Unfolding of diphtheria toxin. Identification of hydrophobic sites exposed on lowering of pH by photolabeling.
We report here the use of a hydrophobic photoactivable reagent, 2-[3H]diazofluorene (DAF), to map the hydrophobic sites exposed when the pH is lowered in diphtheria toxin (DT). The reagent binds to DT, and on photolysis with light of wavelength >350 nm, it covalently attaches itself to DT. The labeling was observed to increase considerably when the pH was lowered from 7.4 to 5.2. Although both A- and B-chains were labeled to a similar degree at pH 7.4, at lower pH (5.2), B-chain was labeled to a much higher extent. Subsequent chemical and enzymatic fragmentation of DT followed by separation indicated that the putative transmembrane domain was labeled to its maximum extent at pH 5.2, with the bulk of labeling associated with residues 340-459. Protein sequencing analysis indicated that the two buried hydrophobic helices, identified in the crystal structure and suggested to insert and span the membrane bilayer, corresponding to residues 326-347 and 358-376, are strongly labeled. The Pro-345 residue was observed to be labeled maximally at lower pH values. Finally, the DAF labeling pattern indicated that the parent structural motifs are retained at low pH, suggesting that the low pH conformation of DT corresponds to an equilibrium molten globule state.
D'Silva
P R
PR
Biomembrane Laboratory, Department of Chemistry, and Biotechnology Center, Indian Institute of Technology Bombay, Powai, Bombay 400 076, India.
Lala
A K
AK
D000345:Affinity Labels; D003979:Diazonium Compounds; D004167:Diphtheria Toxin / Q000737:chemistry*; D005449:Fluorenes; D006863:Hydrogen-Ion Concentration; D010449:Peptide Mapping; D010777:Photochemistry; D017510:Protein Folding
D000345:Affinity Labels; D003979:Diazonium Compounds; D004167:Diphtheria Toxin; D005449:Fluorenes; C052770:2-diazofluorene