Abstract

The objective of this Proposal is to relate the mechanism of Ca2+ transport in cardiac sarcoplasmic reticulum [SR] to the physical structure, both of Ca2+-ATPase and its regulatory protein, phospholamban [PLB]. Two different microscopies will be used to address rather different aspects of molecular structure. To study the structure of PLB, 2-D cocrystals containing both PLB and Ca2+-ATPase will be made either from native cardiac SR or from a reconstituted system. Crystallization will be induced by vanadate, which has previously been shown to make crystals in cardiac SR and which has been used for 3-D reconstruction of skeletal Ca2+-ATPase. The presence of PLB in cocrystals will be verified by immunogold labelling and, after iodination, by elemental mapping techniques developed by Project 5. Cocrystals will then be rapidly frozen and imaged by frozen-hydrated electron microscopy. These images will be used for 3-D reconstruction at ~15Angstroms resolution, which will allow visualization (a) of the structure of cardiac Ca2+-ATPase, (b) of the oligomeric state of PLB, and (c) of their interaction. To study the dynamics of Ca2+-ATPase itself, atomic force microscopy [AFM] will be used to image Ca2+ATPase under a variety of hydrated conditions known to stabilize different conformational states. Particular conditions will stabilize five different reaction intermediates and include: (a) the presence and absence of Ca2+, (b) phosphorylation by inorganic phosphate or incubation with vanadate, and (c) phosphorylation with either CrATP or Co(NH3)4PO4. A number of specimen preparation methods, which will be developed as part of Project 2, will be used. Crystalline specimens will be imaged, first: after Fourier averaging and comparison with crystal structures from electron microscopy, these images will be used to familiarize us with the surface structure of Ca2+-ATPase. Thereafter, non-crystalline specimens will be imaged at ~10Angstroms resolution, and comparisons amongst the various non- crystalline conditions will allow us to link the reaction cycle to specific shape changes in Ca2+-ATPase, and thus, to construct a physical model for the mechanism of Ca2+ transport.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Program Projects (P01)
Project #
5P01HL048807-05
Application #
2346713
Study Section
Project Start
Project End
Budget Start
1995-10-01
Budget End
1996-09-30
Support Year
5
Fiscal Year
1996
Total Cost
Indirect Cost

  Institution

Name
University of Virginia
Department
Type
DUNS #
001910777
City
Charlottesville
State
VA
Country
United States
Zip Code
22904

  Publications

Utepbergenov, Darkhan; Derewenda, Urszula; Olekhnovich, Natalya et al. (2012) Insights into the inhibition of the p90 ribosomal S6 kinase (RSK) by the flavonol glycoside SL0101 from the 1.5 Å crystal structure of the N-terminal domain of RSK2 with bound inhibitor. Biochemistry 51:6499-510
Hoofnagle, Mark H; Neppl, Ronald L; Berzin, Erica L et al. (2011) Myocardin is differentially required for the development of smooth muscle cells and cardiomyocytes. Am J Physiol Heart Circ Physiol 300:H1707-21
Jin, Li; Gan, Qiong; Zieba, Bartosz J et al. (2010) The actin associated protein palladin is important for the early smooth muscle cell differentiation. PLoS One 5:e12823
Khromov, Alexander; Choudhury, Nandini; Stevenson, Andra S et al. (2009) Phosphorylation-dependent autoinhibition of myosin light chain phosphatase accounts for Ca2+ sensitization force of smooth muscle contraction. J Biol Chem 284:21569-79
Jin, Li; Hastings, Nicole E; Blackman, Brett R et al. (2009) Mechanical properties of the extracellular matrix alter expression of smooth muscle protein LPP and its partner palladin; relationship to early atherosclerosis and vascular injury. J Muscle Res Cell Motil 30:41-55
Cierpicki, Tomasz; Bielnicki, Jakub; Zheng, Meiying et al. (2009) The solution structure and dynamics of the DH-PH module of PDZRhoGEF in isolation and in complex with nucleotide-free RhoA. Protein Sci 18:2067-79
Jin, Li; Yoshida, Tadashi; Ho, Ruoya et al. (2009) The actin-associated protein Palladin is required for development of normal contractile properties of smooth muscle cells derived from embryoid bodies. J Biol Chem 284:2121-30
Zheng, Meiying; Cierpicki, Tomasz; Momotani, Ko et al. (2009) On the mechanism of autoinhibition of the RhoA-specific nucleotide exchange factor PDZRhoGEF. BMC Struct Biol 9:36
Jelen, Filip; Lachowicz, Pawel; Apostoluk, Wlodzimierz et al. (2009) Dissecting the thermodynamics of GAP-RhoA interactions. J Struct Biol 165:10-8
Freitas, Maria Regina; Eto, Masumi; Kirkbride, Jason A et al. (2009) Y27632, a Rho-activated kinase inhibitor, normalizes dysregulation in alpha1-adrenergic receptor-induced contraction of Lyon hypertensive rat artery smooth muscle. Fundam Clin Pharmacol 23:169-78

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