The goal of this proposal is to elucidate the conformation of N-terminal polypeptides of increasing length (belonging to the sequence of an all-alpha-helical single domain model protein) in the presence of the cotranslationally active chaperone Hsp70. In order to obtain information at amino-acid specific resolution, multidimensional NMR in the presence of isotopically labeled peptide and unlabeled chaperone will be employed. Other biophysical methods such as isothermal titration calorimetry, size exclusion chromatography and fluorescence spectroscopy will also be used. The work focuses on elongating N-terminal polypeptides derived from apomyoglobin, a relatively small and extremely well characterized system which serves as an excellent model for all-alpha-helical proteins. The proposed investigations will explore whether Hsp70 merely prevents interchain aggregation by holding a statistical coil status, or it also acts by inducing specific polypeptide conformations. This study is not intended to directly mimic intracellular cotranslational and immediately posttranslational folding events. On the other hand, it aims at providing a first order in vitro approximation to how Hsp70 is able to affect the conformational space of elongating polypeptides. The expected influence of specific cell-related effects such as polypeptide tethering (to the ribosome exit channel) and molecular crowding are discussed in the proposal and will be addressed by separate additional experiments. Very little is known about the mechanisms by which Hsp70, the main cotranslationally active chaperone, influences the course of protein folding. Yet, progress is urgently needed in this area since defective action (or insufficient bioavailable amount) of cotranslational chaperones has been linked to the formation of incorrectly folded self-associated species such as those involved in a number of deadly diseases. These include cystic fibrosis, inflammatory heart disease, Crohn disease, P53-related cancers, and several neurodegenerative disorders such as Huntington's and Alzheimer's disease. Experiments to be carried out include (a) high resolution secondary structure mapping of isotopically labeled polypeptides by NMR in the presence of unlabeled Hsp70 chaperone; (b) hydrogen/deuterium exchange pulse labeling kinetic experiments to detect the mechanisms of structure formation in the presence of Hsp70; (c) additional studies in the presence of the Hsp40 and Hsp70-nucleotide exchange factor cochaperones.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM068535-05S1
Application #
7650506
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Wehrle, Janna P
Project Start
2004-04-01
Project End
2010-03-31
Budget Start
2008-04-01
Budget End
2010-03-31
Support Year
5
Fiscal Year
2008
Total Cost
$24,491
Indirect Cost
Name
University of Wisconsin Madison
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Fedyukina, Daria V; Cavagnero, Silvia (2011) Protein folding at the exit tunnel. Annu Rev Biophys 40:337-59
Sekhar, Ashok; Cavagnero, Silvia (2009) 1H photo-CIDNP enhancements in heteronuclear correlation NMR spectroscopy. J Phys Chem B 113:8310-8
Sekhar, Ashok; Cavagnero, Silvia (2009) EPIC- and CHANCE-HSQC: two 15N-photo-CIDNP-enhanced pulse sequences for the sensitive detection of solvent-exposed tryptophan. J Magn Reson 200:207-13
Chen, Zhongjing; Kurt, Nese; Rajagopalan, Senapathy et al. (2006) Secondary structure mapping of DnaK-bound protein fragments: chain helicity and local helix unwinding at the binding site. Biochemistry 45:12325-33
Vega, Carolina A; Kurt, Nese; Chen, Zhongjing et al. (2006) Binding specificity of an alpha-helical protein sequence to a full-length Hsp70 chaperone and its minimal substrate-binding domain. Biochemistry 45:13835-46
Kurt, Nese; Rajagopalan, Senapathy; Cavagnero, Silvia (2006) Effect of hsp70 chaperone on the folding and misfolding of polypeptides modeling an elongating protein chain. J Mol Biol 355:809-20
Cavagnero, Silvia; Jungbauer, Lisa M (2005) Painting protein misfolding in the cell in real time with an atomic-scale brush. Trends Biotechnol 23:157-62
Kurt, Nese; Cavagnero, Silvia (2005) The burial of solvent-accessible surface area is a predictor of polypeptide folding and misfolding as a function of chain elongation. J Am Chem Soc 127:15690-1