The three dimensional structure of a protein is important for its function. When misfolded, a protein may be rendered inactive or adapt a conformation that could be toxic. Studying protein folding requires an understanding of protein conformation. Traditionally, protein conformation has been studied using x-ray crystallography and nuclear magnetic resonance (NMR). X-ray crystallography is limited in the analysis of crystallized proteins and is computationally intensive. NMR deals with proteins in solution but reports only an average of conformation and the technique severely suffers from spectral overlapping due to the thousands of resonances of the protein. More recently, mass spectrometry has been employed not only to elucidate primary structures but also gather information on the three-dimensional conformation of proteins.
In this study, a mass spectrometric-based approach is used to study the changes in conformation of cytochrome c and the green fluorescent protein when subjected to aqueous-organic solvent systems. The technique involved trypsin digestion and generation of peptide mass maps.
For cytochrome c, the experiments were done with ethanol, methanol and acetonitrile to gain insights on naturation and denaturation. An apparent solvent effect to the rate of digestion and propensity for missed cleavages attributed to weakening of hydrophobic interactions and strengthening of intramolecular hydrogen bonding was observed.
For the green fluorescent protein, sulfolane, a known supercharging agent, was used to gain insights on the effect of supercharging to protein conformation. Addition of 2.0% sulfolane shifted the charge state envelope of the protein towards lower m/z while adding lower amounts of sulfolane enhanced lower charge states while broadening the charge state envelope. The time course study showed different patterns of digestion dependent on solvent conditions implying changes in conformation. Furthermore, absorbance and fluorescence measurements suggested that addition of sulfolane protects the fluorophore from quenching. The activity of trypsin is not affected by addition of low amounts of sulfolane.
| Identifer | oai:union.ndltd.org:tamu.edu/oai:repository.tamu.edu:1969.1/149503 |
| Date | 03 October 2013 |
| Creators | Tuvilla, Mavreen Rose |
| Contributors | Russell, David H, Liu, Wenshe, Capareda, Sergio |
| Source Sets | Texas A and M University |
| Language | English |
| Detected Language | English |
| Type | Thesis, text |
| Format | application/pdf |
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