Abstract
HDX-MS has emerged as a powerful tool to interrogate the structure and dynamics of proteins and their complexes. Recent advances in the methodology and instrumentation have enabled the application of HDX-MS to membrane proteins. Such targets are challenging to investigate with conventional strategies. Developing new tools are therefore pertinent for improving our fundamental knowledge of how membrane proteins function in the cell. Importantly, investigating this central class of biomolecules within their native lipid environment remains a challenge but also a key goal ahead. In this short review, we outline recent progresses in dissecting the conformational mechanisms of membrane proteins using HDX-MS. We further describe how the use of computational strategies can aid the interpretation of experimental data and enable visualisation of otherwise intractable membrane protein states. This unique integration of experiments with computations holds significant potential for future applications.
Highlights
Membrane proteins play critical roles in higher organisms and are responsible for diverse cellular functions such as signalling and molecular transport
While detergent micelles remain the primary choice for membrane protein solubilisation, recent advances have enabled the interrogation of membrane protein dynamics in lipid-based environments, reminiscent of their natural conditions [22,26,32]
HDX is tolerant of lipids and other complex environments, interfacing lipids with the fast separation and digestions required for HDX-MS can lead to fouling of the liquid chromatography (LC) and digestion columns
Summary
HDX-MS has emerged as a powerful tool to interrogate the structure and dynamics of proteins and their complexes. MD simulations were performed to investigate the backbone dynamics of dimeric C99 in the presence of lipids and revealed a flexible hinge point between the dimerization and cleavage sites These successful studies demonstrate the possibility for molecular simulations to add valuable structural insights to HDX-MS experiments, quantitative interpretation of HDX-MS is often fraught with difficulty owing to the multiple sources of uncertainty (experimental, structural sampling, and predictive model) involved. In developing the method they show that the well-used phenomenological model for predicting HDX protection factors can discern between protein conformations with a high degree of structural fidelity, including in situations with limited experimental peptide coverage or low resolution They exemplify the approach by re-interpreting the LeuT experimental HDX-MS data of Adhikary et al and again identify outward- and inward-facing conformational populations of wild-type and mutant LeuT in agreement with other biophysical data. We suggest a number of excellent reviews [33,60,61,62]
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