Cofactors may be important determinants, acting as nucleation points limiting the conformational search, for the folding rates of cofactor-binding proteins. To date, folding kinetics of proteins with b-sheet structure has not been as thoroughly investigated as for helical proteins. This research program aims towards probing the role of two inorganic and one organic cofactor in the folding of three proteins with mostly b-sheet structure. The targeted proteins are azurin, a b-barrel protein with a copper-ion cofactor, flavodoxin, a protein with an a/b doubly-wound topology coordinating an organic flavin mononucleotide (FMN), and cytochrome f, a b-sheet protein covalently linked to a heme. Equilibrium biophysical characterization (circular dichroism, fluorescence, absorption, EXAFS, NMR, and various biochemical methods) will aid in revealing the effect of each cofactor on its corresponding protein stability and unfolded polypeptide structure. A recent technique in which folding is initiated by photochemical electron-transfer will be used to probe rapid events during formation of the native-states of the proteins. To allow wide denaturant- and time- ranges to be investigated (and to study the apo proteins), time-resolved experiments will also be performed using stopped-flow mixing.
The specific aims are to: 1. Characterize cofactor- coordination and residual structures created by the cofactors in the unfolded states, 2. Investigate how copper, FMN and heme (bound to the unfolded polypeptides) affect the polypeptide folding kinetics and, finally, 3. Probe early events (starting on the us time scale) during the formation -of native azurin, flavodoxin and cytochrome.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM059663-01A2
Application #
6254770
Study Section
Metallobiochemistry Study Section (BMT)
Program Officer
Wehrle, Janna P
Project Start
2000-12-18
Project End
2005-11-30
Budget Start
2000-12-18
Budget End
2001-11-30
Support Year
1
Fiscal Year
2001
Total Cost
$157,180
Indirect Cost
Name
Tulane University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
City
New Orleans
State
LA
Country
United States
Zip Code
70118
Zong, Chenghang; Wilson, Corey J; Shen, Tongye et al. (2007) Establishing the entatic state in folding metallated Pseudomonas aeruginosa azurin. Proc Natl Acad Sci U S A 104:3159-64
Luke, Kathryn A; Higgins, Catherine L; Wittung-Stafshede, Pernilla (2007) Thermodynamic stability and folding of proteins from hyperthermophilic organisms. FEBS J 274:4023-33
Zong, Chenghang; Wilson, Corey J; Shen, Tongye et al. (2006) Phi-value analysis of apo-azurin folding: comparison between experiment and theory. Biochemistry 45:6458-66
Luke, Kathryn; Wittung-Stafshede, Pernilla (2006) Folding and assembly pathways of co-chaperonin proteins 10: Origin of bacterial thermostability. Arch Biochem Biophys 456:8-18
Wilson, Corey J; Apiyo, David; Wittung-Stafshede, Pernilla (2006) Solvation of the folding-transition state in Pseudomonas aeruginosa azurin is modulated by metal: Solvation of azurin's folding nucleus. Protein Sci 15:843-52
Muralidhara, B K; Rathinakumar, Ramesh; Wittung-Stafshede, Pernilla (2006) Folding of Desulfovibrio desulfuricans flavodoxin is accelerated by cofactor fly-casting. Arch Biochem Biophys 451:51-8
Luke, Kathryn; Perham, Michael; Wittung-Stafshede, Pernilla (2006) Kinetic folding and assembly mechanisms differ for two homologous heptamers. J Mol Biol 363:729-42
de los Rios, Miguel A; Muralidhara, B K; Wildes, David et al. (2006) On the precision of experimentally determined protein folding rates and phi-values. Protein Sci 15:553-63
Chen, Mingzhi; Wilson, Corey J; Wu, Yinghao et al. (2006) Correlation between protein stability cores and protein folding kinetics: a case study on Pseudomonas aeruginosa apo-azurin. Structure 14:1401-10
Perham, Michael; Liao, Jue; Wittung-Stafshede, Pernilla (2006) Differential effects of alcohols on conformational switchovers in alpha-helical and beta-sheet protein models. Biochemistry 45:7740-9

Showing the most recent 10 out of 49 publications