BDB -BiopanningDataSet Details

Biopanning Data

BiopanningDataSet ID: 1670

Peptides:

Sequences
MSQTKRLDDQLYWWEYL

Sequences Features:

Motif:

Unique Sequence Number:

Experimental Method:

mRNA display (subtractive panning)

Sequencing Platform:

Sequencing Method:

Sanger sequencing

Rounds of Panning:

Reference:

PMID: 15248784

Target Name:

Guanine nucleotide-binding protein G(i) subunit alpha-1

Template Name:

Not determined.

Structure of Target-Template Complex:

Not determined.

Structure of Target-Peptide Complex:

Not determined.

Library Name:

GPR X(6) mRNA display library

Affinity Measurement Method:

Surface plasmon resonance (SPR)

Affinity Measurement Description:

The dominant peptide from the selection, R6A (MSQTKRLDDQLYWWEYL), retain high affinity (KD = 60 nM) for the GDP-bound state of GiR1, as measured by surface plasmon resonance.

Brief Description:

The affinity matrix for selection was prepared by rotating Nb- and/or Cb-GiR1(~10μg each) with ~20μL streptavidin agarose (Immobilized NeutrAvidin on Agarose, Pierce) in buffer A (20 mM HEPES-KOH at pH 7.5, 200 mM NaCl, 5 mM MgCl2, 1 mM EDTA, and 0.05% Tween 20) at 4 °C for >1 h. The slurry was supplemented with 1 mMD-biotin (~0.1 mM final concentration) and rotated for an additional 10 min to block biotin-binding sites. After washing thoroughly with buffer A2 [buffer A supplemented with 2μM GDP, 1 mM β-mercaptoethanol, 0.2% (w/v) BSA, and 1μg/mL yeast tRNA (Roche Diagnostics Corp., Indianapolis, IN)], reverse-transcribed fusions were rotated with the affinity matrix in 1 mL of buffer A2 at 4°C for 1 h. A total of 6 rounds of selection were performed on a mixture of immobilized Nb- and Cb-G iR1 to reduce the effects of bias or steric hindrance with either terminus immobilized. Detergent, bovine serum albumin (BSA), and salt were included in selection buffers to minimize recovery of nonspecific binding peptides.

BiopanningDataSet Comments:

DNA sequencing of the 6th round pool revealed a dominant peptide sequence (MSQTKRLDDQLYWWEYL). 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 (MSQTKRLDDQLYWWEYL), 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(iR1), as measured by surface plasmon resonance.

Curator:

Curation Date:

0000-00-00

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