We are exploiting ferrocene peptide conjugates (see Foldamer Design) for the detection of protein binding and for screening of small molecules that compete with the protein-peptide interactions. Electrochemical methods are used to probe the interactions. We have projects in the area of HIV sensor systems and are actively working towards other protein sensors and electrochemical screening tools.
Protein Kinase Activity Detection and Inhibitor Screening
In the cellular communication network, many enzymes and receptors are switched ‘‘on’’ or ‘‘off’’ in other terms ‘‘phosphorylated’’ and ‘‘dephosphorylated’’ by the protein kinases. During phosphorylation, a phosphoryl group from ATP is transferred to specific serine, threonine, or tyrosine residue of the protein. As a result of these modifications, the function orthe function or localization of the protein may change, which in some cases may lead to the formation of the oncoproteins. Abnormal protein phosphorylation is a cause of major diseases, including cancer, diabetes and chronic inflammatory diseases. Using ferrocene labeled ATP as a co-substrate in protein kinase catalyzed phosphorylations of peptides, we were able to follow the progress of the reaction and evaluate the kinetics of the process under real-to-life conditions. Our approach relies on the use of surface-immobilized substrate peptides which are selectively phosphorylated. The Fc group attached to the peptide is electrochemically detected and quantified.
We are also interested in understanding the biochemical processes at the molecular level. To probe the intearctions between protein kinases and peptide substrates we turned to Fc-ATP as a structural probe. The structural variations in Fc-ATP bioconjugates was recently investigated by changing the alkyl linker length (C2-C10) and probing the phosphorylation reactions on the surface. Interestingly, Fc-ATP co-substrates containing the long alkyl spacers produced a significant electrochemical signal..
By further modifying ATP with variety of Fc-groups containing polar and non-polar linkers the outcome of the phosphorylation reaction was modulated. For optimal electrochemical response, the long non-polar alkyl linkers in Fc-ATP are ideal because they produce the least amount of steric hindrance and biomolecular interactions in the catalytic pocket of protein kinase.