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.

Keywords: crystallography; electron microscopy; membrane protein; structural biology.

Figure 1

(a) Cumulative tally of unique membrane protein structures deposited in the Protein Data Bank (PDB) adapted from the Membrane Proteins of Known 3D Structure database [4]. (b) Membrane Protein Laboratory (MPL) projects classified by target origin in 2008/9 (29 projects) and 2018/19 (46 projects). (c) Number of membrane protein structures solved each year by electron diffraction (ED), electron microscopy (EM), X-ray crystallography and nuclear magnetic resonance spectroscopy (NMR) (d) accepted MPL projects by requested technology platform in 2008/9 (29 projects) and 2018/19 (46 projects). Cloning and expression were not supported in 2008/9.

Figure 2

Example sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and fluorescence size exclusion chromatography (FSEC) results from a small-scale detergent screen for a bacterial transporter (a–c) and bacterial membrane kinase (d–f). A SRT-300 (Sepax Technologies Inc.) high-pressure liquid chromatography column was used for FSEC. This has the advantage of tolerating higher pressures than standard SEC columns, enabling faster flow rates at 4 °C. 96 samples can be processed in 24 h. (a) Purification in 12 different extraction conditions of a monomeric bacterial transporter (35 kDa) using Ni-NTA resin. (b) Purification of the same bacterial transporter using Talon resin (cobalt). (c) FSEC profiles using tryptophan fluorescence (excitation at 290 nm, emmission at 350 nm) for purifications shown in panel b. (d) Purification in 12 extraction conditions of a multimeric bacterial histidine kinase. SDS-PAGE gel imaged using GFP fluorescence and (e) Coomassie staining. (f) FSEC profile using GFP fluorescence (excitation at 490 nm, emission at 510 nm) of purification in panels (d) and (e). Peaks correspond to monomeric and dimeric protein.

Figure 3

Thermal unfolding of vcINDY in the presence of different substrates measured by intrinsic tryptophan fluorescence and backscatter. (a) Thermal denaturation curves depicting fraction of protein unfolded. (b) First derivative of (a) reporting maximum T_m for each additive. (c) Scattering curves for each additive. (d) Derived scattering curves with the maximum reporting T_agg.

Figure 4

MIR spectrum of six different membrane protein purifications in octyl glucose neopentyl glycol (OGNG) with cholesteryl hemisuccinate (CHS) from ZMPSTE24 (solid lines) and a membrane receptor (dashed lines). (a) Full MIR spectra and (b) magnified MIR spectra (3050–2700 cm⁻¹). Using a standard curve for a given detergent the amount of detergent in a sample can be quantified. In four of these cases (blue, yellow, grey and orange) detergent content is low (between 0.25% and 0.5%) and at the detection limit for the method. Low detergent readings are expected for these optimised purifications where detergent content is minimised throughout the purification. In the case of the membrane receptor, detergent concentrations are much higher ~2% (dark blue and green) as indicated by the peak at 2850 cm⁻¹.