In vitro selection of state-specific peptide modulators of G protein signaling using mRNA display.
The G protein regulatory (GPR) motif is a approximately 20-residue conserved domain that acts as a guanine dissociation inhibitor (GDI) for G(i/o)(alpha) subunits. Here, we describe the isolation of peptides derived from a GPR consensus sequence using mRNA display selection libraries. Biotinylated G(i)(alpha)(1), modified at either the N or C terminus, serves as a high-affinity binding target for mRNA-displayed GPR peptides. In vitro selection using mRNA display libraries based on the C terminus of the GPR motif revealed novel peptide sequences with conserved residues. Surprisingly, selected peptides contain mutations to a highly conserved Arg in the GPR motif, previously shown to be crucial for binding and inhibition activities. The dominant peptide from the selection, R6A, and a minimal 9-mer peptide, R6A-1, do not contain Arg residues yet retain high affinity (K(D) = 60 and 200 nM, respectively) and specificity for the GDP-bound state of G(i)(alpha)(1), as measured by surface plasmon resonance. The selected peptides also maintain GDI activity for G(i)(alpha)(1), inhibiting both the exchange of GDP in GTPgammaS binding assays and the AlF(4)(-)-stimulated enhancement of intrinsic tryptophan fluorescence. The kinetics of GDI activity, however, are different for the selected peptides and demonstrate biphasic kinetics, suggesting a complex mechanism for inhibition. Like the GPR motif, the R6A and R6A-1 peptides compete with G(betagamma) subunits for binding to G(i)(alpha)(1), suggesting their use as activators of G(betagamma) signaling.
Ja
William W
WW
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA.
Roberts
Richard W
RW
D001120:Arginine; D003001:Cloning, Molecular; D016384:Consensus Sequence; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D019204:GTP-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D015723:Gene Library; D006801:Humans; D007700:Kinetics; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology*; D011485:Protein Binding; D012333:RNA, Messenger; D015398:Signal Transduction / Q000187:drug effects*
D010446:Peptide Fragments; D012333:RNA, Messenger; D001120:Arginine; D019204:GTP-Binding Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go