Membrane proteins in four acts: function precedes structure determination.

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TitleMembrane proteins in four acts: function precedes structure determination.
Publication TypeJournal Article
Year of Publication2011
AuthorsCramer, WA, Zakharov, SD, S Hasan, S, Zhang, H, Baniulis, D, Zhalnina, MV, Soriano, GM, Sharma, O, Rochet, JC, Ryan, C, Whitelegge, J, Kurisu, G, Yamashita, E
Date Published2011 Dec
Keywordsalpha-Synuclein, Bacterial Outer Membrane Proteins, Crystallization, Crystallography, X-Ray, Cyanobacteria, Cytochrome b6f Complex, Enzyme Assays, Escherichia coli, Escherichia coli Proteins, Humans, Membrane Transport Proteins, Models, Molecular, NADPH Dehydrogenase, Porins, Protein Structure, Secondary, Protein Structure, Tertiary, Protein Subunits

Studies on four membrane protein systems, which combine information derived from crystal structures and biophysical studies have emphasized, as a precursor to crystallization, demonstration of functional activity. These assays have relied on sensitive spectrophotometric, electrophysiological, and microbiological assays of activity to select purification procedures that lead to functional complexes and with greater likelihood to successful crystallization: (I), Hetero-oligomeric proteins involved in electron transport/proton translocation. (1) Crystal structures of the eight subunit hetero-oligomeric trans-membrane dimeric cytochrome b(6)f complex were obtained from cyanobacteria using a protocol that allowed an analysis of the structure and function of internal lipids at specific intra-membrane, intra-protein sites. Proteolysis and monomerization that inactivated the complex and prevented crystallization was minimized through the use of filamentous cyanobacterial strains that seem to have a different set of membrane-active proteases. (2) An NADPH-quinone oxido-reductase isolated from cyanobacteria contains an expanded set of 17 monotopic and polytopic hetero-subunits. (II) β-Barrel outer membrane proteins (OMPs). High resolution structures of the vitamin B(12) binding protein, BtuB, solved in meso and in surfo, provide the best example of the differences in such structures that were anticipated in the first application of the lipid cubic phase to membrane proteins [1]. A structure of the complex of BtuB with the colicin E3 and E2 receptor binding domain established a "fishing pole" model for outer membrane receptor function in cellular import of nuclease colicins. (III) A modified faster purification procedure contributed to significantly improved resolution (1.83Å) of the universal porin, OmpF, the first membrane protein for which meaningful 3D crystals have been obtained [2]. A crystal structure of the N-terminal translocation domain of colicin E3 complexed to OmpF established the role of OmpF as an import channel for colicin nuclease cytotoxins. (IV) α-Synuclein, associated with the etiology of Parkinson's Disease, is an example of a protein, which is soluble and disordered in solution, but which can assume an ordered predominantly α-helical conformation upon binding to membranes. When subjected in its membrane-bound form to a trans-membrane electrical potential, α-synuclein can form voltage-gated ion channels. Summary of methods to assay functions/activities: (i) sensitive spectrophotometric assay to measure electron transfer activities; (ii) hydrophobic chromatography to deplete lipids, allowing reconstitution with specific lipids for studies on lipid-protein interactions; (iii) microbiological screen to assay high affinity binding of colicin receptor domains to Escherichia coli outer membrane receptors; (iv) electrophysiology/channel analysis (a) to select channel-occluding ligands for co-crystallization with ion channels of OmpF, and (b) to provide a unique description of voltage-gated ion channels of α-synuclein.

Alternate JournalMethods
PubMed ID22079407
PubMed Central IDPMC3282554
Grant ListR01 GM038323-24 / GM / NIGMS NIH HHS / United States
R01 GM018457-35 / GM / NIGMS NIH HHS / United States
R01 GM018457 / GM / NIGMS NIH HHS / United States
1 P50 GM088499 / GM / NIGMS NIH HHS / United States
R56 GM038323 / GM / NIGMS NIH HHS / United States
P50 GM088499 / GM / NIGMS NIH HHS / United States
GM018457 / GM / NIGMS NIH HHS / United States
R01 GM038323 / GM / NIGMS NIH HHS / United States
P50 GM088499-02 / GM / NIGMS NIH HHS / United States
GM038323 / GM / NIGMS NIH HHS / United States