Year of Award

2019

Document Type

Dissertation

Degree Type

Doctor of Philosophy (PhD)

Degree Name

Biochemistry & Biophysics

Department or School/College

Department of Chemistry and Biochemistry

Committee Chair

Bruce E. Bowler

Commitee Members

Stephen Lodmell, J.B. Alexander Ross, Stephen Sprang, Scott Wetzel, Keith Parker

Keywords

denatured state, instrinsic disorder, kinetics, protein folding, residual structure, thermodynamics

Publisher

University of Montana

Abstract

Protein misfolding is associated with several life threatening diseases – Alzheimer’s, Parkinson’s, Cystic fibrosis, Sickle cell anemia, and the Transmissible spongiform encephalopathies, to include Chronic Wasting disease, Scrapie, and Creutzfeldt-Jakob disease. To provide insight into protein misfolding, it is beneficial to understand how an amino acid sequence encodes structure. Once thought to behave as random coil polymers under denaturing conditions, an abundance of recent evidence suggests denatured proteins retain ‘non-random’ or residual structure, which may bias a protein chain to fold efficiently to its native conformer. Probing energetics of residual structure in denatured proteins is challenging, given the shortage of available methods to test such questions. Using the metalloprotein Cytochrome c (Cytc) as a scaffold, we fuse three highly divergent amino acid sequences with helical topology to the N-terminus of Cytc. Using site-directed mutagenesis, we engineer histidine into highly solvent accessible positions in each of these three folds, creating panels of single histidine variants within each protein sequence. In denaturing conditions, the engineered histidine in each variant serves as ligand to the heme in Cytc, forming a histidine-heme (His-heme) loop with a specific size. Two of the three helical proteins are foldable domains excised from human DNA excision repair protein HHR23A, Ubiquitin-associated domain 1 (UBA(1)) and Ubiquitin-associated domain 2 (UBA(2)). The UBA domains function in Ubiquitin/proteasome signaling. The third protein is mouse cAMP-responsive element binding protein (CBP), an intrinsically disordered protein that serves as a general transcription coactivator involved in hormonal signaling. When bound to its signaling partner activator for thyroid hormone and retinoid receptor (ACTR), CBP folds to an α-helical topology. For UBA(1)-Cytc, UBA(2)-Cytc, and CBP-Cytc fusion proteins, guanidine hydrochloride (GuHCl) denaturation show three state equilibrium unfolding, with the Cytc domain unfolding first. Furthermore, engineered histidine residues in UBA(2) and UBA(1) destabilize the Cytc domain, while CBP has modest effect on Cytc. Equilibrium and kinetic His-heme loop formation measurements in the denatured state at 4 and 6 M GuHCl show that loop stability decreases as the size of the His-heme loop increases, in accord with the Jacobson-Stockmayer equation. However, we observe that the His27-heme loop in UBA(2)-Cytc, the His15-heme & His31-heme loops in UBA(1)-Cytc, are more stable than expected, and break more slowly than expected, relative to His-heme loops exhibiting random coil behavior in the denatured state. Thus, these loops are indicative of non-random behavior in the denatured state. We observe unusual behavior in the biophysical properties of CBP in the denatured state. These results indicate local sequence near His27 in UBA(2), and His15 & His31 in UBA(1), are prone to persistent interactions in the denatured state. When mapped onto their native structures, these regions with denatured state residual structure localize to reverse turns in the UBA domains, and unstructured regions in CBP. Our results on turn bias persisting in the denatured state are consistent with previous findings for the four-helix bundle protein Cytc’, where reverse turns may help establish the gross topology of helix-bundles via biasing the conformational distribution of the denatured state. These results could assist the biotechnology and protein engineering fields in developing therapeutics to aid protein misfolding diseases.

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© Copyright 2019 Moses Joseph Leavens