HDX-guided EPR spectroscopy to interrogate membrane protein dynamics

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Summary

Solvent accessibilities of and distances between protein residues measured by pulsed-EPR approaches provide high-resolution information on dynamic protein motions. We describe protocols for the purification and site-directed spin labeling of integral membrane proteins. In our protocol, peptide-level HDX-MS is used as a precursor to guide single-residue resolution ESEEM accessibility measurements and spin labeling strategies for EPR applications. Exploiting the pentameric MscL channel as a model, we discuss the use of cwEPR, DEER/PELDOR, and ESEEM spectroscopies to interrogate membrane protein dynamics.

For complete details on the use and execution of this protocol, please refer to Wang et al. (2022).

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Protocols for an integrated EPR-based approach to study membrane protein dynamics
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Instructions for the sample preparation of spin-labeled membrane proteins
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Used HDX-MS as a precursor to guide spin labeling strategies for EPR methods
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Probed solvent accessibility at the single-residue level by ESEEM

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Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

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An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol

Huanting Liu, James Naismith (2008)

Background Mutagenesis plays an essential role in molecular biology and biochemistry. It has also been used in enzymology and protein science to generate proteins which are more tractable for biophysical techniques. The ability to quickly and specifically mutate a residue(s) in protein is important for mechanistic and functional studies. Although many site-directed mutagenesis methods have been developed, a simple, quick and multi-applicable method is still desirable. Results We have developed a site-directed plasmid mutagenesis protocol that preserved the simple one step procedure of the QuikChange™ site-directed mutagenesis but enhanced its efficiency and extended its capability for multi-site mutagenesis. This modified protocol used a new primer design that promoted primer-template annealing by eliminating primer dimerization and also permitted the newly synthesized DNA to be used as the template in subsequent amplification cycles. These two factors we believe are the main reasons for the enhanced amplification efficiency and for its applications in multi-site mutagenesis. Conclusion Our modified protocol significantly increased the efficiency of single mutation and also allowed facile large single insertions, deletions/truncations and multiple mutations in a single experiment, an option incompatible with the standard QuikChange™. Furthermore the new protocol required significantly less parental DNA which facilitated the DpnI digestion after the PCR amplification and enhanced the overall efficiency and reliability. Using our protocol, we generated single site, multiple single-site mutations and a combined insertion/deletion mutations. The results demonstrated that this new protocol imposed no additional reagent costs (beyond basic QuikChange™) but increased the overall success rates.

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DEER distance measurements on proteins.

Gunnar Jeschke (2012)

Distance distributions between paramagnetic centers in the range of 1.8 to 6 nm in membrane proteins and up to 10 nm in deuterated soluble proteins can be measured by the DEER technique. The number of paramagnetic centers and their relative orientation can be characterized. DEER does not require crystallization and is not limited with respect to the size of the protein or protein complex. Diamagnetic proteins are accessible by site-directed spin labeling. To characterize structure or structural changes, experimental protocols were optimized and techniques for artifact suppression were introduced. Data analysis programs were developed, and it was realized that interpretation of the distance distributions must take into account the conformational distribution of spin labels. First methods have appeared for deriving structural models from a small number of distance constraints. The present scope and limitations of the technique are illustrated.

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Membrane proteins bind lipids selectively to modulate their structure and function

Arthur Laganowsky, Eamonn Reading, Timothy Allison … (2014)

Previous studies have established that the folding, structure and function of membrane proteins are influenced by their lipid environments 1-7 and that lipids can bind to specific sites, for example in potassium channels 8 . Fundamental questions remain however regarding the extent of membrane protein selectivity toward lipids. Here we report a mass spectrometry (MS) approach designed to determine the selectivity of lipid binding to membrane protein complexes. We investigate the mechanosensitive channel of large conductance (MscL), aquaporin Z (AqpZ), and the ammonia channel (AmtB) using ion mobility MS (IM-MS), which reports gas-phase collision cross sections. We demonstrate that folded conformations of membrane protein complexes can exist in the gas-phase. By resolving lipid-bound states we then rank bound lipids based on their ability to resist gas phase unfolding and thereby stabilize membrane protein structure. Results show that lipids bind non-selectively and with high avidity to MscL, all imparting comparable stability, the highest-ranking lipid however is phosphatidylinositol phosphate, in line with its proposed functional role in mechanosensation 9 . AqpZ is also stabilized by many lipids with cardiolipin imparting the most significant resistance to unfolding. Subsequently, through functional assays, we discover that cardiolipin modulates AqpZ function. Analogous experiments identify AmtB as being highly selective for phosphatidylglycerol prompting us to obtain an X-ray structure in this lipid membrane-like environment. The 2.3Å resolution structure, when compared with others obtained without lipid bound, reveals distinct conformational changes that reposition AmtB residues to interact with the lipid bilayer. Overall our results demonstrate that resistance to unfolding correlates with specific lipid-binding events enabling distinction of lipids that merely bind from those that modulate membrane protein structure and/or function. We anticipate that these findings will be influential not only for defining the selectivity of membrane proteins toward lipids but also for understanding the role of lipids in modulating function or drug binding.

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Christos Pliotas

Journal

Journal ID (nlm-ta): STAR Protoc

Journal ID (iso-abbrev): STAR Protoc

Title: STAR Protocols

Publisher: Elsevier

ISSN (Electronic): 2666-1667

Publication date PMC-release: 18 July 2022

Publication date Collection: 16 September 2022

Publication date (Electronic): 18 July 2022

Volume: 3

Issue: 3

Electronic Location Identifier: 101562

Affiliations

[1 ]Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK

[2 ]School of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK

[3 ]School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK

[4 ]School of Physics and Astronomy, University of St Andrews, St Andrews KY16 9SS, UK

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[∗ ]Corresponding author c.pliotas@ 123456leeds.ac.uk

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Publisher Item ID: S2666-1667(22)00442-7 Publisher ID: 101562

DOI: 10.1016/j.xpro.2022.101562

PMC ID: 9304679

PubMed ID: 35874470

SO-VID: 5ec7ee0e-cc72-431e-b01b-ca69b80da615

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This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

HDX-guided EPR spectroscopy to interrogate membrane protein dynamics

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Most cited references 47

An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol

DEER distance measurements on proteins.

Membrane proteins bind lipids selectively to modulate their structure and function

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