Mapping protein domains involved in macromolecular interactions: a novel protein footprinting approach.
A novel direct approach, analogous to DNA footprinting, for mapping protein domains involved in macromolecular interactions is presented in this paper and applied to cAMP receptor protein (CRP) interactions with the allosteric ligand (cAMP) and DNA. In this approach, a protein-macromolecule complex is subjected to a nonspecific cleavage by Fe-EDTA. The cleavage products are resolved by SDS-PAGE and transferred to a PVDF membrane. Transferred polypeptides are visualized by immunostaining with antibodies specific to the N-terminal peptide of the protein. The mobility of the bands visualized in such a way is directly proportional to the distance of the cleavage sites from the N-terminus, and thus the positions of the sites protected from cleavage by a bound macromolecule can be determined. Thus, protein domains involved in macromolecular interactions can be mapped. In the case of CRP, the cleavage conditions were established which resulted in, on the average, less than one cleavage event/protein molecule and which preserved satisfactory levels of protein and DNA activity. When applied to CRP-DNA interactions, the protein footprinting approach correctly identified domains of CRP that were known to be involved in the recognition of DNA. The obtained results showed also that the binding of CRP to the DNA binding site perturbed the region of CRP involved in intersubunit interactions. An allosteric ligand (cAMP) appeared to perturb the same region of CRP. This stresses out the importance of intersubunit interactions in cAMP modulation of protein DNA binding affinity. The protein footprinting methodology presented in this paper should be broadly generalizable to any protein-macromolecule system.
Heyduk
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Department of Biochemistry and Molecular Biology, St. Louis University, Medical School, Missouri 63104.
Heyduk
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D001671:Biochemistry / Q000379:methods; D000242:Cyclic AMP / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D015151:Immunoblotting; D007502:Iron Chelating Agents / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D010449:Peptide Mapping / Q000379:methods*; D011485:Protein Binding; D011953:Receptors, Cyclic AMP / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D007502:Iron Chelating Agents; D010446:Peptide Fragments; D011953:Receptors, Cyclic AMP; D004247:DNA; D004492:Edetic Acid; D000242:Cyclic AMP