Intermediate filaments (IPs) are present in essentially every human cell. However, the inability to achieve crystal structure for intact IF proteins or IFs means that our understanding of IF structure is based in large part on predictions made from primary sequence and from limited cross- linking studies. Thus, most features of commonly presented models of IF structure are experimentally untested, or based on data which is open to alternative interpretation. This places serious limits on our ability to understand normal IF function, and the changes in IF assembly and structure that are caused by the many disease-causing mutations that have been identified in human IF genes. We have demonstrated that site directed spin labeling and electron paramagnetic resonance can provide excellent structural information from intact IFs in physiologic conditions. We propose here to conduct an exhaustive study of the Type III IF protein vimentin, assembling the first comprehensive map of an intact intermediate filament. We will then conduct similar experiments on another Type III IF protein, desmin, and on cytokeratin IFs made from K8 and K18 to determine the degree to which IF assembly and structure are conserved among the three most common forms of intermediate filaments. These studies will map the location and boundaries of alpha-helical, coiled-coil domains, linker regions, the conserved """"""""stutter"""""""" found in all IF proteins, the boundaries of the rod domain, the surfaces of apposition between monomers in dimers, and between dinners in intact filaments, the registry and orientation of these dimers, and the sites of IF proteins that are available at the surface of the IF for interactions with other cellular proteins/structures. Sites within the head and tail domains that interact with one another, and with the rod domain will be mapped as well. Finally, we will explore the impact of known disease-causing mutations on IF assembly and structure.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY015560-05
Application #
7769871
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Araj, Houmam H
Project Start
2006-04-01
Project End
2011-12-31
Budget Start
2010-03-01
Budget End
2011-12-31
Support Year
5
Fiscal Year
2010
Total Cost
$292,232
Indirect Cost
Name
University of California Davis
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
047120084
City
Davis
State
CA
Country
United States
Zip Code
95618
Liu, Lishan; Hess, John; Sahu, Indra D et al. (2016) Probing the Local Secondary Structure of Human Vimentin with Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy. J Phys Chem B 120:12321-12326
Hess, John F; Budamagunta, Madhu S; Aziz, Atya et al. (2013) Electron paramagnetic resonance analysis of the vimentin tail domain reveals points of order in a largely disordered region and conformational adaptation upon filament assembly. Protein Sci 22:47-55
Aziz, Atya; Hess, John F; Budamagunta, Madhu S et al. (2012) The structure of vimentin linker 1 and rod 1B domains characterized by site-directed spin-labeling electron paramagnetic resonance (SDSL-EPR) and X-ray crystallography. J Biol Chem 287:28349-61
Yoon, Kyoung-hye; FitzGerald, Paul G (2009) Periplakin interactions with lens intermediate and beaded filaments. Invest Ophthalmol Vis Sci 50:1283-9
Pittenger, Josh T; Hess, John F; Budamagunta, Madhu S et al. (2008) Identification of phosphorylation-induced changes in vimentin intermediate filaments by site-directed spin labeling and electron paramagnetic resonance. Biochemistry 47:10863-70
Pittenger, Joshua T; Hess, John F; Fitzgerald, Paul G (2007) Identifying the role of specific motifs in the lens fiber cell specific intermediate filament phakosin. Invest Ophthalmol Vis Sci 48:5132-41
Hess, John F; Budamagunta, Madhu S; Shipman, Rebecca L et al. (2006) Characterization of the linker 2 region in human vimentin using site-directed spin labeling and electron paramagnetic resonance. Biochemistry 45:11737-43