The fine art of integral membrane protein crystallisation

Abstract

Integral membrane proteins are among the most fascinating and important biomolecules as they play a vital role in many biological functions. Knowledge of their atomic structures is fundamental to the understanding of their biochemical function and key in many drug discovery programs. However, over the years, structure determination of integral membrane proteins has proven to be far from trivial, hence they are underrepresented in the protein data bank. Low expression levels, insolubility and instability are just a few of the many hurdles one faces when studying these proteins. X-ray crystallography has been the most used method to determine atomic structures of membrane proteins. However, the production of high quality membrane protein crystals is always very challenging, often seen more as art than a rational experiment. Here we review valuable approaches, methods and techniques to successful membrane protein crystallisation.

Changes in Membrane Protein Structural Biology

“…The data from these detectors can be used to calculate the oligomeric state of an MP, the amount of detergent associated, and the amount of free detergent by The compositional stability of an MP in detergent is often assessed early in the purification process via FSEC. More recent approaches such as nanoDSF and in situ dynamic light scattering can also be used to access the effect of encapsulation reagents on the stability of an MP [102,170,171]. Negative stain is another method that can be used to assess the compositional stability of MPs but is limited by sample throughput but does have the advantage of using nanograms of sample.…”

“…This includes protein and detergent as well as any post-translational modifications such as glycosylation. By using a co-polymer analysis, the amount of protein, glycosylation, protein-associated detergent as well as the free detergent micelles can be calculated [171,178,179].…”

“…Type II crystals also tend to diffract to higher resolution due to the formation of additional crystal contacts in the membrane spanning regions. Consequently, type II crystals have a lower solvent content and are, therefore, more robust [171]. Selection of the right detergents for direct crystallisation is necessary to obtain well diffracting crystals [94,102,185].…”

“…MP must be removed from its native environment. Crystals grown in crystallisation trays, mounted in a loop and cryo-cooled in liquid nitrogen [171].…”

Integrative Modeling of Membrane-associated Protein Assemblies

“…Solid-state NMR spectroscopy, and especially cryo-electron microscopy (cryo-EM), reaching near-atomic resolution, have become central tools to study membraneassociated protein complexes 5,6 . However, experimental conditions such as low expression profiles and/or high instability outside the native membrane still makes their structural characterization challenging 7 . Despite their large representation in the proteome (in human, nearly a quarter of the genome encodes for MPs 8 ), roughly only 1% of all deposited protein structures in the Protein Data Bank 9 11 .…”