Design of SH3 domain ligands using novel arginine mimetics and development of tunable EF-hand sensors of post translational modifications

Scheuermann, Michael
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University of Delaware
Guanidinium groups are widely used in contacts of proteins with other biological molecules, via both electrostatic interactions and hydrogen bonds. However, arginine residues in peptide ligands can potentially lead to a decrease in specificity of the ligand to its binding site because of the flexibility of the linear alkyl side chain in an arginine residue. Arginine mimetics address this issue by using additional functional groups in tandem with the guanidinium group, potentially increasing specificity in arginine mediated recognition. Novel arginine mimetics were synthesized by coupling of alpha-amino acids to the side chain or main chain of the peptide. Guanylation of the amino acid gave the necessary guanidinium group. This design allows for large variation of functionality, via modification of the coupled amino acid, of the arginine mimetic. In order to test these arginine mimetics, the arginine in a Src homology 3 (SH3) domain ligand was replaced by a Dap residue coupled to one of the following guanylated residues: L-Val, D-Val, L-Phe, D-Phe, L-Trp, D-Trp or Gly. These arginine mimetics were stereospecifically incorporated into peptide ligands as determined by NMR. Depending on the alpha-guandino acid coupled to the Dap residue, binding to the SH3 domain of Src and Grb was able to be modulated. Guanylated L-Trp gave an improvement in binding over the parent arginine containing peptide, and exhibited paralog specificity by binding Src preferentially over Grb. Maintaining homeostasis of intracellular redox conditions is crucial to maintaining cell viability, as increasing the oxidation potential puts the cell under oxidative stress. Complex pathways have been developed to maintain homeostasis of redox conditions, many of which rely on glutathione for keeping redox conditions in balance. S-Glutathionylation is a post- translational modification to cysteine residues which plays important roles in cellular signaling pathways and apoptosis. The exact mechanism of S-glutathionylation remains unknown, but it is known that it is tied to cellular redox conditions. To understand more about glutathionylation, a peptide based on the metal binding EF-hand motif was designed in which a Glu-9 residue is replaced with a Cys-8. The glutathionylated and non-glutathionylated peptides were tested for their terbium (III) binding affinity by analyzing their terbium luminescence. After identifying the peptide with greatest differentiation in both terbium binding affinity and fluorescence intensity, conversion studies were run in varying oxidation conditions to examine extent of glutathionylation in the peptides. The extent of glutathionylation in the peptides was analyzed by HPLC and terbium luminescence. Glutathionylation of the peptide occurred via disulfide exchange with oxidized glutathione. This marks the first designed peptide whose structure and fluorescence are dependent on tis glutathionylation state. Tyrosine sulfation is a post-translational modification of tyrosines in membrane-bound and secretory peptides and proteins. Tyrosine sulfated proteins play integral roles in numerous processes, such as cellular signaling, molecular recognition, and blood coagulation. Sulfation of tyrosines is enzymatically performed in the trans-Golgi apparatus by a class of enzymes known as tyrosylprotein sulfotransferases. To date, the sulfoproteome remains largely unexplored, owing to the difficulty in identification and purification of sulfotyrosine-containing proteins. To address questions about tyrosine sulfation, a peptide based on the metal binding EF-hand motif was designed in which a highly conserved Glu12 residue is replaced by a sulfated tyrosine at residue 11. These peptides were tested for their terbium (III) binding and their terbium luminescence. Two peptides were identified that exhibit terbium binding when the tyrosine was sulfated, but poor binding in the absence of the sulfate, showing good differentiation in terbium luminescence between the sulfated and non-sulfated states. These peptides are the first known encodable sensors capable of directly detecting states of tyrosine sulfation based upon luminescence properties. These peptides also represent the first examples of designed proteins dependent on tyrosine sulfation.