Poster abstracts
Poster number 16 submitted by Allyson Langley
High-Throughput Methods of Analyzing Beta-Sheet Protein Stability
Allyson Langley (Chemistry)
Abstract:
Numerous diseases are caused by loss-of-function or destabilizing mutations in a protein, leading to the disruption of a biological pathway. From a drug-discovery perspective, stabilization of these mutant proteins seems like a logical solution. Unfortunately, how the amino acid sequence influences a protein’s structure, function, and stability remains a fundamental, unanswered question in biochemistry. We present research using protein GB1 as a model beta-sheet protein because it is well-characterized, highly stable, and binds Immunoglobulin G allowing affinity changes to be studied. We have developed high-throughput (HT) methods to increase the efficiency of purifying and characterizing two libraries; namely, how do constraints in two loops or beta-strand side chain interactions influence stability and binding. These libraries were cloned by randomizing four positions in the respective areas. Stabilities were then measured with fluorescence and circular dichroism experiments to find the changes in Gibbs free energy. Both libraries produced variants that were up to 6 kcal/mol destabilized, but more wild-type-like stability variants were seen in the loop-library. We were surprised to see increased destabilization overall with the beta-strand library, indicating this structural element may be of greater importance to the overall stability. These HT experiments have also been coupled with phage display to increase the size of the library that can be screened. By looking at a larger number of variants, we can start to gain more insight into what factors control beta-sheet protein stability and make advancements toward answering this question that is crucial to the advancement of biochemical research.
Keywords: proteins, stability, combinatorial