The Effects of Buried Ionizable Amino Acids on the Stability of Ribonuclease Sa

The aim of this study was to investigate the stability contribution of buried ionizable amino acids in proteins. To study the stability contribution of a naturally occurring buried aspartic acid, two stabilized forms of RNase Sa designated 7S and 8S were used. In 7S, aspartic acid 79 has an elevated pK of 7.4 due to its location in the hydrophobic protein core. The stability contribution of this buried anion was calculated by comparing the Δ(ΔG) of 7S at pH 6, 7, and 9 with that of 8S. The stability contribution of ionized Asp79 in RNase Sa 7S is estimated to be -1.8 kcal/mol. To investigate the stability contribution of non-native buried ionizable groups, we introduce aspartic acid, lysine, and alanine residues at positions 70, 71, and 92 in 7S and 8S, and measure the change in stability, Δ(ΔG). Positions 70 and 92 are in close proximity to Asp 79, whereas position 71 is further away and partially shielded by a β-sheet. All mutants were less stable than the parental protein, and the magnitude of the stability change is dependent on the specific location in the protein. Since structural changes can account for differences in the environment of buried charges, it is important to determine whether buried charge mutations alter the structure of our mutant proteins. To date, the structures of the 7S I71A and 8S I71D variants have been resolved by X-ray crystallography. Using software to align crystal structures based on geometries of the residue side-chains, we find that 7S I71A and 8S I71D are comparable in structure to both RNase Sa WT and to each other. Crystal structure analysis indicates that the ionizable groups of the mutant residues are isolated from aqueous solvent. The differences in stabilities of variants were measured in 7S and 8S over a pH range to determine pK values of the mutant ionizable residues. In instances where the pK of buried ionizable mutant side chains are shifted, there is an apparent positive correlation between the magnitude of the pK shift and the magnitude of the change in stability. Thus, the buried ionizable mutants that are the least likely to be charged at physiological pH were observed to have the largest stability contribution. The calculated pK values were then used to assign charge values to the ionizable groups. Once charge values were assigned, the stability contribution of electrostatic interactions was calculated using Coulomb’s law. We calculated the difference in stabilities due to electrostatic effects in the presence or absence of Asp79 in 7S and 8S, respectively. Coulombic interactions were estimated in a range between -0.9 – 1.8 kcal/mol. Lastly, we investigate the localized effect of buried ionizable mutants on the dielectric constant. We find that introducing buried Asp mutants in 7S increases the dielectric constant, whereas making buried Lys mutations decreases the dielectric constant at each location.

Name thesis:degree_name

Doctor of Philosophy

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Biochemistry

Grantor

Texas A & M University

Year dc:date.issued

2014

Author dc:creator

• Everett, Anthany Laurence

Advisors dc:contributor.advisor

• Scholtz, John M.
• Pace, Carlos N.

Committee members dc:contributor.committeemember

• Bondos, Sarah E.
• Straight, Paul D.

• Ribonuclease Sa
• buried charges
• ionizable amino acids
• protein stability
• pKa shift
• thermal denaturation
• circular dichroism

Language dc:language.iso

en

Handle dc:identifier.uri

https://hdl.handle.net/1969.1/153960

OAI record oai:identifier

https://oaktrust.library.tamu.edu/server/oai/request?verb=GetRecord&metadataPrefix=dim&identifier=oai%3Aoaktrust.library.tamu.edu%3A1969.1%2F153960