Rational Design, Synthesis and Self-assembly Studies of Facially Amphiphilic Proteins (FAPs)

Abstract

Nature has evolved to make a diverse set of protein architectures to perform the complex fundamental life processes such as transcription, translation, catalysis, metabolism, and transport. These naturally occurring protein assemblies serve as an inspiration for the design of synthetic and semi-synthetic protein assemblies. This is primarily achieved through two complementary technologies, such as genetic and chemical methods. In the past decade, genetic methods have matured as robust technology for accurate design of protein assemblies with a defined geometry. Compared to the genetic method, chemical methods are in their infancy. Most of the studies in this area are largely focused on a couple of model proteins. More importantly, there are no robust methods for purification, and therefore synthesized protein assemblies lack detailed analytical and biophysical characterization. My thesis work is directed towards developing new chemical strategies to convert monomeric proteins into protein complexes. This method is based on the generation of a simple scaffold, which we call "Facially Amphiphilic Proteins (FAPs)" from native proteins. The generation of FAP was achieved using a micelle-assisted protein labeling (MAPLab) technology.

First, using serine proteases, we have demonstrated that monomeric proteins can be converted into protein complexes of bigger sizes. This was done by targeting active site Ser using fluorophosphonate chemistry. The simple design allowed us to systematically study the scaffold (FAP) with respect to individual units, i.e., protein, linker, and hydrophobic tail length/branching. This study also provided us with an opportunity to control the molecular weights, oligomeric states, and dimensions of the synthesized protein complexes precisely. Then, using a similar design and MAPLab Technology, we have also designed stimuli-responsive protein complexes. Further, we used the same method to make protein-synthetic peptide conjugates. Finally, in order to make this method more universal, we extended this technology for site-specific labeling of N-terminus. In addition, the same technology was explored for site-specific thiol bioconjugation.