Membrane proteins in four acts: function precedes structure determination.

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) ß-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.

Structure-function of the cytochrome b6f complex.

The structure and function of the cytochrome b6f complex is considered in the context of recent crystal structures of the complex as an eight subunit, 220 kDa symmetric dimeric complex obtained from the thermophilic cyanobacterium, Mastigocladus laminosus, and the green alga, Chlamydomonas reinhardtii. A major problem confronted in crystallization of the cyanobacterial complex, proteolysis of three of the subunits, is discussed along with initial efforts to identify the protease. The evolution of these cytochrome complexes is illustrated by conservation of the hydrophobic heme-binding transmembrane domain of the cyt b polypeptide between b6f and bc1 complexes, and the rubredoxin-like membrane proximal domain of the Rieske [2Fe-2S] protein. Pathways of coupled electron and proton transfer are discussed in the framework of a modified Q cycle, in which the heme c(n), not found in the bc1 complex, but electronically tightly coupled to the heme b(n) of the b6f complex, is included. Crystal structures of the cyanobacterial complex with the quinone analogue inhibitors, NQNO or tridecyl-stigmatellin, show the latter to be ligands of heme c(n), implicating heme c(n) as an n-side plastoquinone reductase. Existing questions include (a) the details of the shuttle of: (i) the [2Fe-2S] protein between the membrane-bound PQH2 electron/H+ donor and the cytochrome f acceptor to complete the p-side electron transfer circuit; (ii) PQ/PQH2 between n- and p-sides of the complex across the intermonomer quinone exchange cavity, through the narrow portal connecting the cavity with the p-side [2Fe-2S] niche; (b) the role of the n-side of the b6f complex and heme c(n) in regulation of the relative rates of noncyclic and cyclic electron transfer. The likely presence of cyclic electron transport in the b6f complex, and of heme c(n) in the firmicute bc complex suggests the concept that hemes b(n)-c(n) define a branch point in bc complexes that can support electron transport pathways that differ in detail from the Q cycle supported by the bc1 complex.

Identification of a spectrally stable proteolytic fragment of human neutrophil flavocytochrome b composed of the NH2-terminal regions of gp91(phox) and p22(phox).

A heme-bearing polypeptide core of human neutrophil flavocytochrome b(558) was isolated by applying high performance, size exclusion, liquid chromatography to partially purified Triton X-100-solubilized flavocytochrome b that had been exposed to endoproteinase Glu-C for 1 h. The fragment was composed of two polypeptides of 60-66 and 17 kDa by SDS-polyacrylamide gel electrophoresis and retained a native heme absorbance spectrum that was stable for several days when stored at 4 degrees C in detergent-containing buffer. These properties suggested that the majority of the flavocytochrome b heme environment remained intact. Continued digestion up to 4.5 h yielded several heme-associated fragments that were variable in composition between experiments. Digestion beyond 4.5 h resulted in a gradual loss of recoverable heme. N-Linked deglycosylation and reduction and alkylation of the 1-h digestion fragment did not affect the electrophoretic mobility of the 17-kDa fragment but reduced the 60-66-kDa fragment to 39 kDa. Sequence and immunoblot analyses identified the fragments as the NH(2)-terminal 320-363 amino acid residues of gp91(phox) and the NH(2)-terminal 169-171 amino acid residues of p22(phox). These findings provide direct evidence that the primarily hydrophobic NH(2)-terminal regions of flavocytochrome b are responsible for heme ligation.