Nonspecific protein-DNA interactions: complexation of alpha-chymotrypsin with a genomic DNA.
In this contribution, we report studies on nonspecific protein-DNA interactions of an enzyme protein bovine pancreatic alpha-chymotrypsin (CHT) with genomic DNA (from salmon testes) using two biologically common fluorescent probes: 1-anilinonaphthalene-8-sulfonate (ANS) and 2,6-p-toluidinonaphthalene sulfonate (TNS). TNS molecules that are nonspecifically bound to positively charged basic residues at the surface sites, not in the hydrophobic cavities of the protein, are preferentially displaced upon complexation of TNS-labeled CHT with DNA. The time-resolved fluorescence anisotropy of TNS molecules bound to hydrophobic cavities/clefts of CHT reveals that global tumbling motion of the protein is almost frozen in the protein-DNA complex. A control study on TNS-labeled human serum albumin (HSA) upon interaction with DNA clearly indicates that the ligands in the deep pockets of the protein cannot be displaced by interaction with DNA. We have also found that ANS, which binds to a specific surface site of CHT, is not displaced by DNA. The intactness of the ANS binding in CHT upon complexation with DNA offers the opportunity to measure the distance between the ANS binding site and the contact point of the ethidium bromide (EB)-labeled DNA using the Förster resonance energy transfer (FRET) technique. Enzymatic activity studies on CHT on a substrate (Ala-Ala-Phe 7-amido-4-methyl coumarin) reveal that the active site of the enzyme remains open for the substrate even in the protein-DNA complex. Circular dichroism (CD) studies on CHT upon complexation with DNA confirm the structural integrity of CHT in the complex. Our studies have attempted to explore an application of nonspecific protein-DNA interactions in the characterization of ligand binding of a protein in solution.
Narayanan
S Shankara
SS
Unit for Nano Science & Technology, Department of Chemical, S. N. Bose National Centre for Basic Sciences, Block JD, Sector III, Salt Lake, Kolkata, India.
Pal
Samir Kumar
SK
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002918:Chymotrypsin / Q000737:chemistry*; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry*; D004996:Ethidium / Q000737:chemistry; D006801:Humans; D008297:Male; D009282:Naphthalenesulfonates / Q000737:chemistry; D011485:Protein Binding; D012474:Salmon; D012709:Serum Albumin / Q000737:chemistry
D000817:Anilino Naphthalenesulfonates; D009282:Naphthalenesulfonates; D012709:Serum Albumin; C027132:1-anilino-8-naphthalenesulfonate; C009497:2-(4-toluidino)-6-naphthalenesulfonic acid; D004247:DNA; D002918:Chymotrypsin; D004996:Ethidium