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Applied Spectroscopy

Applied Spectroscopy

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  • Vol. 64, Iss. 9 — Sep. 1, 2010
  • pp: 967–972

Label-Free Screening of Drug–Protein Interactions by Time-Resolved Fourier Transform Infrared Spectroscopic Assays Exemplified by Ras Interactions

Carsten Kötting, Yan Suveyzdis, Ravi S. Bojja, Nils Metzler-Nolte, and Klaus Gerwert

Applied Spectroscopy, Vol. 64, Issue 9, pp. 967-972 (2010)


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Abstract

Time-resolved Fourier transform infrared (FT-IR) spectroscopy can reveal molecular details of protein interactions. Analysis of difference spectra selects the absorptions of respective protein groups involved in an interaction against the background of the whole sample. By comparison of the same difference spectrum with and without a small molecule, one can determine whether the small molecule interferes with the protein or not. Usually a marker band of a specific residue of the protein is monitored. Here, we show three different time-resolved FT-IR assays detecting interactions of potential small molecules for molecular therapy with the GTPase Ras as an example for small GTPase binding proteins. Ras regulates signal transduction processes through a switching mechanism, cycling between an active “on” GTP-bound form and an inactive “off” GDP-bound state. Molecular defects in Ras can impair the ability of Ras and the Ras–RasGAP complex to hydrolyze GTP, contributing to uncontrolled cell growth and cancer. Oncogenic mutated Ras is found in about 30% of all cancer cells. We show in vitro assays, indicating (I) the shift of Ras into its “off” conformation, which inhibits the Ras pathway; (II) down-regulation of Ras signaling by changes in the Ras–Raf effector interaction; and (III) down-regulation of Ras signaling pathway by catalyzing GTP hydrolysis. Since almost all molecules have characteristic marker bands in the infrared, time-resolved FT-IR spectroscopy can be used label-free. No artificial nucleotides that could influence the interaction are needed. Both, sample preparation and evaluation can be automated in order to allow for high-throughput screening.

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Citation
Carsten Kötting, Yan Suveyzdis, Ravi S. Bojja, Nils Metzler-Nolte, and Klaus Gerwert, "Label-Free Screening of Drug–Protein Interactions by Time-Resolved Fourier Transform Infrared Spectroscopic Assays Exemplified by Ras Interactions," Appl. Spectrosc. 64, 967-972 (2010)
http://www.opticsinfobase.org/as/abstract.cfm?URI=as-64-9-967


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