Designing FRET Assays to Study Electrostatic Interactions Pertaining to the Binding of Intrinsically Disordered Proteins

Fibroblast growth factor receptor plays a major role in several biological processes. Without FGFR, a human cannot live. FGFR is involved in cell differentiation and wound healing. Of course, if FGFR signaling becomes unregulated, it causes severe distress in the body. Several cancers are contributed to high signaling levels, as well as developmental conditions like rickets and Kallmann’s syndrome. FGFR is thought to undergo an auto-inhibition (or self-regulatory) process in order to try to facilitate regulation. The exact method of this inhibition is currently unknown, but is proposed to involve the unstructured acid box region of FGFR. We developed a simple model system in order to further investigate current models of inhibition that FGFR may undergo. By using our model system, which contains two 15-mer homopolypeptides of polyE and polyK that mimic the acid box region and its binding site respectively, we were able to use a combination of ITC, CD, NMR, and FRET to show that one model from the literature contains flaws. We are able to characterize the binding of our polypeptide system under varying ionic conditions and pH. This model system also provides a platform to better understand general principles of charge-charge interactions in proteins, which are often characterized by FRET. One of the important findings from this study is that 15-mers of polyE and polyK bind in a parallel arrangement. One of the hurdles in applying FRET to such systems is determining the role that the attached FRET dyes play in the charge-charge interactions. Our model system allowed us to use the preference of the charged polypeptides to bind in a parallel arrangement to determine the size, charge and structural effects of the attached FRET dyes on how peptides bind under electrostatic interaction conditions and to quantify how the attached fluorescent dyes are quenched by both the charged amino acids as well as by the FRET acceptor.