PubMed · 6546871
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G Büldt. 1984. Specific deuteration and membrane structures.. https://doi.org/10.1007/978-1-4899-0375-4_11
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The structure of the nicotinic acetylcholine receptor (nAChR) has been studied using a novel combination of hydrogen/deuterium exchange and attenuated total reflectance Fourier transform infrared spectroscopy. Fourier transform infrared spectra show marked changes in both the amide I and amide II bands upon exposure of the nAChR to 2H2O. The substantial decrease in intensity of the amide II band reflects the exchange of roughly 30% of the peptide hydrogens within seconds of exposure to 2H2O, 50% after 30 min, 60% after 12 h, and 75% after prolonged exposure for several days at room temperature or lower temperatures. The 30% of peptide hydrogens that exchange within seconds is highly exposed to solvent and likely involved in random and turn conformations, whereas the 25% of exchange-resistant peptide hydrogens is relatively inaccessible to solvent and likely located in the transmembrane domains of the nAChR. Marked changes occur in the amide I contour within seconds of exposure of the nAChR to 2H2O as a result of relatively large downshifts in the frequencies of amide I component bands assigned to turns and random structures. In contrast, only subtle change occur in the amide I contour between 3 min and 12 h after exposure to 2H2O as a result of slight downshifts in the frequencies of alpha-helix and beta-sheet vibrations. It is demonstrated that the time courses and relative magnitudes of the amide I component band shifts can be used both as an aid in the assignment of component bands to specific secondary structures and as a probe of the exchange rates of different types of secondary structures in the nAChR. Significantly, the intensities of the band shifts reflecting the exchange of alpha-helical secondary structures are relatively weak indicating that a large proportion of the 25% exchange resistant peptides adopt an alpha-helical conformation. Conversely, no evidence is found for the existence of a large number of exchange-resistant beta-strands. The exchange kinetics suggest a predominantly alpha-helical secondary structure for the transmembrane domains of the nAChR.
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H2-forming N5,N10-methylenetetrahydromethanopterin dehydrogenase from methanogenic Archaea, which is a novel hydrogenase containing neither nickel nor iron-sulfur clusters, catalyzes the reversible reduction of N5,N10-methenyltetrahydomethanopterin (CH identical to H4MPT+) with H2 to N5,N10-methylenetetrahydromethanopterin (CH2 = H4MPT) and a proton (delta G degree' = -5.5 kJ/mol). The enzyme also catalyzes a CH identical to H4MPT(+)-dependent H2/H+ exchange. We report here on kinetic deuterium isotope effects in these reactions. When CH identical to H4MPT+ reduction was performed with D2 instead of H2, Vmax and the Km did not change. A primary isotope effect of 1 was found at all pH and temperatures tested and independent of whether H2O or D2O was the solvent. The findings indicate that a step other than the activation of H2 was rate-determining in CH identical to H4MPT+ reduction with H2. This was substantiated by the observation that also the CH identical to H4MPT(+)-dependent H2/H+ exchange reaction did not exhibit an appreciable deuterium isotope effect. Vmax for CH2 = H4MPT dehydrogenation to CH identical to H4MPT+ and H2 was only 2-3 times higher than for CD2 = H4MPT dehydrogenation to CD identical to H4MPT+ and HD. Such a small primary isotope effect indicates that the breakage of the C-H bond in the methylene group of CH2 = H4MPT was only rate-limiting when hydrogen was substituted by a deuterium.