pH-induced transitions in cholera toxin conformation: a fluorescence study.
Determination of the ratio of intrinsic fluorescence with dibrominated Bry 96 (F) relative to that with unbrominated Bry 96 (F0), at neutral pH and in the presence of 0.2 M NaCl, reveals that the A subunit of cholera toxin (CT A) has a somewhat higher affinity for this mild detergent than intact cholera toxin (CT) and its B subunit (CT B). Receptor (GM1 or oligo-GM1) binding has no influence on the very low detergent binding of CT and CT B. Activation of CT A by treatment with dithiothreitol (20 mM) also does not affect detergent binding. The weak hydrophobic nature of CT A is also reflected by the negative modulatory action of anionic phospholipids and deoxycholate on its mono-ADP-ribosyltransferase activity and the ability of the former to decrease its intrinsic fluorescence intensity in a salt-resistant way. Detergent binding of CT A is only slightly pH dependent whereas, upon lowering the pH, detergent binding to CT or CT B becomes significant. In the pH range 6.5-4.2 a gradual increase in detergent binding to CT and CT B occurs. In the narrow pH range 4.2-4.0 a sharp and time-dependent enhancement of brominated Bry 96 quenching is observed. The increase in detergent binding upon lowering the pH is fully reversible, salt dependent, and complete within 10 min (t1/2 = 2 min at 25 degrees C). Solute quenching experiments with the neutral polar quencher acrylamide reveal that upon lowering the pH to 5.0 a marked increase in the exposure of the lone Trp-88 residue in each beta-polypeptide chain of CT B occurs.(ABSTRACT TRUNCATED AT 250 WORDS)
D020106:Acrylamide; D000178:Acrylamides / Q000494:pharmacology; D002772:Cholera Toxin / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D010743:Phospholipids / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D012492:Salts / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology
D000178:Acrylamides; D003902:Detergents; D010743:Phospholipids; D012492:Salts; D020106:Acrylamide; D012965:Sodium Chloride; D002772:Cholera Toxin