Protein design was originally intended to demonstrate our understanding of how proteins fold. However, over the last three decades, advances in design principles and algorithms have paved the way for possibilities in medicinal, environmental, or enzymatic framework. While biological or drug-like applications for these proteins have been demonstrated, existing computational techniques are generally not able to predict whether a design will be highly or only moderately functional. Here, we have used computational simulation and modeling to study the mini fluorescence activating-protein (mFAP) that was designed to function as a fluorescent marker when bound to an exogenous chromophore, 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI). mFAPs stabilize DFHBI in the cis-planar Z conformation, increasing quantum yields 10 to 100-fold relative to free DFHBI. However, when compared to eGFP they are about 5-fold less efficient. In our latest work, we have built a predictive model that accurately captures the photophysical behavior DFHBI in the binding pocket of 13 mFAP designs via molecular dynamics (MD) simulations. MD analysis of mFAP-A detailed in this thesis shows that certain side-chain rotamers are strongly associated with keeping DFHBI in a cis-planar Z conformation and thus a biasing the mFAP-A/DFHBI complex towards brighter macrostates. We opted to use Rosetta design calculations to reshape the rough energy landscape towards these brighter macrostates and to find mutants that increase the existence of these rotamers.
"},{"label":"Degree","value":["Bachelor of Arts with Departmental Honors"]},{"label":"Author-supplied Keywords","value":["Protein design","Computational biophysics","Fluorescent proteins"]},{"label":"Administrative Note","value":"Drupal Node ID: 33981