Use of SPR to study the interaction of G7-18NATE peptide with the Grb7-SH2 domain

Menachem J Gunzburg, Nigus D Ambaye, Jack T Hertzog, Mark P Del Borgo, Stephanie C Pero, David N Krag, Matthew CJ Wilce, Marie Isabel Aguilar, Patrick Perlmutter, Jacqueline A Wilce

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Abstract

Abstract Surface plasmon resonance (SPR) is a useful biosensor technique for the study of biomolecular interactions, with the potential for high-throughput screening of ligand interactions with drug targets. The key to its successful use, however, is in the appropriate design of the experiment, including the mode of immobilization to the biosensor chip. We report an investigation of the use of SPR for measuring the affinity of the G7-18NATE peptide ligand for its Grb7-SH2 domain target involved in the migratory and proliferative potential of cancer cells. Previous studies have shown that the cyclic non-phosphorylated peptide, G7-18NATE, inhibits Grb7 interactions with upstream binding partners and is able to inhibit both cell migration and proliferation of cancer cells. We report the synthesis of a biotinylated G7-18NATE covalently attached to a linker (G7-18NATE-ASASASK-Biotin) and compare its interaction with the Grb7-SH2 domain by SPR using three different immobilization strategies; immobilisation of the peptide via streptavidin, immobilization of glutathione S-transferase (GST)-Grb7-SH2 domain via anti-GST antibody, and immobilization of biotinylated Grb7-SH2 domain via streptavidin. This revealed that sensorgrams free from non-specific binding and displaying simple kinetics were most readily achieved by immobilising the protein rather than the peptide, in spite of the lower response associated with this method. KD values of 300 lM were determined for both strategies at pH 7.4. This compared with a KD value of 4.4 uM at pH 6 demonstrating the importance of pH on this interaction. Overall, the immobilised protein systems are most suitable for future comparative screening efforts using SPR.
Original languageEnglish
Pages (from-to)177 - 184
Number of pages8
JournalInternational Journal of Peptide Research and Therapeutics
Volume16
Issue number3
DOIs
Publication statusPublished - 2010

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