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      Membrane proteins bind lipids selectively to modulate their structure and function

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          Abstract

          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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          Most cited references55

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          Protein Identification and Analysis Tools on the ExPASy Server

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            Ion mobility-mass spectrometry analysis of large protein complexes.

            Here we describe a detailed protocol for both data collection and interpretation with respect to ion mobility-mass spectrometry analysis of large protein assemblies. Ion mobility is a technique that can separate gaseous ions based on their size and shape. Specifically, within this protocol, we cover general approaches to data interpretation, methods of predicting whether specific model structures for a given protein assembly can be separated by ion mobility, and generalized strategies for data normalization and modeling. The protocol also covers basic instrument settings and best practices for both observation and detection of large noncovalent protein complexes by ion mobility-mass spectrometry.
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              Collision cross sections of proteins and their complexes: a calibration framework and database for gas-phase structural biology.

              Collision cross sections in both helium and nitrogen gases were measured directly using a drift cell with RF ion confinement inserted within a quadrupole/ion mobility/time-of-flight hybrid mass spectrometer (Waters Synapt HDMS, Manchester, U.K.). Collision cross sections for a large set of denatured peptide, denatured protein, native-like protein, and native-like protein complex ions are reported here, forming a database of collision cross sections that spans over 2 orders of magnitude. The average effective density of the native-like ions is 0.6 g cm(-3), which is significantly lower than that for the solvent-excluded regions of proteins and suggests that these ions can retain significant memory of their solution-phase structures rather than collapse to globular structures. Because the measurements are acquired using an instrument that mimics the geometry of the commercial Synapt HDMS instrument, this database enables the determination of highly accurate collision cross sections from traveling-wave ion mobility data through the use of calibration standards with similar masses and mobilities. Errors in traveling-wave collision cross sections determined for native-like protein complexes calibrated using other native-like protein complexes are significantly less than those calibrated using denatured proteins. This database indicates that collision cross sections in both helium and nitrogen gases can be well-correlated for larger biomolecular ions, but non-correlated differences for smaller ions can be more significant. These results enable the generation of more accurate three-dimensional models of protein and other biomolecular complexes using gas-phase structural biology techniques.

                Author and article information

                Journal
                0410462
                6011
                Nature
                Nature
                Nature
                0028-0836
                1476-4687
                30 June 2014
                5 June 2014
                05 December 2014
                : 510
                : 7503
                : 172-175
                Affiliations
                [1 ]Department of Chemistry, University of Oxford, South Parks Road, Oxford, OX1 5QY, UK
                [2 ]Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, Maryland
                Author notes
                [* ]Correspondence and requests for materials should be addressed to A.L. ( art.laganowsky@ 123456chem.ox.ac.uk ) or C.V.R. ( carol.robinson@ 123456chem.ox.ac.uk )

                Author Contributions: A.L., E.R., and C.V.R. designed the research. A.L. and E.R. performed the experiments. T.A. assisted A.L. and E.R. in protein expression and purification. M.B.U. and M.T.D. carried out molecular dynamics. M.T.D. A.J.B. and A.L. performed post molecular dynamics analyses. A.L., E.R. T.A. and A.J.B developed IM-MS analysis software. A.L. and E.R. analyzed the data. A.L., E.R., and C.V.R. wrote the paper with input from the other authors.

                Article
                EMS58316
                10.1038/nature13419
                4087533
                24899312
                a4b06345-7352-4cda-aded-389a11c746d3
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