A growing toolbox of techniques for studying β-barrel outer membrane protein folding and biogenesis.

Sheena E Radford,Jim E Horne

doi:10.1042/bst20160020

Abstract

Great strides into understanding protein folding have been made since the seminal work of Anfinsen over 40 years ago, but progress in the study of membrane protein folding has lagged behind that of their water soluble counterparts. Researchers in these fields continue to turn to more advanced techniques such as NMR, mass spectrometry, molecular dynamics (MD) and single molecule methods to interrogate how proteins fold. Our understanding of β-barrel outer membrane protein (OMP) folding has benefited from these advances in the last decade. This class of proteins must traverse the periplasm and then insert into an asymmetric lipid membrane in the absence of a chemical energy source. In this review we discuss old, new and emerging techniques used to examine the process of OMP folding and biogenesis in vitro and describe some of the insights and new questions these techniques have revealed.

Highlights

The study of protein folding underpins a goal to understand the function of biological systems in terms of the structures, properties and interactions of the molecules which orchestrate many of life’s essential processes
The field of protein folding sits at an intersection between scientific disciplines and requires a plethora of complementary techniques to be combined to answer the question “How do proteins fold?” many of the techniques and underlying principles learned from over 40 years of studies on the folding of water soluble proteins [1,2] can be applied to membrane proteins, the introduction of the lipid bilayer and its steric and physicochemical properties necessarily alters the forces that guide protein folding when coupled with insertion into the bilayer itself
The outer membranes (OM) provides a fundamentally different folding environment compared with the inner membrane: the bilayer is asymmetric as it is enriched in lipopolysaccharide in the outer leaflet, it is densely packed with outer membrane protein (OMP), and diffusion is restricted [7,8]

Summary

Introduction

The study of protein folding underpins a goal to understand the function of biological systems in terms of the structures, properties and interactions of the molecules which orchestrate many of life’s essential processes. Cold SDS-PAGE is useful for the analysis of the formation of native OMPs since these structures are resistant to denaturation in SDS without heating This method involves initiating a folding reaction, taking samples at particular time points and quenching further folding with SDS, running the samples on an SDS-PAGE gel without boiling. 1H–1H NOEs along with a number of complementary NMR experiments have been used to show that in 8 M urea OmpX is globally denatured, but contains locally structured regions [53] These locally structured regions are formed in an area of hydrophobic clustering around a tryptophan residue which may be relevant for early intermediate stages of folding involving membrane adsorption [31,54]. This may indicate a requirement for folding to proceed from a high entropy, low enthalpy unfolded state

Computational methods MD

Findings

Conclusions