The structural basis for the conversion of chemical energy to mechanical movements is a major unresolved problem of muscle contraction and myosin based motility. Using the newly developed structural method of cryo atomic force microscopy, which can provide nm resolution with macromolecular complexes without metal coating, we focus on three major aspects of smooth muscle contraction in this project. The first objective is to search for the predicted structural changes and rearrangements in both isolated single myosin molecules and actomyosin complexes, under the conditions corresponding to each major biochemical (ATP hydrolysis) event during the crossbridge cycle.
We aim at obtaining structural information at 2 nm or higher without using averaging methods, and elucidating the crossbridge cycle at the molecular level. The second objective is the interaction of smooth muscle phosphatase with myosin, where the binding stoichiometry will be examine. These studies are expected to provide a structural basis for understanding the function of this important enzyme in smooth muscle regulation. The third objective is the structure of smooth muscle thin filament which plays a pivotal role in the regulation of contraction, yet, very little information is available about its structure, especially when other regulatory components, such as caldesmon and calponin, are present. The problems to be studied here have been central for our understanding of muscle contraction, and have not been resolved, partly because of the limitations of available structural methods. This is the first attempt to apply the cryo atomic force microscopic method to a well defined biological problem, and many of the current technical limitations can be circumvented, providing a unique opportunity to explore the basic properties of mice. In combination with well established smooth muscle physiology, this new approach is well poised to make fundamental contributions to muscle contraction.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Program Projects (P01)
Project #
5P01HL048807-08
Application #
6327730
Study Section
Project Start
2000-07-01
Project End
2001-06-30
Budget Start
1997-10-01
Budget End
1998-09-30
Support Year
8
Fiscal Year
2000
Total Cost
$187,536
Indirect Cost
Name
University of Virginia
Department
Type
DUNS #
001910777
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
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

Showing the most recent 10 out of 107 publications