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DUP ATP Synthase: What Does It Take to Make a Rotary Enzyme? Stan Dunn Department of Biochemistry Schulich School of Medicine & Dentistry 24 April 2013.

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Presentation on theme: "DUP ATP Synthase: What Does It Take to Make a Rotary Enzyme? Stan Dunn Department of Biochemistry Schulich School of Medicine & Dentistry 24 April 2013."— Presentation transcript:

1 DUP ATP Synthase: What Does It Take to Make a Rotary Enzyme? Stan Dunn Department of Biochemistry Schulich School of Medicine & Dentistry 24 April 2013

2 DUP ATP (Adenosine TriPhosphate) Is the Primary Biological Energy Currency Cell maintenance, protein and nucleic acid biosynthesis Active transport Muscle contraction Food CO 2 and H 2 O Oxidative phosphorylation

3 DUP The Mechanism of Oxidative Phosphorylation Respiratory Complexes I, III, IV ATP synthase, aka Complex V 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 + P i into ATP

4 DUP F-ATP Synthase: two motors sharing one driveshaft/rotor F 1 peripheral ATP synthesis/ hydrolysis chemical rotary motor ADP + P i ATP The stator: 3 3 ab 2 The stator stalk: b 2 F o integral ab 2 c 10 H + conduction electrical rotary motor C 10 b2b2 a H+H+ H+H+ The rotor: c 10 A common driveshaft Image courtesy of Achim Weber

5 DUP Analysis of E. coli b sol Dimer Highly extended, not globular Binds to F 1 sector, through subunit Highly -helical Sequence shows heptad repeat pattern So we expected b sol to form a left-handed coiled coil 1 23 53 122 156 Membrane Tether Dimerization F 1 -Binding b sol Properties of b sol Left-handed coiled coil

6 DUP Arrangement of helices in dimerization domain Disulfide formation between introduced cysteine residues Our conclusion: b 2 forms an atypical coiled coil 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 Ala to Cys mutation changes the side chain from –CH 3 to –CH 2 SH

7 DUP Troubles are good for you. - Efraim Racker, Cornell University

8 DUP Work hard, dont have fun, and save your money for your old age. - Leon Heppel, Cornell University

9 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

10 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?

11 DUP b C b N i 68(h) x 72(a) ii 72(a) x 79(h) iii 79(h) x 83(a) iv 83(a) x 90(h) Offset right-handed coiled coil model of b These disulfide-linked forms had shapes and stabilities characteristic of native proteins They also interacted strongly with F 1 -ATPase

12 DUP Structure of Thermus thermophilus EG Danielle Stock and co-workers, NSMB 2010 alanine zipper

13 DUP Functional relevance of right-handedness of b 2 : structural stabilization to torque on stator

14 DUP Acknowledgments Yumin Bi Paul Del Rizzo Derek McLachlin Greg Gloor Brian Shilton Graduate students Ardy Goliaei Kristi Wood Dan Cipriano Lee-Ann Briere Matt Revington Undergraduates Carla Busnello Karen Dunkerley Nancy Wang Kevin Talbott Chelsea Botsford Stahs Pripotnev Western James Choy Gary Shaw Greg Gloor Eric Ball Lindi Wahl Mike Strong Canada Quim Madrenas Liz Meiering International Brian Cain Stephan Wilkens Wolfgang Junge Michael B ӧ rsch Gabriele Deckers- Hebestreit Paola Turina Masamitsu Futai

15 DUP Does b 2 have a function beyond simply holding F 1 on the membrane?

16 DUP What is the structure of ATP synthase and how does it utilize an H + gradient to make ATP? Electron micrograph of mitochondrial membranes Purified E. coli ATP synthase

17 DUP Right-handed vs. left-handed coiled coils Characterized by heptad pattern where a, d positions are usually hydrophobic, knobs-into-holes packing of side chains, in-register helices Expected to have a hendecad (11-residue) pattern, but such a structure had never been seen, it was only theoretical!

18 DUP MSA and periodicity search by Fourier transform using REPPER FTwin support 11-residue repeat Peak periodicity is at 3.68 (11 residues/3 turns of helix, right-handed) Left-handed periodicity would be 3.5 (7 residues/2 turns of helix)

19 DUP Mitochondria are derived from a bacterium Mitochondria: subcellular organelles Escherichia coli: a bacterial model

20 DUP Stalk structures in distantly related ATPases Archaeal A-type Kish-Trier and Wilkens, JBC 2009 Eukaryotic V-type Zhang et al., JBC, 2008

21 DUP How stiff is the stator stalk relative to the rotor? (collaboration with Wolfgang Junge) Single molecule analysis -motions of parts of enzyme were constrained by engineered disulfide bonds -a short fluorescently labeled actin filament or magnetic bead was attached -torsional stiffness ( ) of various sections was determined by observing thermal fluctuations of the bead or filament Normally this setup will measure the torsional stiffness of the rotor; when a disulfide link is made between the a subunit and the c ring, b 2 will restrict the fluctuations

22 DUP The Results The Results

23 DUP Glycolysis occurs in cytoplasm, generates a little bit of ATP Citric acid cycle and oxidative phosphorylation occur in mitochondria, generate a lot of ATP through ATP Synthase ATP Synthase

24 DUP Functional effects of softening the stator stalk I Oxidative phosphorylation in vivo

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