2009 OSU Molecular Life Sciences
Interdisciplinary Graduate Programs Symposium
The inability to accurately decipher the relationship between protein sequence and structural stability presents a major difficulty in predicting the thermodynamic effects of mutation on protein folding. Our studies focus on the sequence-stability-function relationship of the four-helix bundle protein Rop by combinatorial repacking of the hydrophobic core. Using an in vivo screen that utilizes GFP as a reporting phenotype, we are able to screen large libraries for functional variants, representing Rop mutants that are able to achieve an active, folded conformation. These functional variants from a modestly repacked library have been sequenced via high-throughput colony sequencing technology to accumulate a data set of over 200 unique Rop variants differing only in packing of the hydrophobic core. To gain insight into the thermodynamic consequences of core packing, we have developed a high-throughput thermal scanning (HTTS) assay to assess the relative stabilities of the sequenced active variants. This robust data set of sequence and stability information suggests that the packing of the hydrophobic core of Rop is lenient in regards to function, but stringent in regards to stability and native-like structure. Interestingly, a large portion of the functional variants are molten globular in structure as a result of poor core packing, and large differences in stability are evident even with very small differences in primary sequence. These results suggest that packing of the hydrophobic core in stable, native-like proteins is akin to fitting together of a jig-saw puzzle. This has obvious implications for in silico effective energy functions aimed at structure prediction. To this end, we are currently using our empirical data to compare and contrast with different predictive EEFs. In addition, we are using biophysical methods such as circular dichroism, 1H-15N HSQC, and x-ray crystallography to provide detailed information of the structure of core variants of interest.
Keywords: protein folding, protein engineering, protein biophysics