Peptides that bind to a target of interest can be selected from a phage-displayed peptide library, where they are fused to a coat protein on the bacteriophage. The identification of the binding peptides follows directly from sequencing the viral DNA, making phage display particularly convenient as a combinatorial method. Phage display has been used previously to probe the enzyme RNase A, comprised of the S-protein (104 aa) and S-peptide (20 aa) domains. Even when the two domains are separated by proteolysis, they assemble with high affinity, and RNA hydrolysis is maintained. When a hexapeptide library was panned against S-protein by G.P. Smith, et al., peptides were selected that bear little resemblance to S-peptide, but acted as antagonists against it. We seek to select peptides that replace S-peptide to recreate the active site by panning against the S-protein in the presence of a transition state analog. Ribonucleoside vanadyl complex mimics the 5-coordinate phosphorus-containing transition state that arises during RNA hydrolysis. We used commercially available phage display libraries (Ph.D.-7, 12, and C7C) to pan against S-protein in the presence and absence of the vanadyl complex. Our preliminary results suggest that SPSKFIK is strongly selected whether or not the complex is present. However, when the complex is included, additional peptides were identified, e.g., AIMPTRL, ATSKITF, YPGSWRI. Interestingly, all these latter clones contain mutations in the 5’-untranslated region of gene II, putatively conferring a propagation-related advantage to the phage that leads to the selection of target-unrelated peptides. In contrast, SPSKFIK has no mutation, suggesting that it was selected purely based on its binding to S-protein. We continue to vary panning conditions to seek peptides that specifically bind in the presence of the transition state analog, with the ultimate goal of reconstituting the enzyme with a novel active site between the S-protein and the selected peptide.

Reconstitution of RNase A as a hybrid of the S-protein and a peptide selected from a phage-displayed peptide library

ACS Spring 2022

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