Year of Award

2021

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

Klára Briknarová, Nigel D. Priestley, J. B. Alexander Ross, Stephen R. Sprang

Keywords

chemical shift, denatured state, NMR, protein folding, residual structure

Publisher

University of Montana

Abstract

Protein folding is the natural process through which a linear polypeptide, originally encoded by a gene, adopts a distinct, three-dimensional structure capable of biological function. Over the last several decades, the denatured state ensemble (DSE), one of the two major populations adopted by a protein along with the native state, has been a topic of interest. Classically, the DSE has been interpreted as a collection of random-coil conformations devoid of any structural features. However, numerous studies have demonstrated that residual structure persists in the denatured state of proteins and may dictate the earliest stages of the protein folding process. Characterization of residual structure in the DSE can facilitate our understanding of proteopathies, such as amyloid and prion diseases, and intrinsically disordered proteins (IDPs), which are integral to cell signaling and regulation and are capable of pathological aggregation.

To build upon other well-studied determinants of residual structure, we have investigated the influence of interhelical turns on defining residual structure, thereby providing evidence to the importance of the DSE in accelerating folding or determining fold topology. Here, the folding properties of a three-helix bundle protein, the first ubiquitin-associated (UBA(1)) domain of HHR23A, have been investigated. Equilibrium thermal and chemical denaturation experiments, along with low-temperature stopped-flow folding kinetics experiments in GdnHCl, demonstrate that UBA(1) is a modestly-stable, monomeric protein with a folding time of 77 μs, on par with fast-folding proteins. The guanidine-denatured state of UBA(1), defined for our purposes at denaturant concentrations at and above 4 M GdnHCl, was studied by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies. Both techniques provide evidence that residual α-helical structure persists under strongly denaturing conditions, and chemical shift analysis demonstrates that transient helices are native-like. CD spectra also indicate polyproline-II helix population is enhanced at higher GdnHCl concentrations, an effect antithetical to the observed population of α-helices. We predict the denatured state of UBA(1) to occupy 17-33% helical structure under folding conditions, determined by extrapolation of NMR data to native buffer conditions. A helix 1 peptide of UBA(1) was generated and similarly iii characterized. An overall decrease in α-helical structure is observed for the peptide, indicating transient tertiary structure persists in the denatured state to stabilize helix 1 of UBA(1).

Mutations to UBA(1) have been generated with particular focus on perturbing interhelical turn sequences and hydrophobic core packing. For each mutation of a hydrophobic residue, the free energy of unfolding is found to be inversely related to that residue’s relative sequence conservation within the UBA protein family. Residues near or at helix caps exhibit changes in stability in accord with previously established residue-specific frequencies for helix caps. Residual structure has been characterized for three single mutations near helix caps, Y175V, S187A, and N190A, as well as one hydrophobic core mutation, V180A, with the strongest effects at 4 M GdnHCl. Mutations Y175V and N190A elicit a gradient-like destabilization of residual helical structure, focused near the sites of mutation and diminishing along the length of an adjacent helix in a zipper-like fashion. Likewise, both mutations exhibit destabilization among all three helical regions in the denatured state. Effects of S187A on residual helical structure are more heterogeneous, wherein the two adjacent helices are stabilized and the proximal helix is instead destabilized. The mutation V180A, a probe of hydrophobic packing, shows unusually insubstantial effects on residual helical structure in the guanidine-denatured state of UBA(1). Overall, our work provides compelling evidence that interhelical turn residues retain persistent residual structure in the chemically denatured state of a helical protein.

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