Stability and Dynamics Studies of Apo-Azurin from Pseudomonas aeruginosa

Author: Luo, Ji-Ye

Year: 1997

Degree: Dissertation (Ph.D.)

Advisor: Mayo, Stephen L.

Committee Members: Chan, Sunney I.; Richards, John H.; Rees, Douglas C.; Mayo, Stephen L.

Option: Chemistry; Biology

DOI: 10.7907/je80-hr40

Abstract

The solution stability and dynamics of apo-azurin from Pseudomonas aeruginosa have been studied. First, the unfolding stability was measured for 20 apo-azurin mutants mutating at T124 in the context of a β-sheet host-guest environment. The unfolding free energy difference was taken as a measure of the relative β-sheet propensity for the 20 naturally occurring amino acids. These data, when combined with data from other experimental studies and compared to a statistical analysis of the protein structure data base, strongly support intrinsic secondary structure preference as the major determinant of β-sheet propensity. Particularly, residues Val, Thr, Ile, Phe, and Tyr as a group are intrinsically favored for β-sheet formation, and residues Pro, Gly, and Asp as a group are intrinsically disfavored. The relative contribution of the intrinsic propensity and local context to the β-sheet formation was further discussed with the results from mutating S34 to Val and Thr. The S34T mutant was more stable than the wild-type protein, but the S34V mutant was less stable. Secondly, partially perturbed states of different forms of azurin at low pH's were observed. Apo-azurin at pH 2.9 is a molten globule-like state, while holo-azurin at pH's as low as 2.6 is only partially perturbed. Third, backbone 1H and 15N chemical shift assignments and solution dynamics of apo-azurin were studied. The overall correlation time of apo-azurin at 30 °C was determined to be 5.8 ns, and order parameters were mostly at 0.8-0.95. Residues around the Cu(II) binding site exhibited low order parameters and significant 1H chemical shift difference from those of holo-azurin. Last, the osmolyte stabilizing effect was studied on RNase A using Hydrogen/Deuterium exchange. Two types of H/D exchange behavior were observed. The H/D exchange rates of type I residues become slower in the presence of high concentrations of glycine, as expected from the global stability change; the H/D exchange rates of type II residues are not affected, however.

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