X-ray diffraction reveals the intrinsic difference in the physical properties of membrane and soluble proteins

Xavier Robert,Nicoletta Ceres,Pierre Falson,Patrice Gouet,Randy J. Read,Josiane Kassis-Sahyoun,Vincent Chaptal,Juliette Martin,Michael R. Sawaya

doi:10.1038/s41598-017-17216-1

Abstract

Membrane proteins are distinguished from soluble proteins by their insertion into biological membranes. This insertion is achieved via a noticeable arrangement of hydrophobic amino acids that are exposed at the surface of the protein, and renders the interaction with the aliphatic tails of lipids more energetically favorable. This important difference between these two categories of proteins is the source of the need for a specific handling of membrane proteins, which transpired in the creation of new tools for their recombinant expression, purification and even crystallization. Following this line, we show here that crystals of membrane proteins display systematically higher diffraction anisotropy than those of soluble proteins. This phenomenon dramatically hampers structure solution and refinement, and has a strong impact on the quality of electron-density maps. A farther search for origins of this phenomenon showed that the type of crystallization, and thus the crystal packing, has no impact on anisotropy, nor does the nature or function of the membrane protein. Membrane proteins fully embedded within the membrane display equal anisotropy compared to the ones with extra membranous domains or fusions with soluble proteins. Overall, these results overturn common beliefs and call for a specific handling of their diffraction data.

Highlights

Membrane proteins are the link between the cell and its environment, being the means of exchange through a lipid bilayer that is otherwise impermeable
We show that membrane proteins behave differently than soluble proteins towards X-ray diffraction, and confirmed that they display more anisotropy
We chose to explore this field of research with a special emphasis on membrane proteins, because they carry the advantage of having an intrinsic orientation; the membrane insertion defines a plane to which protruding portions are perpendicular

Summary

Introduction

Membrane proteins are the link between the cell and its environment, being the means of exchange through a lipid bilayer that is otherwise impermeable. Diffraction anisotropy is defined from a 2003 study from Popov & Bourenkov who reported the diffraction of 72 macromolecule crystals made of proteins of different molecular weights and folds, and belonging to distinct space groups, that diffracted X-rays to 0.9 Å resolution[7] They showed that the observed reflections follow a typical intensity falloff as a function of resolution. The pattern of diffraction intensities is not homogeneously distributed in spherical shells depending on scattering angles and can be described as ellipsoids[2,9] These experimental X-rays observations are generally linked to defects in crystal packing and/or anisotropic vibrations in the crystallized macromolecules. We were faced with many cases where crystals diffracted very anisotropically but without any obvious absence of contacts in the crystal packing, which goes against the above assumption

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Results

Conclusion