Characterisation of Conformational and Ligand Binding Properties of Membrane Proteins Using Synchrotron Radiation Circular Dichroism (SRCD)

Abstract

Membrane proteins are notoriously difficult to crystallise for use in X-ray crystallographic structural determination, or too complex for NMR structural studies. Circular dichroism (CD) is a fast and relatively easy spectroscopic technique to study protein conformational behaviour in solution. The advantage of synchrotron radiation circular dichroism (SRCD) measured with synchrotron beamlines compared to the CD from benchtop instruments is the extended spectral far-UV region that increases the accuracy of secondary structure estimations, in particular under high ionic strength conditions. Membrane proteins are often available in small quantities, and for this SRCD measured at the Diamond B23 beamline has successfully facilitated molecular recognition studies. This was done by probing the local tertiary structure of aromatic amino acid residues upon addition of chiral or non-chiral ligands using long pathlength cells (1–5 cm) of small volume capacity (70 μl–350 μl). In this chapter we describe the use of SRCD to qualitatively and quantitatively screen ligand binding interactions (exemplified by Sbma, Ace1 and FsrC proteins); to distinguish between functionally similar drugs that exhibit different mechanisms of action towards membrane proteins (exemplified by FsrC); and to identify suitable detergent conditions to observe membrane protein-ligand interactions using stabilised proteins (exemplified by inositol transporters) as well as the stability of membrane proteins (exemplified by GalP, Ace1). The importance of the in solution characterisation of the conformational behaviour and ligand binding properties of proteins in both far- andnear-UV regions and the use of high-throughput CD (HT-CD) using 96- and 384-well multiplates to study the folding effects in various protein crystallisation buffers are also discussed.

High mobility group box 1 (HMGB1) acts as an “alarmin” to promote acute myeloid leukaemia progression

“…SRCD measurements were performed using 0.2 µM of samples in a 1 cm path length cell of 3 mm aperture diameter and 60 µl capacity using a Module B instrument with a 1 nm increment, 1 s integration time, 1.2 nm bandwidth at 23 °C. 19-24 Temperature denaturation measurements were collected over the temperatures 20°C – 95°C (in 5°C increments) for HMGB1, Tim-3 and the 1:1 mixture. The results obtained were processed using CDApps 25 and OriginPro®.…”

Measuring Protein Aggregation and Stability Using High-Throughput Biophysical Approaches

“…The application of CD can be significantly broadened by the elevated photon flux of a synchrotron, which improves the signal-to-noise levels considerably, giving rise to a technique known as synchrotron radiation CD (SRCD). SRCD is capable of measuring at lower wavelengths, greatly improving the analysis of beta-sheet content and fold motifs, as well as at lower sample volumes and concentrations ( Wallace et al, 2011 ; Hussain and Siligardi, 2016 ), which is a significant advantage when studying membrane proteins, for which high concentrations and volumes are difficult and costly to produce. Additionally, higher ionic solutions can be measured (up to 500 mM NaCl) due to the higher flux, which extends the buffer screening capability of conventional CD ( Hussain and Siligardi, 2016 ).…”

“…SRCD is capable of measuring at lower wavelengths, greatly improving the analysis of beta-sheet content and fold motifs, as well as at lower sample volumes and concentrations ( Wallace et al, 2011 ; Hussain and Siligardi, 2016 ), which is a significant advantage when studying membrane proteins, for which high concentrations and volumes are difficult and costly to produce. Additionally, higher ionic solutions can be measured (up to 500 mM NaCl) due to the higher flux, which extends the buffer screening capability of conventional CD ( Hussain and Siligardi, 2016 ). For example, beamline B23 at Diamond Light Source operates in a HTP manner (also referred to as HT-CD), with 96 or 384-well suprasil quartz plates available for data collection, in which protein samples of just 15 ul at 0.5 mg/ml can be measured allowing users to screen a wide range of buffers and/or ligands without the laborious task of changing and washing each cuvette ( Hussain et al, 2016 ).…”

Selection of Biophysical Methods for Characterisation of Membrane Proteins

“…Conformational behaviour of membrane proteins induced under different environmental conditions can significantly affect protein function including drug-binding affinity. Therefore, high-throughput screening of many buffer conditions, including crystallisation buffers and a variety of ligands, using SRCD offers a significant advantage compared to benchtop CD [86]. For example, HTCD in the far-UV region of the bacterial transporter LacY measured in a 96-cell plate revealed that only four buffer mixtures out of the initial MemGold2 98 screen formulations (), induced 100% α-helical content, consistent with the crystallographic structure [87].…”