand,Danny W Lin,Matthew A Stetz,Bryan S Marques

doi:10.1002/anie.202003527

Abstract

The internal motions of integral membrane proteins have largely eluded comprehensive experimental characterization. Here the fast side‐chain dynamics of the α‐helical sensory rhodopsin II and the β‐barrel outer membrane protein W have been investigated in lipid bilayers and detergent micelles by solution NMR relaxation techniques. Despite their differing topologies, both proteins have a similar distribution of methyl‐bearing side‐chain motion that is largely independent of membrane mimetic. The methyl‐bearing side chains of both proteins are, on average, more dynamic in the ps–ns timescale than any soluble protein characterized to date. Accordingly, both proteins retain an extraordinary residual conformational entropy in the folded state, which provides a counterbalance to the absence of the hydrophobic effect. Furthermore, the high conformational entropy could greatly influence the thermodynamics underlying membrane‐protein functions, including ligand binding, allostery, and signaling.

Highlights

The motions of amino acid side chains of proteins are important for understanding the connection between energetics, structure and function in these complex macromolecules
outer membrane protein W (OmpW) can be refolded in high yield and the traditional approach of expression during growth on bulk D2O media permitted a standard labeling strategy for 15N, 13CH3 ILV labeling in a background of carbon bonded deuterium (Fig. S1B & S1D). proteins: sensory rhodopsin II (pSRII) and OmpW were solubilized for NMR experiments in both detergent micelles as well as DMPC lipid bilayers in the form of q ~ 1-1.1 bicelles where q is the molar ratio of long-chain DMPC and short chain DHPC
In summary, the first comprehensive studies of fast side chain motion in integral membrane proteins presented here have revealed the existence of extensive sampling of microstates and point to a relatively high residual conformational entropy in pSRII and OmpW

Summary

Introduction

The motions of amino acid side chains of proteins are important for understanding the connection between energetics, structure and function in these complex macromolecules. Membrane mimetic has little impact on fast internal motions of integral membrane proteins Methyl cross-correlated relaxation experiments[15] were interpreted in the context of the LipariSzabo formalism,[21] yielding squared generalized order parameter of the methyl group symmetry axis (O2axis) for nearly every resolved methyl resonance in pSRII and OmpW.

Results

Conclusion