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S O Smith

Publications and source records attributed to S O Smith.

64 records · Page 4Linked to original sources
PubMed1985

Determination of retinal chromophore structure in bacteriorhodopsin with resonance Raman spectroscopy.

The analysis of the vibrational spectrum of the retinal chromophore in bacteriorhodopsin with isotopic derivatives provides a powerful "structural dictionary" for the translation of vibrational frequencies and intensities into structural information. Of importance for the proton-pumping mechanism is the unambiguous determination of the configuration about the C13=C14 and C=N bonds, and the protonation state of the Schiff base nitrogen. Vibrational studies have shown that in light-adapted BR568 the Schiff base nitrogen is protonated and both the C13=C14 and C=N bonds are in a trans geometry. The formation of K625 involves the photochemical isomerization about only the C13=C14 bond which displaces the Schiff base proton into a different protein environment. Subsequent Schiff base deprotonation produces the M412 intermediate. Thermal reisomerization of the C13=C14 bond and reprotonation of the Schiff base occur in the M412------O640 transition, resetting the proton-pumping mechanism. The vibrational spectra can also be used to examine the conformation about the C--C single bonds. The frequency of the C14--C15 stretching vibration in BR568, K625, L550 and O640 argues that the C14--C15 conformation in these intermediates is s-trans. Conformational distortions of the chromophore have been identified in K625 and O640 through the observation of intense hydrogen out-of-plane wagging vibrations in the Raman spectra (see Fig. 2). These two intermediates are the direct products of chromophore isomerization. Thus it appears that following isomerization in a tight protein binding pocket, the chromophore cannot easily relax to a planar geometry. The analogous observation of intense hydrogen out-of-plane modes in the primary photoproduct in vision (Eyring et al., 1982) suggests that this may be a general phenomenon in protein-bound isomerizations. Future resonance Raman studies should provide even more details on how bacterio-opsin and retinal act in concert to produce an efficient light-energy convertor. Important unresolved questions involve the mechanism by which the protein catalyzes deprotonation of the L550 intermediate and the mechanism of the thermal conversion of M412 back to BR568. Also, it has been shown that under conditions of high ionic strength and/or low light intensity two protons are pumped per photocycle (Kuschmitz & Hess, 1981). How might this be accomplished?(ABSTRACT TRUNCATED AT 400 WORDS)

Bacteriorhodopsins↗
PubMed1985

Resonance Raman spectra of the acidified and deionized forms of bacteriorhodopsin.

The 568-nm absorption band of light-adapted bacteriorhodopsin (BR) shifts to 605 nm at pH 2, forming BR605A, and it shifts back to 565 nm at pH 0, forming BR565A. We have obtained resonance Raman spectra of BR605A and BR565A using purple membrane samples that have been suspended in a rotating Raman cell with a polyacrylamide gel. Raman spectra were also obtained of purple membrane in deionized solutions (BR605D). The spectra of BR605A and BR605D are very similar, and they correspond closely with the Raman spectrum of dark-adapted BR, which contains an approximately equal mixture of 13-cis and all-trans retinal protonated Schiff-base chromophores. This shows that BR605A and BR605D are not homogeneous molecular species but contain a mixture of pigment molecules with both 13-cis and all-trans retinal isomers. The Raman spectrum of BR565A is nearly identical to that of light-adapted BR, demonstrating that BR565A contains an all-trans protonated Schiff-base chromophore. These data provide constraints on the possible structural changes that can be invoked to explain the spectral shifts induced in the acid and deionized species.

Bacteriorhodopsins↗
PubMed1985

Vibrational analysis of the all-trans retinal protonated Schiff base.

We have obtained Raman spectra of a series of all-trans retinal protonated Schiff-base isotopic derivatives. 13C-substitutions were made at the 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 positions while deuteration was performed at position 15. Based on the isotopic shifts, the observed C--C stretching vibrations in the 1,100-1,400 cm-1 fingerprint region are assigned. Normal mode calculations using a modified Urey-Bradley force field have been refined to reproduce the observed frequencies and isotopic shifts. Comparison with fingerprint assignments of all-trans retinal and its unprotonated Schiff base shows that the major effect of Schiff-base formation is a shift of the C14--C15 stretch from 1,111 cm-1 in the aldehyde to approximately 1,163 cm-1 in the Shiff base. This shift is attributed to the increased C14--C15 bond order that results from the reduced electronegativity of the Schiff-base nitrogen compared with the aldehyde oxygen. Protonation of the Schiff base increases pi-electron delocalization, causing a 6 to 16 cm-1 frequency increase of the normal modes involving the C8--C9, C10--C11, C12--C13, and C14--C15 stretches. Comparison of the protonated Schiff base Raman spectrum with that of light-adapted bacteriorhodopsin (BR568) shows that incorporation of the all-trans protonated Schiff base into bacterio-opsin produces an additional approximately 10 cm-1 increase of each C--C stretching frequency as a result of protein-induced pi-electron delocalization. Importantly, the frequency ordering and spacing of the C--C stretches in BR568 is the same as that found in the protonated Schiff base.

Retinaldehyde↗
PubMed1984

Structure of the retinal chromophore in the hR578 form of halorhodopsin.

Halorhodopsin is a retinal-containing pigment that is thought to function as a light-driven chloride ion pump in the cell membrane of Halobacterium halobium. To address the role of the retinal chromophore in chloride ion transport, resonance Raman spectra have been obtained of the hR578 form of chromatographically purified halorhodopsin (hR). The close similarity of the frequencies and intensities of the hR578 Raman bands with those of light-adapted bacteriorhodopsin (bR568) shows that the chromophore in hR578 has an all-trans configuration and that the protein environment around the chromophore in these two pigments is very similar. In addition, hR578 exhibits a Raman line at 1633 cm-1 which is assigned as the stretching vibration of a protonated Schiff base linkage to the protein based on its shift to 1627 cm-1 in D2O. The reduced frequency of the Schiff base stretching vibration compared with bR568 (1640 cm-1) is shown to result from a reduction of its coupling with the NH in-plane rock. This may be due to a reduction in hydrogen-bonding between the Schiff base proton and an electronegative counterion in halorhodopsin.

Bacteriorhodopsins↗
PubMed1984

Solid-state 13C NMR studies of retinal in bacteriorhodopsin.

Solid-state 13C magic-angle sample spinning (MASS) NMR has been used to study lyophilized dark-adapted purple membrane containing 13C-labeled retinals. C-10-, C-11-, and C-12-labeled derivatives each showed two lines, assigned to the coexisting 13-cis and all-trans isomers. The isotropic chemical shifts, particularly of C-11, indicate that the Schiff base is protonated. Shift anisotropies are also similar to those of model compounds, indicating that this part of the chromophore is rigid and immobile and possesses the same degree of in-plane bending as crystalline retinal derivatives. Purple membrane samples labeled on the C-19- and C-20-methyl groups both give single lines from the retinal, upfield shifted by 2.1 and 1.0 ppm, respectively, from model compounds. In all cases, high-quality spectra were obtained from approximately 50-mg samples in modest signal-averaging times. These results suggest that it is now practical to exploit the enormous potential of MASS NMR for structural studies of 13C-labeled membrane proteins.

Bacteriorhodopsins↗
PubMed1984

Dark-adapted bacteriorhodopsin contains 13-cis, 15-syn and all-trans, 15-anti retinal Schiff bases.

13C NMR spectra of lyophilized dark-adapted [14-13C]retinyl-labeled bacteriorhodopsin show a large anomalous upfield shift for the 13C-14 resonance assigned to the 13-cis isomer, relative to both the all-trans isomer and model compounds. We attribute this to the so-called gamma effect, which results from a steric interaction between the C-14 retinal proton and the protons on the epsilon CH2 of the lysine. As a consequence of this observation, we infer that dark-adapted bacteriorhodopsin is composed of a mixture of all-trans, 15-anti (trans or E) and 13-cis, 15-syn (cis or Z) isomers. These occur in an approximate 4:6 ratio and are commonly identified as bR568 and bR548. This conclusion is based on an examination of the isotropic and anisotropic chemical shifts and a comparison with 13C shifts of the carbons adjacent to the C = N linkage in protonated ketimines. Other possible origins for the anomalous shift are examined and shown to be insufficient to account for either the size of the shift or the nature of the shift tensor. We discuss the consequences of this finding for the structure and photochemistry of bacteriorhodopsin.

Bacteriorhodopsins↗
PubMed1984

Determination of retinal Schiff base configuration in bacteriorhodopsin.

Resonance Raman spectra of the BR(568), BR(548), K(625), and L(550) intermediates of the bacteriorhodopsin photocycle have been obtained in (1)H(2)O and (2)H(2)O by using native purple membrane as well as purple membrane regenerated with 14,15-(13)C(2) and 12,14-(2)H(2) isotopic derivatives of retinal. These derivatives were selected to determine the contribution of the C(14)-C(15) stretch to the normal modes in the 1100- to 1400-cm(-1) fingerprint region and to characterize the coupling of the C(14)-C(15) stretch with the NH rock. Normal mode calculations demonstrate that when the retinal Schiff base is in the C[unk]N cis configuration the C(14)-C(15) stretch and the NH rock are strongly coupled, resulting in a large ( approximately 50-cm(-1)) upshift of the C(14)-C(15) stretch upon deuteration of the Schiff base nitrogen. In the C[unk]N trans geometry these vibrations are weakly coupled and only a slight (<5-cm(-1)) upshift of the C(14)-C(15) stretch is predicted upon N-deuteration. In BR(568), the insensitivity of the 1201-cm(-1) C(14)-C(15) stretch to N-deuteration demonstrates that its retinal C[unk]N configuration is trans. The C(14)-C(15) stretch in BR(548), however, shifts up from 1167 cm(-1) in (1)H(2)O to 1208 cm(-1) in (2)H(2)O, indicating that BR(548) contains a C[unk]N cis chromophore. Thus, the conversion of BR(568) to BR(548) (dark adaptation) involves isomerization about the C[unk]N bond in addition to isomerization about the C(13)[unk]C(14) bond. The insensitivity of the native, [14,15-(13)C(2)]-, and [12,14-(2)H(2)]K(625) and L(550) spectra to N-deuteration argues that these intermediates have a C[unk]N trans configuration. Thus, the primary photochemical step in bacteriorhodopsin (BR(568) --> K(625)) involves isomerization about the C(13)[unk]C(14) bond alone. The significance of these results for the mechanism of proton-pumping by bacteriorhodopsin is discussed.

Journal Article↗
PubMed1979

Changes in the activities of protein kinase modulators in the cerebellum of mice due to ethanol, caffeine, or phenobarbital administration.

Decreased activities of both the inhibitory modulator of adenosine 3':5'-monophosphate (cAMP)-dependent protein kinase (A-PK) as well as the stimulatory modulator of guanosine 3':5'-monophosphate (cGMP)-dependent protein kinase (G-PK) from the mouse cerebellum were noted due to the administration of excessive doses of ethanol, caffeine, and phenobarbital for up to 28 days. The dose-dependent of the inhibition of A-PK or the stimulation of G-PK was observed as a function of the amount of protein kinase modulators in the cerebellum of mice receiving different doses of ethanol.

Animals↗
PubMedDate not supplied

High-resolution structural studies of the retinal--Glu113 interaction in rhodopsin.

The key to understanding the reaction mechanism of rhodopsin lies in determining the structure of the retinal binding site and in defining the charge interactions between Glu113 and the retinal protonated Schiff base chromophore. We have been using 13C-NMR chemical shift data to determine the location of the Glu113 carboxyl side chain in relation to the retinal. The NMR data constrain one of the carboxylate oxygens of Glu113 to be ca. 3 A from the C12 position of the retinal with the second oxygen oriented away from the conjugated chain. A water molecule forming a hydrogen bond with the Schiff base is incorporated into the model to account for the high C = N stretching frequency [Han et al., Biophys. J., 65 (1993) 899]. In this study, we have refined the counterion position and have shown that it can reproduce the observed chemical shift data as well as the red-shifted absorption maximum of rhodopsin. Furthermore, the retinal binding site geometry derived from the NMR constraints can be readily incorporated into a recent structural model of the apoprotein.

Amino Acid Sequence↗