Role of arginine-82 in fast proton release during the bacteriorhodopsin photocycle: a time-resolved FT-IR study of purple membranes containing 15N-labeled arginine

Arginine-82 has long been recognized as an important residue in bacteriorhodopsin (bR), because its mutation usually results in loss of fast H(+) release, an important step in the normal light-induced H(+) transport mechanism. To help to clarify the structural changes in Arg-82 associated with the H(+)-release step, we have measured time-resolved FT-IR difference spectra of wild-type bR containing either natural-abundance isotopes ((14)N-Arg-bR) or all seven arginines selectively and uniformly labeled with (15)N at the two eta-nitrogens ((15)N-Arg-bR). Comparison of the spectra from the two isotopic variants shows that a 1556 cm(-1) vibrational difference band due to the M photocycle intermediate of (14)N-Arg-bR loses substantial intensity in (15)N-Arg-bR. However, this isotope-sensitive arginine vibrational difference band is only observed at pH 7 and not at pH 4 where fast H(+) release is blocked. These observations support the earlier conclusion, based on site-directed mutagenesis and chemical labeling, that a strong C-N stretch vibration of Arg-82 can be assigned to a highly perturbed frequency near 1555 cm(-1) in the M state of wild-type bR [Hutson et al. (2000) Biochemistry 39, 13189-13200]. Furthermore, alkylguanidine model compound spectra indicate that the unusually low arginine C-N stretch frequency in the M state is consistent with a nearly stoichiometric light-induced deprotonation of an arginine side chain within bR, presumably arginine-82.     

Vibrational spectroscopy of bacteriorhodopsin mutants: I. Tyrosine-185 protonates and deprotonates during the photocycle

The techniques of FTIR difference spectroscopy and site-directed mutagenesis have been combined to investigate the role of individual tyrosine side chains in the proton-pumping mechanism of bacteriorhodopsin (bR). For each of the 11 possible bR mutants containing a single Tyr----Phe substitution, difference spectra have been obtained for the bR----K and bR----M photoreactions. Only the Tyr-185----Phe mutation results in the disappearance of a set of bands that were previously shown to be due to the protonation of a tyrosinate during the bR----K photoreaction [Rothschild et al.: Proceedings of the National Academy of Sciences of the United States of America 83:347, (1986]). The Tyr-185----Phe mutation also eliminates a set of bands in the bR----M difference spectrum associated with deprotonation of a Tyr; most of these bands (e.g., positive 1272-cm-1 peak) are completely unaffected by the other ten Tyr----Phe mutations. Thus, tyrosinate-185 gains a proton during the bR----K reaction and loses it again when M is formed. Our FTIR spectra also provide evidence that Tyr-185 interacts with the protonated Schiff base linkage of the retinal chromophore, since the negative C = NH+ stretch band shifts from 1640 cm-1 in the wild type to 1636 cm-1 in the Tyr-185----Phe mutant. A model that is consistent with these results is that Tyr-185 is normally ionized and serves as a counter-ion to the protonated Schiff base. The primary photoisomerization of the chromophore translocates the Schiff base away from Tyr-185, which raises the pKa of the latter group and results in its protonation.     

Diagnosing Growth Hormone Deficiency: Can a Combined Arginine and Clonidine Stimulation Test Replace 2 Separate Tests?

ObjectiveGiven the large number of false-positive growth hormone deficiency (GHD) diagnoses from a single growth hormone (GH) stimulation test in children, 2 different pharmacologic tests, performed on separate days or sequentially, are required. This study aimed to assess the reliability and safety of a combined arginine-clonidine stimulation test (CACST).MethodsThis was a retrospective, single-center, observational study. During 2017-2019, 515 children aged >8 years underwent GH stimulation tests (CACST: n = 362 or clonidine stimulation test [CST]: n = 153). The main outcome measures used to compare the tests were GH response (sufficiency/deficiency) and amplitude and timing of peak GH and safety parameters.ResultsPopulation characteristics were as follows: median age of 12.2 years (interquartile range [IQR]: 10.7, 13.4), 331 boys (64%), and 282 prepubertal children (54.8%). The GHD rate was comparable with 12.7% for CACST and 14.4% for CST followed by a confirmatory test (glucagon or arginine) (P = .609). Peak GH was higher and occurred later in response to CACST compared with CST (14.6 ng/mL [IQR: 10.6, 19.4] vs 11.4 ng/mL [IQR: 7.0, 15.8], respectively, P < .001; 90 minutes [IQR: 60, 90] vs 60 minutes [IQR: 60, 90], respectively, P < .001). No serious adverse events occurred following CACST.ConclusionOur findings demonstrate the reliability and safety of CACST in detecting GHD in late childhood and adolescence, suggesting that it may replace separate or sequential GH stimulation tests. By diminishing the need for the second GH stimulation test, CACST saves time, is more cost-effective, and reduces discomfort for children, caregivers, and medical staff.     

Vibrational spectra of individual millimeter-size membrane patches using miniature infrared waveguides.

We have used miniature planar IR waveguides, consisting of Ge strips 30-50 microm thick and 2 mm wide, as evanescent-wave sensors to detect the mid-(IR) evanescent-wave absorbance spectra of small areas of biomolecular monolayers and multilayers. Examples include picomolar quantities of an integral transmembrane protein (bacteriorhodopsin) and lipid (dimyristoyl phosphatidylcholine). IR bands due to the protein and lipid components of the plasma membrane of individual 1.5-mm-diameter devitellinized Xenopus laevis oocytes, submerged in buffer and sticking to the waveguide surface, were also detected. A significant improvement in sensitivity was observed, as compared to previous sizes and geometries of evanescent-wave sensors (e.g., commercially available internal reflection elements or tapered optical fibers). These measurements suggest the feasibility of using such miniature supported planar IR waveguides to observe structural changes in transmembrane proteins functioning in vivo in single cells.     

Management of Fully Pubertal Girls With Nonclassical Congenital Adrenal Hyperplasia: Glucocorticoids Versus Oral Contraceptives

ObjectiveTo compare clinical outcomes of 3 treatment regimens—glucocorticoids (GCs), oral contraceptives (OCs), or a combination of both—administered to adolescents and young women diagnosed in childhood with nonclassical congenital adrenal hyperplasia (NCCAH), who had been treated with GCs until their adult height was achieved.MethodsA retrospective study of medical records of 53 female patients with NCCAH followed in 3 tertiary pediatric endocrinology institutes. The 3 treatment groups were compared for the prevalence of hirsutism and acne, standardized body mass index (BMI)-standard deviation score (SDS), and androgen levels at the attainment of adult height (baseline), 1-year later, and at the last documented visit.ResultsAt baseline, there were no significant differences among groups in BMI-SDS, androgen levels, hirsutism prevalence, acne, or irregular menses. From baseline to the last visit, the rate of hirsutism declined significantly only in the OC group (37.5% vs 6.2%, respectively; P = .03). The rate of acne declined in the combined group (50% vs 9%, respectively; P = .03) with a similar tendency in the OC group (50% vs 12.5%, respectively; P = .05). No significant changes were observed in BMI-SDS for the entire cohort or any subgroup during follow-up. A significant rise in androstenedione (P < .001), testosterone (P < .01), and 17-hydroxyprogesterone (P < .01) levels was observed only in the OC group.ConclusionIn girls diagnosed in childhood with NCCAH, who require treatment for hyperandrogenism following completion of linear growth, management should be tailored individually using a patient-centered approach. Treatment with OCs might be better than that with GCs for regression of hirsutism and acne. The long-term effects of elevated levels of androgens associated with this treatment regimen should be further studied.     

An Edible-to-insects Calcium Alginate Gel as a Carrier for Entomopathogenic Nematodes

A carrier for entomopathogenic nematodes based on an edible-to-insects calcium alginate gel was developed. The alginate system was produced by external setting through an interaction between an aqueous sodium alginate mixture and calcium ions under acidic conditions. Sodium hexa-metaphosphate was used to control gel formation. Yeast extract used in the gel as a phagostimulant for Spodoptera littoralis larvae improved the insect's relative consumption rate and digestibility. The nematodes in the gel effectively controlled the larvae in a 24-h leaf bioassay, although nematode survival in the gel was ~ 50%. Gels subjected to 31% relative humidity (RH) prior to larval feeding became desiccated and were inedible to insects. However, gels at 61% RH supported larval feeding, although the water loss from the gel due to evaporation from 200-400-mg gel cubes at this humidity exceeded 50%. The gel might be a useful delivery system for nematodes against insects infesting the plant canopy in greenhouses.     

SARS-CoV-2 spike variants exhibit differential infectivity and neutralization resistance to convalescent or post-vaccination sera

1. Download : Download high-res image (186KB)
2. Download : Download full-size image

     

Detection of fast light-activated H+ release and M intermediate formation from proteorhodopsin.

Background  

Proteorhodopsin (pR) is a light-activated proton pump homologous to bacteriorhodopsin and recently discovered in oceanic γ-proteobacteria. One perplexing difference between these two proteins is the absence in pR of homologues of bR residues Glu-194 and Glu-204. These two residues, along with Arg-82, have been implicated in light-activated fast H⁺ release to the extracellular medium in bR. It is therefore uncertain that pR carries out its physiological activity using a mechanism that is completely homologous to that of bR.     

Conserved amino acids in F-helix of bacteriorhodopsin form part of a retinal binding pocket

A 3-dimensional model for the retinal binding pocket in the light-driven proton pump, bacteriorhodopsin, is proposed on the basis of spectroscopic studies of bacteriorhodopsin mutants. In this model Trp-182, Pro-186 and Trp-189 surround the polyene chain while Tyr-185 is positioned close to the retinylidene Schiff base. This model is supported by sequence homologies in the F-helices of bacteriorhodopsin and the related retinal proteins, halorhodopsin and rhodopsins.