| dc.description.abstract | The process of protein folding involves the transition of a sequence of amino acids in an unfolded polypeptide chain to a unique folded state, called the native state of the protein. Understanding protein folding requires explaining how and why a protein attains a stable native conformation. The “protein folding problem” defines our inability to predict the native state of a given amino acid sequence. This remains one of the most intriguing and fundamental problems in biophysical chemistry. Not only are we unable to predict the relationship between sequence and tertiary structure, but we also cannot reliably predict the effect of a single amino acid substitution on folding, stability, or dynamics.
One way to understand protein folding, dynamics, and stability is through fragment complementation systems-model systems in which two or more fragments of a protein bind each other in solution to form a complex resembling the native protein. Their advantage lies in the ability of unstructured fragments to reassemble into a native like structure under physiological conditions.
Ribonuclease S (RNase S) is a fragment complementation system derived from bovine pancreatic ribonuclease A (RNase A) by subtilisin cleavage between residues 20-21. The resulting fragments-S peptide (residues 1-20) and S protein (residues 21-124)-form the RNase S complex in solution, which closely resembles RNase A. This thesis studies various aspects of protein structure, stability, and dynamics using RNase S as a model system. In addition, the crystal structure of barstar, a ribonuclease inhibitor, was solved and analysed in comparison with the NMR structure of barstar and the structure of barstar bound to barnase.
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Chapter I - Introduction
Reviews the progress in protein folding research relevant to the work described in the thesis.
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Chapter II - Dynamics of RNase A vs RNase S
Three probes were used to compare the dynamics of RNase A and RNase S:
• molecular dynamics simulations
• proteolytic susceptibility
• proton exchange rates by NMR
The analysis indicates that observed dynamic differences between the two proteins arise from the presence of free S protein and free S peptide at equilibrium with the RNase S complex. Because folded RNase S is in equilibrium with its fragments, its dynamics show concentration dependence. A model is proposed for hydrogen exchange occurring through fragment dissociation and re association at equilibrium.
S protein, the larger fragment, is folded in solution but is believed to have a less compact tertiary structure. Since its structure is unknown, crystallization trials were undertaken (Chapter III), both alone and in combination with small molecule inhibitors and truncated S peptides. A structural model of S protein in solution is proposed based on native state hydrogen exchange data of RNase S.
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Chapter IV - Structural and Thermodynamic Effects of Buried Residue Mutations
To understand how core residues stabilize proteins, methionine at position 13 of RNase S was replaced with the isosteric residue norleucine. Thermodynamic data on peptide binding, combined with crystallographic comparisons of Met13, Ile13, and Leu13 variants, reveal how subtle packing differences influence protein stability.
To examine the effect of introducing a cavity, phenylalanine 8 (F8) was replaced with alanine, methionine, or norleucine. Crystal structures show:
• In the F8Ala mutant, the cavity is filled by rotation of F116 into the space.
• In F8Met and F8Nle mutants, the protein accommodates the substitutions with minimal structural perturbation.
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Chapter V - Structural Basis of Protein Denaturation
Proteins are denatured by urea, guanidinium chloride, or low pH, but the structural basis is not fully understood. To investigate this, crystals of RNase S were soaked in increasing concentrations of urea and at low pH, and X ray data were collected.
Findings include:
• Structures in 0-3 M urea show no major local unfolding.
• At 5 M urea, increased RMS deviation and higher B factors indicate global destabilisation.
• At pH 2, large conformational changes occur in loop 65-71, suggesting it may be the initial site of acid induced unfolding.
Crystals were also soaked in four osmolytes-sarcosine, trimethylamine N oxide (TMAO), betaine, and taurine. No evidence was found for osmolyte binding to the native state or significant changes in water structure around the protein.
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Chapter VI - Crystal Structure of Barstar (C82A Mutant)
Barstar, the natural inhibitor of the bacterial ribonuclease barnase, is a small (80 residue) protein widely used as a folding model due to its lack of disulfide bonds. Although the crystal structure of the barnase-barstar complex is known, the free barstar structure had not been solved.
This work reports the 2.8 Å crystal structure of the C82A mutant of barstar, and compares it with:
• the barnase bound state
• the published NMR structure of free barstar
The free crystal structure closely resembles the bound structure, both overall and at the binding surface. In contrast, the NMR derived structure shows significant deviations in packing and overall geometry. A packing density analysis reveals that NMR structures-here and more generally-tend to display anomalously low packing. The crystal derived structure is therefore likely to be a more accurate representation of free barstar.
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Appendix
Contains descriptions of experimental and analytical techniques used throughout the thesis. | |
