Mapping protein-ligand interactions by hydroxyl-radical protein footprinting.
Hydroxyl-radical protein footprinting is a direct method to map protein sites involved in macromolecular interactions. The first step is to radioactively end-label the protein. Using hydroxyl radicals as a peptide backbone cleavage reagent, the protein is then cleaved in the absence and presence of ligand. Cleavage products are separated by high-resolution gel electrophoresis. The digital image of the footprinting gel can be subjected to quantitative analysis to identify changes in the sensitivity of the protein to hydroxyl-radical cleavage. Molecular weight markers are electrophoresed on the same gel and hydroxyl-radical cleavage sites assigned by interpolation between the known cleavage sites of the markers. The results are presented in the form of a difference plot that show regions of the protein that change their susceptibility to cleavage while bound to a ligand.
Loizos
Nick
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Department of Protein Chemistry, ImClone Systems Inc., New York, NY, USA.
D000595:Amino Acid Sequence; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D029968:Escherichia coli Proteins; D006801:Humans; D017665:Hydroxyl Radical / Q000737:chemistry*; D008024:Ligands; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D023302:Protein Footprinting / Q000379:methods*; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism; D035602:Transcriptional Elongation Factors
D029968:Escherichia coli Proteins; C079667:GreB protein, E coli; D008024:Ligands; D046911:Macromolecular Substances; D011506:Proteins; D014157:Transcription Factors; D035602:Transcriptional Elongation Factors; D017665:Hydroxyl Radical