Year of Award


Document Type


Degree Type

Doctor of Philosophy (PhD)

Degree Name


Other Degree Name/Area of Focus


Department or School/College

Department of Chemistry and Biochemistry

Committee Chair

J. B. Alexander Ross

Commitee Members

Bruce E. Bowler, Klara Briknarova, Michael DeGrandpre, Michele A. McGuirl


Aggregation, Denatured State, Protein Folding, Random Coil, TR-FRET


University of Montana


Various diseases result from protein misfolding. Curing these conditions requires understanding the principles governing folding. Efforts toward understanding how proteins fold have focused on the transition state rather than the earliest folding events. We study these initial events using the assumption that protein folding must involve the formation of the most primitive structure possible – a simple loop. Our laboratory has developed a system of studying simple loops in the denatured state using c-type cytochromes. New insights into how the properties of these loops impact the denatured state are outlined in this thesis. First, studies on a 22-residue loop revealed a previously unreported finding that equilibrium loop formation was not strongly affected by sequence composition. While loop formation rates depended only on sequence composition, loop breakage rates also depended on sequence order. Second, thermodynamic and kinetic studies on homopolymeric inserts in “poor” and “good” solvents revealed that homopolymeric non-foldable protein sequences behave like a random coil. However, heteropolymeric foldable sequences have scaling factors higher than those of a random coil, suggesting the presence of residual structure in denatured proteins. Thus, peptide models with homopolymeric sequences do not adequately describe the nature of foldable sequences. Third, we investigated the kinetics of reversible oligomerization in the denatured state using a P25A yeast iso-1-cytochrome c variant. The findings indicated that intermolecular aggregation in a denatured protein is extremely fast – 107-108 M-1s-1 and that the P25A mutation strongly affects intermolecular aggregation. This work suggests that equilibrium control of folding versus aggregation is advantageous for productive protein folding in vivo. Fourth, we use time-resolved FRET to follow compact and extended distributions of a protein under denaturing conditions. Our findings revealed three major populations in the unfolded state when no loop is present whereas only two populations remain when the loop forms. The most extended population is lost upon loop formation showing that simple loop formation dramatically constrains the denatured state. Thus, thermodyamic and kinetic studies on simple loops using a variety of spectroscopic techniques have enhanced understanding of the initial events of protein folding and the role of the denatured state in modulating protein aggregation.



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