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ATP Synthase: What Does It Take to Make a Rotary Enzyme? Stan Dunn Department of Biochemistry Schulich School of Medicine & Dentistry 24 April 2013 DUP ATP (Adenosine TriPhosphate) Is the Primary Biological “Energy Currency” Food Cell maintenance, protein and nucleic acid biosynthesis Active transport Oxidative phosphorylation Muscle contraction CO2 and H2O DUP The Mechanism of Oxidative Phosphorylation Respiratory complexes use the energy released during the oxidation of food to drive H+ ions across the inner mitochondrial membrane, producing an electrochemical H+ gradient containing potential energy; ATP synthase allows the H+ to return, using the released energy to convert ADP + Pi into ATP Respiratory Complexes I, III, IV ATP synthase, aka Complex V DUP F-ATP Synthase: two motors sharing one driveshaft/rotor d F1 • peripheral • a3b3gde • ATP synthesis/ hydrolysis •chemical rotary motor Fo a b2 a b ADP + Pi The stator: a3b3gdab2 ATP g H+ • integral •ab2c10 •H+ conduction •electrical rotary motor The stator stalk: b2d e C10 The rotor: gec10 A common driveshaft Image courtesy of Achim Weber a H+ DUP Analysis of E. coli bsol 1 23 Membrane 53 Tether 122 Dimerization 156 F1-Binding bsol Properties of bsol • • • • • Dimer Highly extended, not globular Binds to F1 sector, through d subunit Highly a-helical Sequence shows heptad repeat pattern So we expected bsol to form a left-handed coiled coil Left-handed coiled coil DUP Arrangement of helices in dimerization domain Disulfide formation between introduced cysteine residues Ala to Cys mutation changes the side chain from –CH3 to –CH2SH Heptad: b c d e f g a b c d Residue: 59 60 61 62 63 64 65 66 67 68 WT res: A S A T D Q L K K A Our conclusion: b2 forms ”an atypical coiled coil” DUP “Troubles are good for you.” -Efraim Racker, Cornell University DUP “Work hard, don’t have fun, and save your money for your old age.” -Leon Heppel, Cornell University DUP The dimerization domain of b •Dimer in solution, but monomer in crystal (crystallization from MPD/isopropanol/water) •Panel A shows hydrophobic face with alanines in orange, branched aliphatics in green. •Panel B highlights alanines along the face •Panel C shows a model dimerized about this face: this will produce a right-handed coiled coil DUP Selective disulfide bond formation between a and h positions Observation: preferential disulfide formation between an (a) position from one helix and the (h) from the adjacent helix. This result implies that (a) & (h) positions are at the helix-helix interface … but how can 79 link to 72 and 83? DUP Offset right-handed coiled coil model of b bC bN i 68(h) x 72(a) ii 72(a) x 79(h) iii 79(h) x 83(a) iv 83(a) x 90(h) bC bN A79(h) A83(a) iii These disulfide-linked forms had shapes and stabilities characteristic of native proteins They also interacted strongly with F1-ATPase A79(h) A72(a) ii A68(h) i A72(a) DUP Structure of Thermus thermophilus EG alanine zipper Danielle Stock and co-workers, NSMB 2010 DUP Functional relevance of right-handedness of b2: structural stabilization to torque on stator DUP Acknowledgments Derek McLachlin Paul Del Rizzo Graduate students Ardy Goliaei Kristi Wood Dan Cipriano Lee-Ann Briere Matt Revington Canada Quim Madrenas Liz Meiering Yumin Bi Undergraduates Carla Busnello Karen Dunkerley Nancy Wang Kevin Talbott Chelsea Botsford Stahs Pripotnev Brian Shilton Greg Gloor International Western Brian Cain James Choy Gary Shaw Stephan Wilkens Wolfgang Junge Greg Gloor Michael Bӧrsch Eric Ball Lindi Wahl Gabriele DeckersHebestreit Mike Strong Paola Turina Masamitsu Futai DUP