Changes in Membrane Protein Structural Biology

Abstract

Membrane proteins are essential components of many biochemical processes and are important pharmaceutical targets. Membrane protein structural biology provides the molecular rationale for these biochemical process as well as being a highly useful tool for drug discovery. Unfortunately, membrane protein structural biology is a difficult area of study due to low protein yields and high levels of instability especially when membrane proteins are removed from their native environments. Despite this instability, membrane protein structural biology has made great leaps over the last fifteen years. Today, the landscape is almost unrecognisable. The numbers of available atomic resolution structures have increased 10-fold though advances in crystallography and more recently by cryo-electron microscopy. These advances in structural biology were achieved through the efforts of many researchers around the world as well as initiatives such as the Membrane Protein Laboratory (MPL) at Diamond Light Source. The MPL has helped, provided access to and contributed to advances in protein production, sample preparation and data collection. Together, these advances have enabled higher resolution structures, from less material, at a greater rate, from a more diverse range of membrane protein targets. Despite this success, significant challenges remain. Here, we review the progress made and highlight current and future challenges that will be overcome.

The high-throughput production of membrane proteins

“…His-tags enable affinity purification and comprise six to ten histidine residues. Background contamination can be high when using his-tags [ 54 ] and we recommend a reverse purification step. For one-step purification we prefer twin-strep-tags [ 55 ] which are ideal for HTP screening platforms [ 30 ].…”

“…GFPtags also give a crude indication of protein folding and localisation, and enable assessment of monodisperisty and stability, by F-SEC, in different encapsulation reagents. However, GFP tags cause both false positives and negatives as GFP is highly stable and can reduce expression levels [ 54 ]. A recently developed multivalent nitrilotriacetic acid fluorescent probe [ 68 ] enables GFP-free screening but background histidine rich proteins can interfere.…”

Lipid Membrane Mimetics in Functional and Structural Studies of Integral Membrane Proteins

“…In the last decade, EM and single-particle cryoEM in particular have made historic progress in studying detergent-solubilized IMPs by expanding this technique’s applications to diverse families of IMPs and by determining these proteins’ 3D structure at high resolution down to ca. 3 Å [ 21 , 95 ]. In contrast to X-ray crystallography, EM does not require protein-crystal formation and has much more potential to deal with conformationally heterogeneous proteins and protein complexes.…”

“…In contrast to X-ray crystallography, EM does not require protein-crystal formation and has much more potential to deal with conformationally heterogeneous proteins and protein complexes. Nevertheless, successful IMP structure determination via EM requires high stability and proper folding of the detergent-solubilized protein [ 95 ]. For this reason, detergents are screened similarly to the crystallization of IMPs.…”

Structural Biology and Structure–Function Relationships of Membrane Proteins

“…James Birch, Harish Cheruvara, Nadisha Gamage, Peter J. Harrison, Ryan Lithgo and Andrew Quigley [ 11 ] provide a compressive review on the most recent advances in the field of membrane protein structural biology. The review takes the reader through each step in the process of solving the atomic resolution structure of a membrane protein.…”