Date of Completion

5-8-2013

Embargo Period

5-8-2014

Advisors

Carolyn Teschke, Arlene Albert

Field of Study

Molecular and Cell Biology

Degree

Master of Science

Open Access

Open Access

Abstract

The bacteriophage P22 I-domain structure contains a 6-stranded b-barrel folding motif, a 3-stranded b-sheet, and a small a-helical region. This study describes the dynamics of the I-domain determined by 15N NMR relaxation and native-state hydrogen exchange experiments. Relaxation data suggests that the I-domain is experiencing the majority of its motion on the ps-ns timescale. S2 order parameters show two loop regions, which are poorly defined in the NMR structure, are the most flexible regions. The thermodynamic stability of the I-domain to unfolding, as a function of denaturant, has been studied with circular dichroism (CD) and native-state hydrogen exchange (nsHX) experiments. The CD experiments show ∆Gspec values for unfolding to be about 6.2 kcal/mol at pH 6.5. By contrast, nsHX experiments showed a variety of ∆GHX values ranging from 7.28 to 11.0 kcal/mol. This spread in stability indicates that the protein is undergoing sub-global unfolding, as opposed to global or local unfolding reactions. The b-barrel is shown to be the most stable region of the protein, and includes some super-stable residues, which have ∆GHX values that are higher than the average of 8.3 kcal/mol. ∆GHX values on average are about 2 kcal/mol higher than the estimated ∆Gspec value obtained from CD. This discrepancy can be due to the fact that nsHX experiments are measured at an individual residue level, and CD experiments monitor global unfolding transitions of the protein. ∆GHX showed increased stability for the structured b-strand regions; however, the a-helical region of the I-domain showed no protection to exchange, indicating that this secondary structure is only marginally stable. Taken together, the relaxation and nsHX results suggest that the structurally conserved b-barrel core is the most stable and resistant to denaturation.

Major Advisor

Andrei Alexandrescu

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