Actin and tubulin play a central role in motility and the control of cell form. Given the crucial role of these proteins in both health and disease, the quaternary structures of actin and tubulin are important, since structural information can elucidate many questions of function. Specifically, this proposal is aimed at understanding the conformational changes that accompany actin polymerization and the hydrolysis of ATP, and the macromolecular organization of tubulin-tektin ribbons that appear to be key determinants of axonemal doublet microtubules. The main techniques that we will be used are electron microscopy and image analysis. An atomic structure for the actin monomer provides the basis for most of the actin research that is proposed. It is clear that the technique of combining x-ray and EM data will become an increasingly important tool in understanding the structure of complex macromolecular polymers and assemblies. Actin is a dynamic system, both in conformational changes that must occur within the protein during assembly as well as in terms of the ability of the assembled filament to undergo flexing and torsion. The techniques of electron microscopy and image analysis can be very useful in deriving dynamic information from static images. The application of these methods to actin should be able to shed light on the molecular motions that result from the hydrolysis of ATP after filament assembly, as well as on factors that appear to modulate the torsional and flexural rigidity of actin. However, electron microscopy cannot directly establish the time scales on which such motions occur, and collaborative spectroscopic studies to obtain this information are planned. While no crystal structure exists for tubulin, the proposed project on the tubulin-tektin ribbons, isolated from axonemes, stands on its own in the absence of high-resolution structures. The quaternary structure will be determined of these """"""""ribbons"""""""", and such information will be of great relevance to understanding the assembly of the complicated axoneme which contains over 100 different proteins. Further, such studies may help explicate the structural organization of tektin within these ribbons, and provide a model for the folding of this protein family.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR042023-04
Application #
2006300
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
1993-12-01
Project End
1997-11-30
Budget Start
1996-12-01
Budget End
1997-11-30
Support Year
4
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
168559177
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
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Shvetsov, Alexander; Galkin, Vitold E; Orlova, Albina et al. (2008) Actin hydrophobic loop 262-274 and filament nucleation and elongation. J Mol Biol 375:793-801
Benchaar, Sabrina A; Xie, Yongming; Phillips, Martin et al. (2007) Mapping the interaction of cofilin with subdomain 2 on actin. Biochemistry 46:225-33
Cherepanova, Olga; Orlova, Albina; Galkin, Vitold E et al. (2006) Xin-repeats and nebulin-like repeats bind to F-actin in a similar manner. J Mol Biol 356:714-23
Kudryashov, D S; Galkin, V E; Orlova, A et al. (2006) Cofilin cross-bridges adjacent actin protomers and replaces part of the longitudinal F-actin interface. J Mol Biol 358:785-97
Galkin, Vitold E; Orlova, Albina; Fattoum, Abdellatif et al. (2006) The CH-domain of calponin does not determine the modes of calponin binding to F-actin. J Mol Biol 359:478-85
Galkin, Vitold E; Orlova, Albina; Koleske, Anthony J et al. (2005) The Arg non-receptor tyrosine kinase modifies F-actin structure. J Mol Biol 346:565-75
Galkin, Vitold E; Orlova, Albina; Lukoyanova, Natalya et al. (2003) The location of ubiquitin in Lethocerus arthrin. J Mol Biol 325:623-8
Galkin, Vitold E; Orlova, Albina; VanLoock, Margaret S et al. (2003) Do the utrophin tandem calponin homology domains bind F-actin in a compact or extended conformation? J Mol Biol 331:967-72
Galkin, Vitold E; Orlova, Albina; VanLoock, Margaret S et al. (2002) The utrophin actin-binding domain binds F-actin in two different modes: implications for the spectrin superfamily of proteins. J Cell Biol 157:243-51

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