In Vitro Selection of State-Specific Peptide Modulators of G Protein Signaling Using mRNA Display
The G protein regulatory (GPR) motif is a ∼20-residue conserved domain that acts as a guanine dissociation inhibitor (GDI) for G_(i/oα) subunits. Here, we describe the isolation of peptides derived from a GPR consensus sequence using mRNA display selection libraries. Biotinylated G_(iα1), 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_(iα1), as measured by surface plasmon resonance. The selected peptides also maintain GDI activity for G_(iα1), inhibiting both the exchange of GDP in GTPγS 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_(βγ) subunits for binding to G_(iα1), suggesting their use as activators of G_(βγ) signaling.
Additional Information© 2004 American Chemical Society. Received 22 January 2004. Published online 19 June 2004. Published in print 1 July 2004. This work was supported by grants from the NIH (RO160416) and the Beckman Foundation to R.W.R. W.W.J. was supported in part by a DOD National Defense Science and Engineering Graduate Fellowship. R.W.R. is an Alfred P. Sloan Foundation Research Fellow. We thank Prof. Roger K. Sunahara (University of Michigan) for the G protein expression vector, Dr. David S. Waugh (National Cancer Institute at Frederick) for the in vivo biotinylation vector, Prof. Melvin I. Simon and Valeria Mancino for the polyclonal Gβ antiserum and use of the Flexstation fluorescence plate reader, Prof. Pamela J. Bjorkman and Anthony M. Giannetti for time and technical assistance on their Biacore 2000 instrument, and Prof. Douglas C. Rees and Terry Takahashi for helpful discussions on the kinetics models. Prof. David G. Myszka (University of Utah) generously provided the analysis software, Scrubber and CLAMP. We greatly appreciate the preparative and technical expertise on protein purification provided by Dr. Shuwei Li (University of Texas Southwestern Medical Center) and Christopher T. Balmaseda. We are also grateful to Dr. Judy E. Kim for comments on the paper.
Supplemental Material - bi0498398si20040411_124052.pdf