In vitro selection of state-specific peptide modulators of G protein signaling using mRNA display

Biochemistry
William W Ja, R W Roberts

Abstract

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 ac...Continue Reading

References

Jan 1, 1987·Annual Review of Biochemistry·A G Gilman
Nov 24, 1999·The Journal of Biological Chemistry·A TakesonoS M Lanier
Mar 14, 2003·Trends in Biochemical Sciences·Terry T TakahashiRichard W Roberts
Mar 3, 2004·Molecular Interventions·Randall J KimpleDavid P Siderovski

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Citations

Sep 20, 2007·Science in China. Series C, Life Sciences·Song YanXiuKun Lin
Sep 20, 2006·Biochemistry·Christopher A JohnstonDavid P Siderovski
Dec 16, 2006·ACS Chemical Biology·William W JaRichard W Roberts
Sep 27, 2007·ACS Chemical Biology·Steven W MillwardRichard W Roberts
Jun 5, 2007·Nature Chemical Biology·William W JaRichard W Roberts
Jan 13, 2006·Annual Review of Pharmacology and Toxicology·Motohiko SatoStephen M Lanier
Mar 4, 2014·Annual Review of Biochemistry·Toby PassiouraHiroaki Suga
Jul 26, 2005·Reviews of Physiology, Biochemistry and Pharmacology·M J Cismowski, S M Lanier
Oct 18, 2005·FEBS Letters·Christopher A JohnstonDavid P Siderovski
Mar 15, 2006·FEBS Letters·Kyoung Seob SongS M Lanier
Mar 11, 2008·Journal of Molecular Biology·Ryan J AustinRichard W Roberts
Sep 23, 2014·Scientific Reports·Shannon M HowellRichard W Roberts
Oct 28, 2005·Journal of Molecular Recognition : JMR·Rebecca L Rich, David G Myszka
Sep 10, 2008·Chembiochem : a European Journal of Chemical Biology·Stephen V Fiacco, Richard W Roberts
Jan 24, 2007·Pharmacology & Therapeutics·Joe B BlumerStephen M Lanier
Sep 16, 2006·Neurochemistry International·Harish V ThiagarajKeith K Parker
Apr 20, 2006·Seminars in Cell & Developmental Biology·Mary J Cismowski
Dec 13, 2005·Biochemical and Biophysical Research Communications·Francis S Willard, David P Siderovski
Jun 14, 2005·Trends in Biochemical Sciences·William W Ja, Richard W Roberts
Jul 29, 2016·Chembiochem : a European Journal of Chemical Biology·Stephen V FiaccoRichard W Roberts
Jun 28, 2005·Protein Engineering, Design & Selection : PEDS·William W JaRichard W Roberts
Jan 4, 2013·Chemical Communications : Chem Comm·Takanori UzawaYoshihiro Ito
Jul 29, 2006·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·Achim RotheBarbara E Power
Jul 30, 2005·The Journal of Biological Chemistry·William W JaRichard W Roberts
Jun 25, 2021·Chemical Society Reviews·Golnaz KamaliniaRichard W Roberts
Nov 6, 2018·Chemical Reviews·Yichao HuangHiroaki Suga

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