Abstract:
Nuclear magnetic resonance (NMR) spectroscopy using personal computer-based hardware has the potential of enabling the application of NMR methods to fields where conventional state of the art equipment is either impractical or too costly. With such a strategy for data acquisition and processing, disciplines including civil engineering, agriculture, geology, archaeology, and others have the possibility of utilizing magnetic resonance techniques within the laboratory or conducting applications directly in the field. Another aspect is the possibility of utilizing existing NMR magnets which may be in good condition but unused because of outdated or nonrepairable electronics. Moreover, NMR applications based on personal computer technology may open up teaching possibilities at the college or even secondary school level. The goal of developing such a personal computer (PC)-based NMR standard is facilitated by existing technologies including logic cell arrays, direct digital frequency synthesis, use of PC-based electrical engineering software tools to fabricate electronic circuits, and the use of permanent magnets based on neodymium-iron-boron alloy. Utilizing such an approach, we have been able to place essentially an entire NMR spectrometer console on two printed circuit boards, with the exception of the receiver and radio frequency power amplifier. Future upgrades to include the deuterium lock and the decoupler unit are readily envisioned. The continued development of such PC-based NMR spectrometers is expected to benefit from the fast growing, practical, and low cost personal computer market.
The conformation of retinal bound to the G protein-coupled receptor rhodopsin is intimately linked to its photochemistry, which initiates the visual process. Site-directed deuterium ((2)H) NMR spectroscopy was used to investigate the structure of retinal within the binding pocket of bovine rhodopsin. Aligned recombinant membranes were studied containing rhodopsin that was regenerated with retinal (2)H-labeled at the C(5), C(9), or C(13) methyl groups by total synthesis. Studies were conducted at temperatures below the gel to liquid-crystalline phase transition of the membrane lipid bilayer, where rotational and translational diffusion of rhodopsin is effectively quenched. The experimental tilt series of (2)H NMR spectra were fit to a theoretical line shape analysis [Nevzorov, A. A., Moltke, S., Heyn, M. P., and Brown, M. F. (1999) J. Am. Chem. Soc. 121, 7636-7643] giving the retinylidene bond orientations with respect to the membrane normal in the dark state. Moreover, the relative orientations of pairs of methyl groups were used to calculate effective torsional angles between different planes of unsaturation of the retinal chromophore. Our results are consistent with significant conformational distortion of retinal, and they have important implications for quantum mechanical calculations of its electronic spectral properties. In particular, we find that the beta-ionone ring has a twisted 6-s-cis conformation, whereas the polyene chain is twisted 12-s-trans. The conformational strain of retinal as revealed by solid-state (2)H NMR is significant for explaining the quantum yields and mechanism of its ultrafast photoisomerization in visual pigments. This work provides a consensus view of the retinal conformation in rhodopsin as seen by X-ray diffraction, solid-state NMR spectroscopy, and quantum chemical calculations.
PMID: 843526;Abstract:
The tyrosyl and tryptophyl fluorescence of diisopropylphosphorylsubtilisins Carlsberg and Novo, respectively, is quenched efficiently by I- but is not significantly affected by Cs+. The I- quenching data were analyzed using a modified Stern-Volmer treatment (Lehrer, S. S. (1971), Biochemistry 10, 3254), yielding values for the effective fraction of accessible protein fluorescence of 90-95 and 88-92% for the tyrosyl and tryptophyl emission of diisopropylphosphorylsubtilisins Carlsberg and Novo, respectively. Similar values were obtained at pH 5 and 7. The effective collisional quenching constant depends on pH in a manner suggesting the participation of protein surface charge in the quenching mechanism. Significant singlet energy transfer (efficiency = 0.52) from tyrosyl to tryptophyl residues was inferred from the excitation spectra of diisopropylphosphorylsubtilisin Novo. The very low efficiency of energy transfer to Trp-113 in diisopropylphosphorylsubtilisin Carlsberg suggests that Trp-105 and Trp-241 are the acceptors of tyrosyl emission in the homologous Novo enzyme. The unusually low quantum yield of Trp-113 in diisopropylphosphorylsubtilisin Carlsberg together with the tryptophyl fluorescence quenching behavior of the Novo enzyme suggests that this residue is "buried" and inaccessible to quenching in both enzymes. The tyrosyl quenching behavior of diisopropylphosphorylsubtilisin Carlsberg is consistent with the high degree of solvent exposure of aromatic residues evident in the x-ray model of subtilisin Novo.
