Year of Award


Document Type


Degree Type

Master of Science (MS)

Degree Name

Biochemistry & Biophysics

Department or School/College

Biochemistry and Biophysics Program

Committee Chair

Bruce Bowler

Commitee Members

J. Stephen Lodmell, Klara Briknarova


cytochrome c', residual structure


University of Montana


To expand our understanding of helical propensity and residual structure in the denatured state, two approaches have been taken. In the first project, we have engineered serine in place of alanine near the center of the third helix (positions 83 and 87) in cytochrome c’ (Cytc’) and have measured histidine-heme loop formation in 3 M gdnHCl. A series of thirteen variants that include the A83S/A87S substitutions have been engineered with single surface histidine substitutions to provide a range of His-heme loop sizes from 10 to 111 residues and to provide direct comparison to previous studies with pWT Cytc’ . We observe decreased global stability for most variants and an average decrease of the midpoint of gdnHCl unfolding for A83S/A87S variants compared to pWT. Loop stability versus loop size data yields a scaling exponent of 2.24 ± 0.24, similar to the pWT value of 2.5 ± 0.3, but with a crossover point and values that suggest that loop flexibility and stability increase in loops that contain the A83S/A87S substitutions. Kinetic data shows nonrandom behavior in the denatured state similar to pWT, and also supports the suggestion of helical propensity only being important if the helical segment is contained within the loop, as kf values are slightly faster for loops containing A83S/A87S. Molecular dynamic simulations of the thermal unfolding of Cytc’ show a perhaps surprising amount of structure retention in the third helix, even with the helical propensity lowering A83S/A87S substitutions. In the second project, we combine NMR methods with chemical shift secondary structure analysis on the iso-1 cytochrome c K54H variant in 3 and 6 M gdnHCl. We consider two pH conditions, pH 6.3 where the His54-heme loop is formed and pH 3.6 where the His54-heme loop is broken. A method designed to estimate the secondary structure propensities quantitatively was used to process chemical shift data obtained for each residue. Regions of residual structure were identified in 3 M gdnHCl, a condition at which the protein is fully denatured, as well as in the very harsh denaturant condition of 6 M gdnHCl. The data presented here may contribute to the identification of residues and structural behavior involved in early folding which could lead to better understanding of protein folding pathways.



© Copyright 2013 Travis Arthur Danielson