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Elucidation of the Structural Basis of Protein Kinetic Stability

Development of a Simple Assay for Determining Protein Kinetic Stability

Based on the observation that some proteins are resistant to denaturation by SDS, we hypothesized that this phenomenon was due to kinetic stability. We tested 33 proteins to determine their SDS-resistance by comparing the migration on a gel of boiled and unboiled protein samples containing SDS (Fig 3.). Proteins that migrated to the same location on the gel regardless of whether or not the sample was boiled were classified as not being stable to SDS. Those proteins that exhibited a slower migration when the sample was not heated were classified as being at least partially resistant to SDS. Of the proteins tested, 8 were found or confirmed to exhibit resistance to SDS, including Cu/Zn superoxide dismutase (SOD), streptavidin (STR), transthyretin (TTR), P22 tailspike (TSP), chymopapain (CPAP), papain (PAP), avidin (AVI), and serum amyloid P (SAP) (Manning and Colón 2004)

To probe the kinetic stability of our SDS-resistant proteins, their native unfolding rate constants were obtained by measuring the unfolding rate at different guanidine hydrochloride (GdnHCl) concentrations and extrapolating to 0 M. The native unfolding rate for all the SDS-resistant proteins was found to be very slow, with protein unfolding half-lives ranging from 79 days to 270 years. The results obtained in this study suggest a general correlation between kinetic stability and SDS-resistance, and demonstrate the potential usefulness of SDS-PAGE as a simple method for identifying and selecting kinetically stable proteins (Manning and Colón 2004). We are currently developing a 2D SDS-PAGE method for the high throughput identification of kinetically stable proteins in complex protein mixtures, such as bacterial and eukaryotic cellular extracts and human plasma.

A key to understanding kinetic stability in proteins may lie in determining the physical basis for their structural rigidity, as this appears to be a common property of kinetically stable proteins (Jaswal, et al. 2002; Parsell and Sauer 1989). In our study, the presence of predominantly oligomeric ?-sheet structures emerged as a common characteristic of most of the kinetically stable proteins. Perhaps the higher content of non-local interactions in ?-sheet proteins may allow for higher rigidity than in ?-helical proteins. Clearly, not all oligomeric ?-sheet proteins are kinetically stable/SDS-resistant, indicating that 2° and 4° structure are not the main structural factors determining this property. Clearly, computational analysis of a large database of kinetically stable proteins like the one we are now uniquely able to generate will be required to elucidate the structural basis of kinetic stability.

The assays developed will be used to assess the kinetic stability of a variety of protein types, including those known to be stable (such as certain kinases) and those with lower kinetic stability. Specific mutations of the primary sequence are proposed as a means for creating protein variants with greater or lesser kinetic stability, with the goal of identifying key molecular mechanisms for enhancing stability. Data generated during this study would be utilized to identify specific features of proteins that exhibit enhanced kinetic stability.

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