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Protein-hungry cells 'go fishing,' report Purdue biologists. full story...

Purdue biologists' spotlight solves mysteries of photosynthesis, metabolism. full story...

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Research Interests

Structure-Function of the Cytochrome b6f Complex

Although there are approximately 45,000 independent structures of soluble proteins, and somewhat more than 150 such structures of integral membrane proteins (IMP) in the Protein Data Bank, there are only about 2 dozen structures of hetero-oligomeric oligomeric structures that have been solved to a resolution ≤ 3.0 Å.  Although these membrane proteins are of great interest in health and disease-related studies, most of these hetero-oligomeric structures are of protein complexes that function in electron transfer in energy transducing membranes.  A high resolution (3.0 Å) structure has been obtained of one of the three major hetero-oligomeric membrane protein complexes, the cytochrome b6f complex, the electron and proton transferring complex that connects the two reaction centers in photosynthetic electron transport. The dimeric 220 kDa complex contains 8 polypeptide subunits (Science, 302, 1009-, 2003; Ann. Rev. Biochem., 75, 769-, 2006) 13 trans-membrane helices, and 7 prosthetic groups (4 hemes, 1 FeS cluster, 1 Chl a, 1 beta-carotene) per monomer. Several structure and function aspects of this complex are novel, including the single chlorophyll a, beta-carotene, and unique covalently bound heme cn, which is 5-coordinate, with two waters as the ligand, but unique because there is no amino acid side chain that serves as an axial ligand.  Crystal structures with quinone analogue inhibitors imply that heme cn is the electron donor to plastoquinone on the n- or stromal side of the membrane (Yamashita et al., J. Mol. Biol., 370:59-72, 2007).  The details of the structure are of interest not only for the insight they provide to electron transport and energy transduction, but also for their description of the labyrinthine transfer pathways of the hydrophobic plastoquinone and –quinol across the b6f complex

Cramer lab

Protein Import: The Cytotoxic Colicins

Hypothesesfor the pathway and mechanisms of import into E. coli of the cytotoxic E colicins are partly based on structures of a complex of the colicin with its outer membrane vitamin B12 receptor (BtuB), which the colicins parasitize for import. A 2.75 Å structure of the complex of the receptor-binding domain of the endoribonucleolytic colicin E3 showed the elongate 100 Å long colicin domain to be bound in an oblique mode, in which it appears to be ‘fishing,’ for a second outer membrane receptor-translocator (Nature Structural Biology , 10, 948-954, 2003). A similar structure has been obtained with the receptor binding domain of colicin E2 (Sharma et al., JBC, 2007). Circular dichroism and electrophysiological studies imply that the colicin is first bound tightly to the extracellular surface of BtuB, is unfolded upon binding, and then uses another very abundant outer membrane protein, OmpF, for translocation (Biochemistry, 45:10199-10207, 2006, . Biol. Chem, J. Biol. Chem. 2007. 282: 23163-23170). It is proposed that colicin import across the outer membrane involves formation of a ‘translocon’ between BtuB and OmpF.  A 1.6 Å structure has recently been obtained for OmpF (Yamashita et al., in preparation, 2007). A structure of the BtuB vitamin B12 receptor has recently been obtained, in collaboration with M. Caffrey, by crystallization in the lipid cubic phase (Cherezov et al., 2006). At 1.95 Å resolution, it is the highest resolution structure of a membrane protein not related to bacteriorhodopsin to be obtained by the in meso approach to crystallization.

Alpha-Synuclein

α-Synuclein, a 140 amino acid cytosolic protein implicated in the pathogenesis of Parkinson's Disease (PD), can exert its cellular function through interaction with membranes. Studies that we have carried out with J. - C. Rochet showed that monomeric synuclein in an alpha-helical conformation can form specific ion channels in planar bilayer membranes (Zakharov et al., Biochemistry, 2007). These channels must result from the formation of a trans-membrane oligomer of defined structure. The synuclein channels could have a positive function in the metabolism of synaptic membranes.



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