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-[H-3 ]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 it self to DT. The labeling was observed to increase considerably when th e pH was lowered from 7.4 to 5.2. Although both A- and B-chains were l abeled to a similar degree at pH 7,4, at lower pH (5.2), B-chain was l abeled to a much higher extent. Subsequent chemical and enzymatic frag mentation of DT followed by separation indicated that the putative tra nsmembrane domain was labeled to its maximum extent at pH 5,2, with th e bulk of labeling associated with residues 340-459. Protein sequencin g analysis indicated that the two buried hydrophobic helices, identifi ed in the crystal structure and suggested to insert and span the membr ane bilayer, corresponding to residues 326-347 and 358-376, are strong ly labeled. The Pro-345 residue was observed to be labeled maximally a t lower pH values. Finally, the DAF labeling pattern indicated that th e parent structural motifs are retained at low pH, suggesting that the low pH conformation of DT corresponds to an equilibrium molten globul e state.