NMR studies of the influence of dodecyl sulfate on the amide hydrogen exchange kinetics of a micelle-solubilized hydrophobic tripeptide.
Backbone amide hydrogen exchange measurements are an important source of information about the internal dynamics of proteins. Before such measurements can be interpreted unambiguously, contributions to hydrogen exchange rates from the chemical and physical environment of the amides must be taken into account. Membrane proteins are often solubilized in detergents, yet there have not been any systematic investigations of the possible effects detergents may have on the amide hydrogen exchange rates of proteins. To address this question, we have measured individual backbone and carboxyl-terminal amide exchange rates for the amphipathic tripeptide Leu-Val-Ile-amide dissolved in water and dodecyl sulfate micelles. 1H NMR spectroscopy was used to measure exchange using the direct exchange-out into D2O technique at 5 degrees C and using an indirect steady-state saturation-transfer technique at 25 degrees C. The broadening effect of micelle-incorporated spin-labeled fatty acid (12-doxylstearate) on the 1H NMR spectra of both the detergent and the peptide resonances was used to demonstrate that the tripeptide is intimately associated with the micelle. The resonance from formate ion, which is excluded from the micelle, was unperturbed by the spin label. The detergent did not retard the exchange rates of either the primary (terminal) or secondary (backbone) amides of the tripeptide. This suggests that the micelle/peptide interaction does not restrict access of charged catalysts and water to these amides and shows that the peptide amides are not hydrogen bonded. However, the pH for the exchange minima of these amides in detergent was increased between 1.2 and 1.7 units compared to exchange in water.(ABSTRACT TRUNCATED AT 250 WORDS)
O'Neil
J D
JD
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Sykes
B D
BD
D000577:Amides; D003102:Colloids; D006863:Hydrogen-Ion Concentration; D007532:Isoleucine; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008433:Mathematics; D008823:Micelles; D008954:Models, Biological; D009842:Oligopeptides; D012967:Sodium Dodecyl Sulfate; D014633:Valine
D000577:Amides; D003102:Colloids; D008823:Micelles; D009842:Oligopeptides; D007532:Isoleucine; C059614:leucyl-valyl-isoleucinamide; D012967:Sodium Dodecyl Sulfate; D014633:Valine