The use of tryptophan mutants in identifying the 296 nm transient absorbing species during the photocycle of bacteriorhodopsin

The transient absorption at 296 nm was part of the spectroscopic evidence that initiated the proposal that tyrosinate (Tyr-) is formed during, and important to, the photocycle of bacteriorhodopsin (bR). Recent evidence against such a proposal comes from the results of NMR, UV Raman as well as electron cryo-microscopic structural studies. This makes it credible to assign this absorption to a charge perturbation of the lowest energy absorption of one of the tryptophan (Trp) residues in bR. The transient absorption at 296 nm is examined for each of 8 tryptophan mutants in which Trp is substituted by phenylalanine or cysteine, which absorb at shorter wavelength. It is shown that while all go through the photocycle, all but Trp-182 mutant show this transient absorption. This strongly suggests the assignment of this absorption to a charge perturbaton of the lowest energy absorption of Trp-182 during the photocycle. The chemical identity of the perturbing charge(s) is briefly discussed.     

Cloning and characterization of the Brucella ovis heat shock protein DnaK functionally expressed in Escherichia coli.

The Brucella ovis dnaK gene, homolog to the eukaryotic hsp70 genes, was cloned by using a Drosophila melanogaster probe. Comparison of B. ovis and Escherichia coli sequences revealed a similar organization for the dnaK and dnaJ genes and putative regulatory signals. In E. coli transfected with the cloned fragment, B. ovis hsp70 was expressed at 30 and 50 degrees C apparently under the control of its own promoter. The recombinant protein and a B. ovis native protein displaying the same molecular weight were both recognized by anti-E. coli DnaK serum. Native B. ovis protein was also recognized by sera of sheep either infected or vaccinated with an attenuated Brucella strain, suggesting that Brucella hsp70 could be up-regulated during host colonization. A thermosensitive E. coli dnaK mutant transfected with the cloned fragment recovered colony-forming ability at 42 degrees C, showing that the B. ovis DnaK protein could behave as a functional heat shock protein in E. coli.     

Effects of tryptophan mutation on the deprotonation and reprotonation kinetics of the Schiff base during the photocycle of bacteriorhodopsin.

The rates of deprotonation and reprotonation of the protonated Schiff base (PSB) are determined during the photocycle of nine bacteriorhodopsin mutants in which Trp-10, 12, 80, 86, 137, 138, 182 and 189 are individually substituted by either phenylalanine or cysteine. Of all the mutants, the replacement of Trp-86, Trp-182, and Trp-189 by phenylalanine and Trp-137 by cysteine is found to significantly alter the rate of the deprotonation, but not that of the reprotonation process. As compared with ebR, the Trp-86 mutation dramatically increases the rate of deprotonation of the PSB while the Trp-182 mutation greatly decreases this rate. Temperature dependence studies on the rate constants of the deprotonation demonstrate that the different energetic and entropic effects of the mutation are responsible for the observed different kinetic behavior of the Trp-86 and Trp-182 mutants as compared with that of ebR. In the case of Trp-86 mutant, a large decrease in both energy and entropy of activation suggests that the mutation of this tryptophan residue opens up the protein structure as a result of eliminating the hydrogen-bonding group on its side chain by a phenylalanine substitution. A correlation is observed between the proton pumping yield and the relative amplitudes of the slow deprotonation component but not with rate constants of the rise or decay process at constant pH. These results are best discussed in terms of the heterogeneity model (with parallel cycle) rather than back reaction model.     

Vibrational spectroscopy of bacteriorhodopsin mutants. Evidence for the interaction of aspartic acid 212 with tyrosine 185 and possible role in the proton pump mechanism

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.     

Bladder tissue characterization using probe‐based Raman spectroscopy: Evaluation of tissue heterogeneity and influence on the model prediction

Existing approaches for early‐stage bladder tumor diagnosis largely depend on invasive and time‐consuming procedures, resulting in hospitalization, bleeding, bladder perforation, infection and other health risks for the patient. The reduction of current risk factors, while maintaining or even improving the diagnostic precision, is an underlying factor in clinical instrumentation research. For example, for clinic surveillance of patients with a history of noninvasive bladder tumors real‐time tumor diagnosis can enable immediate laser‐based removal of tumors using flexible cystoscopes in the outpatient clinic. Therefore, novel diagnostic modalities are required that can provide real‐time in vivo tumor diagnosis. Raman spectroscopy provides biochemical information of tissue samples ex vivo and in vivo and without the need for complicated sample preparation and staining procedures. For the past decade there has been a rise in applications to diagnose and characterize early cancer in different organs, such as in head and neck, colon and stomach, but also different pathologies, for example, inflammation and atherosclerotic plaques. Bladder pathology has also been studied but only with little attention to aspects that can influence the diagnosis, such as tissue heterogeneity, data preprocessing and model development. The present study presents a clinical investigative study on bladder biopsies to characterize the tumor grading ex vivo, using a compact fiber probe‐based imaging Raman system, as a crucial step towards in vivo Raman endoscopy. Furthermore, this study presents an evaluation of the tissue heterogeneity of highly fluorescent bladder tissues, and the multivariate statistical analysis for discrimination between nontumor tissue, and low‐ and high‐grade tumor.     

Relationships between Methylobacteria and Glyphosate with Native and Invasive Plant Species: Implications for Restoration

After removing invasive plants, whether by herbicides or other means, typical restoration design focuses on rebuilding native plant communities while disregarding soil microbial communities. However, microbial–plant interactions are known to influence the relative success of native versus invasive plants. Therefore, the abundance and composition of soil microorganisms may affect restoration efforts. We assessed the effect of herbicide treatment on phytosymbiotic pink‐pigmented facultative methylotrophic (PPFM) bacteria and the potential consequences of native and invasive species establishment post‐herbicide treatment in the lab and in a coastal sage scrub (CSS)/grassland restoration site. Lab tests showed that 4% glyphosate reduced PPFM abundance. PPFM addition to seeds increased seedling length of a native plant (Artemisia californica) but not an invasive plant (Hirschfeldia incana). At the restoration site, methanol addition (a PPFM substrate) improved native bunchgrass (Nassella pulchra) germination and size by 35% over controls. In a separate multispecies field experiment, PPFM addition stimulated the germination of N. pulchra, but not that of three invasive species. Neither PPFM nor methanol addition strongly affected the growth of any plant species. Overall, these results are consistent with the hypothesis that PPFMs have a greater benefit to native than invasive species. Together, these experiments suggest that methanol or PPFM addition could be useful in improving CSS/grassland restorations. Future work should test PPFM effects on additional species and determine how these results vary under different environmental conditions.     

The Active and the Inactive Form of the hAT1 Receptor Have an Identical Ligand-Binding Environment: An MPA Study on a Constitutively Active Angiotensin II Receptor Mutant

Several models of activation mechanisms were proposed for G protein-coupled receptors (GPCRs), yet no direct methods exist for their elucidation. The availability of constitutively active mutants has given an opportunity to study active receptor conformations within acceptable limits using models such as the angiotensin II type 1 (AT₁)¹ receptor mutant N111G-hAT₁ which displays an important constitutive activity. Recently, by using methionine proximity assay, we showed for the hAT₁ receptor that TMD III, VI, and VII form the ligand-binding pocket of the C-terminal amino acid of an antagonistic AngII analogue. In the present contribution, we investigated whether the same residues would also constitute the ligand-binding contacts in constitutively activated mutant (CAM) receptors. For this purpose, the same Met mutagenesis strategy was carried out on the N111G double mutants. Analysis of 43 receptors mutants in the N111G-hAT₁ series, photolabeled and CNBr digested, showed that there were only subtle structural changes between the wt-receptor and its constitutively active form.