title: Novel peptide recognized by RhoA GTPase.
abstract: A phage-displayed random 7-mer disulfide bridge-constrained peptide library was used to map the surface of the RhoA GTPase and to find peptides able to recognize RhoA switch regions. Several peptide sequences were selected after four rounds of enrichment, giving a high signal in ELISA against RhoA-GDP. A detailed analysis of one such selected peptide, called R2 (CWSFPGYAC), is reported. The RhoA-R2 interaction was investigated using fluorescence spectroscopy, chemical denaturation, and determination of the kinetics of nucleotide exchange and GTP hydrolysis in the presence of RhoA regulatory proteins. All measurements indicate that the affinity of the R2 peptide for RhoA is in the micromolar range and that R2 behaves as an inhibitor of: i) GDP binding to the apo form of RhoA (Mg2+-and nucleotide-free form of the GTPase), ii) nucleotide exchange stimulated by GEF (DH/PH tandem from PDZRhoGEF), and iii) GTP hydrolysis stimulated by the BH domain of GrafGAP protein.
authors.affiliation: Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, Wrocław, Poland.
authors.forename: Dominika
authors.initials: D
authors.lastname: Drulis-Fajdasz
authors.forename: Filip
authors.initials: F
authors.lastname: Jelen
authors.forename: Arkadiusz
authors.initials: A
authors.lastname: Oleksy
authors.forename: Jacek
authors.initials: J
authors.lastname: Otlewski
mesh_terms: D000595:Amino Acid Sequence; D017104:Bacteriophage M13 / Q000235:genetics; D020558:GTP Phosphohydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000378:metabolism; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D019151:Peptide Library; D010449:Peptide Mapping / Q000379:methods; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
chemical_list: D020690:GTPase-Activating Proteins; D020730:Guanine Nucleotide Dissociation Inhibitors; D020662:Guanine Nucleotide Exchange Factors; D019151:Peptide Library; D010455:Peptides; D011994:Recombinant Proteins; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D020742:rhoA GTP-Binding Protein