NMR studies of the influence of dodecyl sulfate on the amide hydrogen exchange kinetics of a micelle-solubilized hydrophobic tripeptide

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Abstract
Backbone amide hydrogen exchange measurements are an importnat 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.degree. C and using an indirect steady-state saturation-transfer technique at 25.degree. 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 amies 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. This is due to the electrostatic effect of the negative charge on the sulfate groups that concentrate protons in their vicinity, effectively lowering the pH in the microenvironment of the amides. These experiments help to explain the elevated pHmin observed for backbone amides in the sodium dodecyl sulfate solubilized M13 coat protein [O''Neil, J. D. J., and Sykes, B. D. (1988) Biochemistry 27, 2753-2762].