Biomedical subjects
Publications and source records attributed to Y W He.
This paper deals with the extraction, determination and identification of the alkaloids in differently processed products of the seeds of Strychnos nux-vomica. The relationship between processing methods and toxicitys is discussed according to the comparison of acute toxicity.
The M-->N transition in the photocycle of bacteriorhodopsin involves the transfer of a proton from Asp96 to the retinylidene Schiff base, possibly through a network of hydrogen-bonded amino acid residues and water molecules (Rothschild, K. J., He, Y. W., Sonar, S., Marti, T., and Khorana, H. G. (1992) J. Biol. Chem. 267, 1615-1622). A conformational change of the protein backbone is also observed during this transition. In this work, we have investigated the effects of replacing the residue Thr46, which might be part of this chain, with an aspartic acid. Both Fourier transform infrared and resonance Raman spectroscopy show that the chromophore structure of this mutant (T46D) is normal. However, N formation is accelerated and N decay is significantly slowed compared to wild-type bacteriorhodopsin. This effect causes the N intermediate to accumulate under steady-state illumination thereby facilitating spectroscopic studies under normal pH conditions. Fourier transform infrared difference spectroscopy reveals that like native bacteriorhodopsin, N formation in T46D involves deprotonation of Asp96, reprotonation of the Schiff base, and a change in the backbone secondary structure. However, in contrast to bacteriorhodopsin, bands assigned to the C = O stretch mode of the carboxylic acid group of Asp96 are upshifted by 10 cm-1 reflecting a change in the Asp96 environment and a drop in its effective pKa throughout the photocycle. This change in the pKa can directly account for changes in the photocycle kinetics and indicates that Asp96 deprotonation/protonation are the rate limiting steps in the formation and decay of the N intermediate. By studying the effects of H/D exchange, evidence is found that the backbone structural changes involve transmembrane alpha-helices. It is proposed that these structural changes serve to modulate the local environment and protonation state of Asp96 during the photocycle and are also essential for formation of the proton conducting hydrogen bonded network which functions during Schiff base reprotonation.
The role of Thr-46 and Thr-89 in the bacteriorhodopsin photocycle has been investigated by Fourier transform infrared difference spectroscopy and time-resolved visible absorption spectroscopy of site-directed mutants. Substitutions of Thr-46 and Thr-89 reveal alterations in the chromophore and protein structure during the photocycle, relative to wild-type bacteriorhodopsin. The mutants T89D and to a lesser extent T89A display red shifts in the visible lambda max of the light-adapted states compared with wild type. During the photocycle, T89A exhibits an increased decay rate of the K intermediate, while a K intermediate is not detected in the photocycle of T89D at room temperature. In the carboxyl stretch region of the Fourier transform infrared difference spectra of T89D, a new band appears as early as K formation which is attributed to the deprotonation of Asp-89. Along with this band, an intensity increase occurs in the band assigned to the protonation of Asp-212. In the mutant T46V, a perturbation in the environment of Asp-96 is detected in the L and M intermediates which corresponds to a drop in its pK alpha. These data indicate that Thr-89 is located close to the chromophore, exerts steric constraints on it during all-trans to 13-cis isomerization, and is likely to participate in a hydrogen-bonding network that extends to Asp-212. In addition, a transient interaction between Thr-46 and Asp-96 occurs early in the photocycle. In order to explain these results, a previously proposed model of proton transport is extended to include the existence of a transient network of hydrogen-bonded residues. This model can account for the protonation changes of key amino acid residues during the photocycle of bacteriorhodopsin.
Though melioidosis has been reported since 1912, it is still a new problem for most Chinese physicians and medical scientists. Because of the lack of understanding and clinical experiences in melioidosis, none had been reported until 1990 in the mainland of China. In view of China's open policy and health work the natural foci of Pseudomonas pseudomallei in South China, ie. Hainan, Guangdong and Guangxi Provinces, should be given sufficient concern. Efforts should be made in training physicians in the endemic areas to improve their knowledge about the disease and to avoid misdiagnosis.
The role of Asp-96 in the bacteriorhodopsin (bR) photocycle has been investigated by time-resolved and static low-temperature Fourier transform infrared difference spectroscopy. Bands in the time-resolved difference spectra of bR were assigned by obtaining analogous time-resolved spectra from the site-directed mutants Asp-96----Ala and Asp-96----Glu. As concluded previously (Braiman, M. S., Mogi, T., Marti, T., Stern, L. J., Khorana, H. G., and Rothschild, K. J. (1988) Biochemistry 27, 8516-8520) Asp-96 is predominantly in a protonated state in the M intermediate. Upon formation of the N intermediate, deprotonation of Asp-96 occurs. This is consistent with its postulated role as a key residue in the reprotonation pathway leading from the cytoplasm to the Schiff base. A broad band centered at 1400 cm-1, which increases in intensity upon N formation is assigned to the Asp-96 symmetric COO- vibration. The Asp-96----Ala mutation also causes a delay in the Asp-212 protonation which normally occurs during the L----M transition. It is concluded that Asp-96 donates a proton into the Schiff base reprotonation pathway during N formation and that it accepts a proton from the cytoplasm during the N----O or O----bR transition.
The photocycle kinetics of the bacteriorhodopsin mutant Tyr-185----Phe has been investigated by UV-visible transient spectroscopy. Flash-induced spectral changes were measured from 100 ns to 500 ms using a gated optical multichannel analyzer on protein samples that were reconstituted in vesicles with Halobacterium halobium lipids. Tyr-185----Phe exhibits a pH-dependent absorbance spectrum reflecting contributions from two different species. At pH 6, the dominant photocycling species has a lambda max near 610 nm although the absorption maximum of light-adapted Tyr-185----Phe is at 581 nm. This red-shifted species does not form any M-like intermediate and undergoes a photocycle similar to that observed for deionized blue membrane. At pH 8, the dominant photoactive form exhibits a lambda max near 550 nm. This purple species, which is blue shifted 20 nm relative to wild-type bacteriorhodopsin, exhibits a photocycle similar to the wild type. However, M formation occurs in 8 microseconds, approximately three times faster than wild-type bacteriorhodopsin at pH 8. In addition, an unusually long lived intermediate absorbing at 610 nm is observed at high pH. In the UV region, a broad band near 300-310 nm is absent in the mutant relative to wild type, consistent with earlier measurements made at low temperature which suggest that Tyr-185 undergoes a change in protonation. Steady-state proton pumping action spectra indicate that the 550 nm species does transport protons but that the blue species is inactive. These results are discussed in terms of a model that hypothesizes that Tyr-185 is located close to the bacteriorhodopsin chromophore and stabilizes the interaction of helices F and G through formation of a polarizable bond with Asp-212.
The role of Asp-212 in the proton pumping mechanism of bacteriorhodopsin (bR) has been studied by a combination of site-directed mutagenesis and Fourier transform infrared difference spectroscopy. Difference spectra were recorded at low temperature for the bR----K and bR----M photoreactions of the mutants Asp-212----Glu, Asp-212----Asn, and Asp-212----Ala. Despite an increased proportion of the 13-cis form of bR (normally associated with dark adaptation), all of the mutants exhibited a light-adapted form containing as a principal component the normal all-trans retinal chromophore. The absence of a shift in the retinal C = C stretching frequency in these mutants indicates that Asp-212 is not a major determinant of the visible absorption wavelength maximum in light-adapted bR. It is unlikely that Asp-212 is the acceptor group for the Schiff base proton since both the Asp-212----Glu and Asp-212----Ala mutants formed an M intermediate. All of the Asp-212 mutants were missing a Fourier transform infrared difference band that had been assigned previously to protonation changes of Tyr-185. These results are discussed in terms of a model in which Tyr-185 and Asp-212 form a polarizable hydrogen bond and are positioned near the C13-Schiff base portion of the chromophore. These 2 residues may be involved in stabilizing the relative orientation of the F and G helices and isomerizing the retinal in a regioselective manner about the C13 = C14 double bond.
Fourier-transform infrared difference spectroscopy has been used to study the role of the three membrane-embedded proline residues, Pro-50, Pro-91, and Pro-186, in the structure and function of bacteriorhodopsin. All three prolines were replaced by alanine and glycine; in addition, Pro-186 was changed to valine. Difference spectra were recorded for the bR----K and bR----M photoreactions of each of these mutants and compared to those of wild-type bacteriorhodopsin. Only substitutions of Pro-186 caused significant perturbations in the frequency of the C = C and C - C stretching modes of the retinylidene chromophore. In addition, these substitutions reduced bands in the amide I and II region associated with secondary structural changes and altered signals assigned to the adjacent Tyr-185. Pro-186----Val caused the largest alterations, producing a second species similar to bR548 and nearly blocking chromophore isomerization at 78 K but not at 250 K. These results are consistent with a model of the retinal binding site in which Pro-186 and Tyr-185 are located in direct proximity to the chromophore and may be involved in linking chromophore isomerization to protein structural changes. Evidence is also found that Pro-50 may be structurally active during the bR----K transition and that substitution of this residue by glycine preserves the normal protein structural changes during the photocycle.
Structural changes involving bacteriohodopsin proline residues have been investigated by Fourier transform infrared difference spectroscopy. Bacteriohodopsin (bR)-producing Halobacteria halobium were grown on a stringent medium containing either ring-perdeuterated proline or 15N-labeled proline. Comparison of the difference spectra obtained from the photoreactions of these labeled bR samples with those for unlabeled bR has led to the assignment of peaks due to proline vibrations. [proline-N15]bR exhibited a 15-cm-1 isotopic downshift of peaks in the 1420- to 1440-cm-1 region of the bR----K and bR----M difference spectra as well as a similar downshift of peaks found in the absolute absorption spectrum of bR. In contrast, [proline-D7]bR did not cause shifts in this region of the difference spectra. These results indicate that one or more prolines undergo a structural rearrangement during the bR photocycle involving the Xaa-Pro C--N peptide bond. This change may be directly coupled to the light-induced isomerization of the retinal chromophore from all-trans-retinal to 13-cis-retinal.
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