Aggregation is a big problem that prevents researchers from solving protein structures either using nuclear magnetic resonance spectroscopy (NMR) or X-ray crystallography. In order to isolate a protein for NMR or for crystallization, it must be removed from its natural environment. This often results in aggregation. Many factors can cause aggregation, including self-association through strong electrostatic interactions. In our research, we introduce amino acid mutations, and thereby alter protein self-association through electrostatic interactions, without changing the overall structure of the protein. We use Ubiquitin as a model protein because its structure is well characterized, and this allows us to determine if the amino acid substitutions change the protein structure. In this study, we characterize two different mutants of Ubiquitin, in each case replacing the amino acid lysine, which has a positive charge. In the first mutant we replaced lysine with alanine (K6A), which has a neutral charge. In the second mutant we replaced lysine with aspartic acid (K6D), which has a negative charge. Our goal was to determine if changing the electrostatic charge distribution changed the protein solubility, aggregation, stability and structure of the protein. If successful this method might be useful to help solve the structure of many new proteins that suffer aggregation which prevents structure determination using NMR spectroscopy or X-ray crystallography.

Disrupting electrostatic interactions that cause protein aggregation and prevent protein structure determination

243rd National Meeting (2012)

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