Protein denatured states remain poorly understood and yet, as the starting point for protein folding, an understanding of the conformational constraints, acting upon the denatured state, is central to solving the problem of how a protein folds efficiently. Misfolding diseases, such as Alzheimer's and Parkinson's diseases are major health problems, where the causative agents are believed to be non-native and denatured states of proteins. Thus, fundamental research on denatured proteins is essential to new insight into the genesis of these disease states. This laboratory has developed a novel strategy to probe the conformational and thermodynamic properties of unfolded proteins. The propensity for forming loops of different sizes is assessed through histidine-heme loop equilibria. Single surface histidine variants have been produced in yeast iso-1-cytochrome c, allowing loop equilibria for loops of 9 to 83 amino acids to be measured under denaturing conditions. Formation of closed loops are required in the earliest stages of structure accretion when a protein folds. This system has already yielded important insights into the deviation of protein denatured states from random coil behavior. Several key properties of unfolded proteins will be probed with this system. To understand how residual structure affects contact probability in a denatured protein, we will stabilize residual structure with disulfide crosslinks, insert a known stable beta hairpin into iso-1-cytochrome c, and apply our methodology to cytochrome c', which is know to have a much more compact denatured state than iso-1-cytochrome c (specific aim 1). The effect of sequence composition on denatured state conformational properties will be probed by inserting homopolymeric arnino acid sequences into the disordered N-terminal region of iso-1-cytochrome c (specific aim 2), with emphasis on the properties of the flexible amino acid glycine and the rigid amino acid proline. Kinetics studies on loop formation and breakage are planned to probe how loop size, denatured state compactness and residual structure impact the rate at which denatured state contacts form and the factors which cause contacts to persist (specific aim 3). NMR and FRET methods will be used to correlate denatured state thermodynamic with denatured state structural properties (specific aim 4). Our multipronged approach probes key parameters of protein denatured states, expected to be principal modulators of early events in protein folding. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM074750-01
Application #
6956116
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Basavappa, Ravi
Project Start
2005-09-10
Project End
2006-12-31
Budget Start
2005-09-10
Budget End
2006-12-31
Support Year
1
Fiscal Year
2005
Total Cost
$211,650
Indirect Cost
Name
University of Denver
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
007431760
City
Denver
State
CO
Country
United States
Zip Code
80208
Leavens, Moses J; Cherney, Melisa M; Finnegan, Michaela L et al. (2018) Probing Denatured State Conformational Bias in a Three-Helix Bundle, UBA(2), Using a Cytochrome c Fusion Protein. Biochemistry 57:1711-1721
Danielson, Travis A; Bowler, Bruce E (2018) Helical Propensity Affects the Conformational Properties of the Denatured State of Cytochrome c'. Biophys J 114:311-322
Danielson, Travis A; Stine, Jessica M; Dar, Tanveer A et al. (2017) Effect of an Imposed Contact on Secondary Structure in the Denatured State of Yeast Iso-1-cytochrome c. Biochemistry 56:6662-6676
Khan, Md Khurshid Alam; Bowler, Bruce E (2012) Conformational properties of polyglutamine sequences in guanidine hydrochloride solutions. Biophys J 103:1989-99
Bowler, Bruce E (2012) Residual structure in unfolded proteins. Curr Opin Struct Biol 22:4-13
Khan, Md Khurshid A; Miller, Abbigail L; Bowler, Bruce E (2012) Tryptophan stabilizes His-heme loops in the denatured state only when it is near a loop end. Biochemistry 51:3586-95
Finnegan, Michaela L; Bowler, Bruce E (2012) Scaling properties of glycine-rich sequences in guanidine hydrochloride solutions. Biophys J 102:1969-78
Dar, Tanveer A; Schaeffer, R Dustin; Daggett, Valerie et al. (2011) Manifestations of native topology in the denatured state ensemble of Rhodopseudomonas palustris cytochrome c'. Biochemistry 50:1029-41
Tzul, Franco O; Bowler, Bruce E (2010) Denatured states of low-complexity polypeptide sequences differ dramatically from those of foldable sequences. Proc Natl Acad Sci U S A 107:11364-9
Finnegan, Michaela L; Bowler, Bruce E (2010) Propensities of aromatic amino acids versus leucine and proline to induce residual structure in the denatured-state ensemble of iso-1-cytochrome c. J Mol Biol 403:495-504

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