Comparative assessment of immunogenicity of recombinant insulin Aspart from BioGenomics and its originator NovoRapid® in adult patients with type 2 diabetes mellitus.

OBJECTIVES: To assess and compare the immunogenicity of recombinant Insulin Aspart [manufactured by BioGenomics Limited (BGL-ASP)] with its originator NovoRapid® (manufactured by Novo Nordisk) in adult patients with type 2 diabetes mellitus. RESEARCH DESIGN AND METHODS: BGL-IA-CTP301 study was a randomized, open label, parallel group, multicenter phase-III clinical study to compare the efficacy and safety of recombinant Insulin Aspart 100 U/mL [manufactured by BioGenomics Limited (BGL-ASP)] with its reference medicinal product (RMP); NovoRapid® [manufactured by Novo Nordisk], in adult patients with Type 2 diabetes mellitus (T2DM). The primary objective of the study was to compare the immunogenicity of BGL-ASP and RMP; NovoRapid® in patient serum samples collected from phase-III clinical study. Immunogenicity was studied as the incidence of patients positive for anti-insulin Aspart (AIA) antibodies, developed against BGL-ASP/RMP at baseline, end of 12 week and end of 24 week of the treatment period. The changes in incidence of patients positive for AIA antibodies post-baseline were also studied to assess and compare the treatment-emergent antibody response (TEAR) between the treatment groups (BGL-ASP and RMP). Statistical evaluation was done by Fisher's exact test to compare the overall incidence of patients positive for AIA antibodies and the TEAR positives observed post-baseline in both the treated groups. An in-vitro neutralizing antibody assay (Nab assay) was also performed to study the effect of AIA antibodies in neutralizing the biological activity/metabolic function of the insulin. The neutralizing potential of AIA was studied by its effect on %glucose uptake. We also evaluated the association between AIA antibody levels and its impact on biological activity by studying the correlation between them. RESULTS: Analysis of immunogenicity data suggested that the percentage of patients positive for AIA antibodies until week 24 was similar and comparable in both the treatment groups, BGL-ASP and RMP; NovoRapid®. The changes in incidence of patients positive for AIA post-baseline in terms of TEAR positives were also similar and comparable between the treatment groups. The results of the Nab assay with confirmed positive AIA samples from BGL-ASP- and RMP-treated groups did not have any negative impact on %glucose uptake by the cells in Nab assay, confirming the absence of neutralizing antibodies in both the treatment groups. The correlation studies also showed absence of association between AIA antibody levels and percentage glucose uptake in both BGL-ASP and RMP-NovoRapid® treatment groups. CONCLUSIONS: The immunogenicity assessment based on the overall incidence of patients positive for AIA, changes in incidence of patients positive for AIA post-baseline, TEAR rates and absence of neutralizing antibodies, were found to be apparently similar and comparable in both the treatment groups (BGL-ASP and RMP). We conclude from our studies that the immunogenicity of BGL-ASP is similar and comparable to RMP and the observed immunogenicity in terms of anti-insulin Aspart antibody levels had no impact on the biological activity of insulin.

Sistrunk Procedure on Malignant Thyroglossal Duct Cyst.

A thyroglossal duct cyst is a lesion that occurs as a result from failure of the thyroglossal duct to obliterate during fetal development. Malignant progression is a rare event that might occur in less than 1% of all cases. Because of its rarity, there are conflicting opinions regarding the management of the case. In the present study, a 46-year-old male presented with a painless neck mass that had increased in size over the last 6 months. There was no difficulty in swallowing and breathing, change in voice, significant weight loss, or any signs of hyperthyroidism. Laboratory workup showed that results were within normal limits. Thyroid gland ultrasonography and cervical contrast CT scan revealed a complex cystic mass that pointed towards a thyroglossal duct cyst. We performed Sistrunk procedure. Postoperative pathology examination revealed microscopic appearance of the thyroglossal duct cyst with a classic follicular variant of papillary thyroid carcinoma. Our latest follow-up showed no signs of tumor recurrence or any complications following surgery on locoregional status. As a fine needle aspiration biopsy cannot ensure a precise result in all of cases, it is essential to perform a solid physical examination and thorough supporting examination in deciding the precise management for the patient.

Association of CYP2D6*10 (c. 100 C>T) Genotype with Z-END Concentration in Patients with Breast Cancer Receiving Tamoxifen Therapy in Indonesian Population.

BACKGROUND: Tamoxifen (TAM) is a frequently used hormonal prodrug for patients with breast cancer that needs to be activated by cytochrome P450 2D6 (CYP2D6) into Zusammen-endoxifen (Z-END). OBJECTIVE: The purpose of the study was to determine the association between CYP2D6*10 (c.100C>T) genotype and attainment of the plasma steady-state Z-END minimal threshold concentration (MTC) in Indonesian women with breast cancer. METHODS: A cross-sectional study was performed in 125 ambulatory patients with breast cancer consuming TAM at 20 mg/day for at least 4 months. The frequency distribution of CYP2D6*10 (c.100C>T) genotypes (C/C: wild type; C/T: heterozygous mutant; T/T: homozygous mutant) was detected using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), the results of which were subsequently confirmed by sequencing. The genotypes were categorized into plasma Z- END concentrations of <5.9 ng/mL and ≥5.9 ng/mL, which were measured using ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). RESULTS: Percentages of C/C, CT, and T/T genotypes were 22.4%, 29.6%, and 48.8%, respectively. Median (25-75%) Z-END concentrations in C/C, C/T, and T/T genotypes were 9.58 (0.7-6.0), 9.86 (0.7-26.6), and 3.76 (0.9-26.6) ng/mL, respectively. Statistical analysis showed a significant difference in median Z-END concentration between patients with T/T genotype and those with C/C or C/T genotypes (p<0.001). There was a significant association between CYP2D6*10 (c.100C>T) genotypes and attainment of plasma steady-state Z-END MTC (p<0.001). CONCLUSION: There was a significant association between CYP2D6*10 (c.100C>T) and attainment of plasma steady-state Z-END MTC in Indonesian breast cancer patients receiving TAM at a dose of 20 mg/day.

Craniofacial Brown Tumor in Patients with Secondary Hyperparathyroidism to Chronic Renal Failure: Report of Two Cases in Cipto Mangunkusumo Hospital.

Brown tumor is a bone lesion that arises in the setting of excess osteoclast activity in hyperparathyroidism. It consists of fibrous tissue, woven bone, and supporting vasculature, while contains no matrix. The characteristic of brown-colored lesion is a result of hemosiderin deposition into the osteolytic cysts. Two cases of young women aged 26 and 29 years old, respectively, are known with a history of end-stage renal disease (ESRD). Dialysis is performed two times/week over the last 7 years. Our patients presented with an intraoral mass of the hard palate since 12 months ago and decreased body height of 10 cm. The lesion causes difficulties in swallowing and talking. Laboratory workup showed elevated parathormone or PTH (3.391 pg/mL and >5.000 pg/mL). Neck ultrasound showed enlargement of the parathyroid glands. Supporting examination to diagnose brown tumor are neck ultrasound, CT of the neck, and parathyroid sestamibi scan. We performed parathyroidectomy. Pathology revealed hyperplasia of the parathyroid. The tumor regressed significantly within 2 weeks following the surgery, and we still observe tumor regression as well as reduction in PTH level. As clinicians, we should be alert to other possible causes of bony lesions. Clinical examination, laboratory finding, and imaging present important information to diagnose brown tumor.

Clara cells drive eosinophil accumulation in allergic asthma.

Development of allergic asthma is a complex process involving immune, neuronal and tissue cells. In the lung, Clara cells represent a major part of the "immunomodulatory barrier" of the airway epithelium. To understand the contribution of these cells to the inflammatory outcome of asthma, disease development was assessed using an adjuvant-free ovalbumin model. Mice were sensitised with subcutaneous injections of 10 µg endotoxin-free ovalbumin in conjunction with naphthalene-induced Clara cell depletion. Clara epithelial cell depletion in the lung strongly reduced eosinophil influx, which correlated with decreased eotaxin levels and, moreover, diminished the T-helper cell type 2 inflammatory response, including interleukin (IL)-4, IL-5 and IL-13. In contrast, airway hyperresponsiveness was increased. Further investigation revealed Clara cells as the principal source of eotaxin in the lung. These findings are the first to show that Clara airway epithelial cells substantially contribute to the infiltration of eotaxin-responsive CCR3+ immune cells and augment the allergic immune response in the lung. The present study identifies Clara cells as a potential therapeutic target in inflammatory lung diseases such as allergic asthma.

Prospective controlled study of effect of laparoscopic sleeve gastrectomy on small bowel transit time and gastric emptying half-time in morbidly obese patients with type 2 diabetes mellitus.

BACKGROUND: Published data on sleeve gastrectomy (SG) have indicated better remission of type 2 diabetes mellitus (T2DM) and improvement in satiety compared with other restrictive procedures. Mechanisms in addition to rapid, extensive weight loss are responsible for the restoration of the euglycemic state. To prospectively evaluate the role of laparoscopic SG on gastric emptying half-time and small bowel transit time (SBTT) and effect of these on weight loss, satiety, and improvement in T2DM. METHODS: A total of 67 subjects were studied. Of these 67 subjects, 24 were lean controls (body mass index 22.2 +/- 2.84 kg/m(2)), 20 were severely and morbidly obese patients with T2DM who had not undergone SG (body mass index 37.73 +/- 5.35 kg/m(2)), and 23 were severely and morbidly obese patients with T2DM after SG (body mass index 40.71 +/- 6.59 kg/m(2)). All 67 patients were evaluated for gastric emptying half-time and SBTT using scintigraphic imaging. Imaging was performed every 15 minutes up to the ileocecal region. The Three-Factor Eating Questionnaire was administered simultaneously. Fasting blood sugar, postprandial blood sugar, and glycated hemoglobin were assessed. Nonparametric analysis of variance and the Mann-Whitney U test were applied. RESULTS: The mean SBTT was significantly lower (P <.05) in the post-SG group (199 +/- 65.7 minutes) than in the non-SG group (281.5 +/- 46.2 minutes) or control group (298.1 +/- 9.2 minutes). The gastric emptying half-time values were also significantly shorter (P <.05) in the post-SG (52.8 +/- 13.5 minutes) than in the non-SG (73.7 +/- 29.0 minutes) and control (72.8 +/- 29.6 minutes) groups. The glycated hemoglobin, fasting blood sugar, and postprandial sugar were all significantly lower after SG. The Three-Factor Eating Questionnaire findings revealed significantly earlier satiety (29.0 +/- 7.2) for the post-SG patients (P <.05) compared with the non-SG (45.8 +/- 9.0) and control (37.9 +/- 6.2) subjects. CONCLUSION: A decreased gastric emptying half-time and SBTT after SG can possibly contribute to better glucose homeostasis in patients with T2DM.

Nerve growth factor enhances Clara cell proliferation after lung injury.

The lung epithelia facilitate wound closure by secretion of various cytokines and growth factors. Nerve growth factor (NGF) has been well described in airway inflammation; however, its likely role in lung repair has not been examined thus far. To investigate the repair function of NGF, experiments were performed in vitro using cultured alveolar epithelial cells and in vivo using a naphthalene-induced model of Clara epithelial cell injury. Both in vitro and in vivo experiments revealed airway epithelial cell proliferation following injury to be dependent on NGF and the expression of its receptor, tropomyosin-receptor-kinase A. Additionally, NGF also augmented in vitro migration of alveolar type II cells. In vivo, transgenic mice over-expressing NGF in Clara cells (NGFtg) did not reveal any proliferation or alteration in Clara cell phenotype. However, following Clara cell specific injury, proliferation was increased in NGFtg and impaired upon inhibition of NGF. Furthermore, NGF also promoted the expression of collagen I and fibronectin in vitro and in vivo during repair, where significantly higher levels were measured in re-epithelialising NGFtg mice. Our study demonstrates that NGF promotes the proliferation of lung epithelium in vitro and the renewal of Clara cells following lung injury in vivo.

Comparison of adjuvant and adjuvant-free murine experimental asthma models.

INTRODUCTION: The most widely used protocol for the induction of experimental allergic airway inflammation in mice involves sensitization by intraperitoneal (i.p.) injections of the antigen ovalbumin (OVA) used in conjunction with the adjuvant aluminium hydroxide (alum). Although adjuvants are frequently used, there are questions regarding the necessity of alum for murine asthma studies due to the non-physiological nature of this chemical. OBJECTIVE: The objective of this study was to compare experimental asthma phenotypes between adjuvant and adjuvant-free protocols of murine allergic airway inflammation in an attempt to develop a standardized alternative to adjuvant use. METHOD: An adjuvant-free OVA model of experimental asthma was investigated in BALB/c mice using i.p. or subcutaneous (s.c.) sensitization routes. For the s.c. sensitization, beta-galactosidase (beta-gal) was also tested as an antigen. In addition, OVA adjuvant and adjuvant-free sensitization protocols were compared in BALB/c and C57BL/6 mice. Open-field testing was performed to assess the effect of alum on mouse behaviour. RESULTS: Comparison of adjuvant vs. adjuvant-free and i.p. vs. s.c. protocols revealed that both adjuvant use and route of antigen application significantly influenced OVA-specific antibody production. Comparison of adjuvant and adjuvant-free protocols in this study clearly demonstrated the non-requirement of alum for the induction of acute allergic airway inflammation, as both protocols induce a similar disease phenotype. BALB/c mice were significantly more susceptible than C57BL/6 mice to sensitization. Using the improved s.c. adjuvant-free protocol, it was demonstrated that alternative antigens such as beta-gal can also be utilized. Behavioural studies indicated severe distress in mice treated with alum. CONCLUSION: The OVA s.c. adjuvant-free protocol used in this study generates a phenotype comparable to the benchmark adjuvant protocol widely used in the literature. The adjuvant-free alternative avoids the added complication of non-physiological adjuvants that may interfere with asthma treatment or prevention strategies.

Bacterioopsin-triggered retinal biosynthesis is inhibited by bacteriorhodopsin formation in Halobacterium salinarium.

Factors regulating retinal biosynthesis in halobacteria are not clearly understood. In halobacteria, events leading to the biosynthesis of bacteriorhodopsin have been proposed to participate in stringent regulation of retinal biosynthesis. The present study describes a novel approach of in vivo introductions of mRNA and membrane proteins via liposome fusion to test their role in cellular metabolism. Both the bacterioopsin-encoding mRNA and the liposome-encapsulated bacterioopsin (apoprotein) are independently introduced in spheroplasts of the purple membrane-negative strain Halobacterium salinarium that initially contain neither bacterioopsin nor retinal. Isoprenoid analyses of these cells indicate that the expression/presence of bacterioopsin triggers retinal biosynthesis from lycopene, and its subsequent binding to opsin generates bacteriorhodopsin. When bacteriorhodopsin and excess retinal were independently introduced into spheroplasts of purple membrane-negative cells, the introduction of bacteriorhodopsin resulted in an accumulation of lycopene, indicating an inhibition of retinal biosynthesis. These results provide direct evidence that the formation of bacterioopsin acts as a trigger for lycopene conversion to beta-carotene in retinal biosynthesis. The trigger for this event does not lie with either transcription or translation of the bop gene. It is clearly associated with the folded and the membrane-integrated state of bacterioopsin. On the other hand, the trigger signaling inhibition of retinal biosynthesis does not lie with the presence of excess retinal but with the correctly folded, retinal-bound form, bacteriorhodopsin.

Site-directed isotope labeling and FTIR spectroscopy: assignment of tyrosine bands in the bR-->M difference spectrum of bacteriorhodopsin.

Fourier transform infrared difference spectroscopy has been used extensively to probe structural changes in bacteriorthodopsin and other retinal proteins. However, the absence of a general method to assign bands to individual chemical groups in a protein has limited the application of this technique. While site-directed mutagenesis has been successful in special cases for such assignments, in general, this approach induces perturbations in the structure and function of the protein, thereby preventing unambiguous band assignments. A new approach has recently been reported (Sonar et al., Nature Struct. Biol. 1 (1994) 512-517) which involves cell-free expression of bacteriorhodopsin and site-directed isotope labeling (SDIL). We have now used this method to re-examine bands assigned in the bR-->M difference spectrum to tyrosine residues. Our results show that out of 11 tyrosines in bR, only Tyr 185 is structurally active. This work further demonstrates the power of SDIL and FTIR to probe conformational changes at the level of individual amino acid residues in proteins.

Photocleavable biotin derivatives: a versatile approach for the isolation of biomolecules.

While the strong biotin-avidin interaction has been widely used for the detection of biomolecules, its irreversibility complicates their isolation. We report the synthesis of a photocleavable biotin derivative (PCB) which eliminates many limitations of existing methods. This reagent contains a biotin moiety linked through a spacer arm to a photocleavable moiety, which reacts selectively with primary amino groups on any substrate. In experiments using [leucine]-enkephalin as a model substrate, we show that PCB retains its high affinity toward avidin/streptavidin and allows rapid (< 5 min) and efficient (> 99%) photorelease of the substrate in a completely unaltered form. Photocleavable biotins should be useful in numerous applications involving the isolation of proteins, nucleic acids, lipids, and cells.

Site-directed isotope labeling and ATR-FTIR difference spectroscopy of bacteriorhodopsin: the peptide carbonyl group of Tyr 185 is structurally active during the bR-->N transition.

The largest secondary structural change occurs in the bacteriorhodopsin (bR) photocycle during the M-->N transition. In this work site-directed isotope labeling (SDIL) and attenuated total reflection Fourier transform infrared (ATR-FTIR) difference spectroscopy were used to investigate this conformational change. L-Tyrosine containing a 13C isotope at the carbonyl carbon was selectively incorporated at Tyr 57, Tyr 147, and Tyr 185 by SDIL. This involves the cell-free expression of bR in the presence of Escherichia coli suppressor tRNA(CUATyr) aminoacylated with L-[1-13C]Tyr. ATR-FTIR difference spectroscopy reveals that of the 11 tyrosines, only the peptide carbonyl group of Tyr 185 undergoes a significant structural change during the bR-->N transition. Along with other spectroscopic evidence, this result suggests that the Tyr 185-Pro 186 region of the protein is structurally active and may function as a hinge which facilitates the tilt of the cytoplasmic portion of the F-helix in bacteriorhodopsin during the M-->N transition.

Detection of a water molecule in the active-site of bacteriorhodopsin: hydrogen bonding changes during the primary photoreaction.

FTIR-difference spectroscopy in combination with site-directed mutagenesis has been used to investigate the role of water during the photocycle of bacteriorhodopsin. At least one water molecule is detected which undergoes an increase in H-bonding during the primary bR-->K phototransition. Bands due to water appear in the OH stretch region of the bR-->K FTIR-difference spectrum which downshift by approximately 12 cm-1 when the sample is hydrated with H2(18)O. In contrast to 2H2O, the H2(18)O-induced shift is not complete, even after 24 h of hydration. This indicates that even though water is still able to exchange protons with the outside medium, it is partially trapped in the interior of the protein. In the mutant Y57D, these bands are absent while a new set of bands appear at much lower frequencies which undergo H2(18)O-induced shifts. It is concluded that the water molecule we detect is located inside the bR active-site and may interact with Tyr-57. The change in its hydrogen-bonding strength is most likely due to the photoinduced all-trans-->13-cis isomerization of the retinal chromophore and the associated movement of the positively charged Schiff base during the bR-->K transition. In contrast, a second water molecule, whose infrared difference bands are not affected by the Y57D mutation, appears to undergo a decrease in hydrogen bonding during the K-->L and L-->M transitions.

A redirected proton pathway in the bacteriorhodopsin mutant Tyr-57-->Asp. Evidence for proton translocation without Schiff base deprotonation.

Light-driven proton pumping in bacteriorhodopsin involves deprotonation of the retinylidene Schiff base during M formation and reprotonation during N formation as key steps. This study reports on the spectroscopic characterization of the bacteriorhodopsin mutant Tyr-57-->Asp (Y57D). The results reveal that although formation of the M intermediate and Schiff base deprotonation is blocked, the mutant still exhibits a significant level of light-driven proton translocation. The photocycle of Y57D involves formation of K and L intermediates accompanied by the normal chromophore isomerization and changes in the hydrogen bonding of Asp-96 and Asp-115. However, an additional Asp residue deprotonates during formation of the L intermediate along with a transmembrane alpha-helical structural change that normally occurs upon N formation. We postulate that proton transport in Y57D occurs through a redirected pathway that does not involve the deprotonation of the Schiff base. Chromophore isomerization, which normally results in the transfer of a proton from the Schiff base to Asp-85, instead causes the deprotonation of Asp-57 in Y57D, most likely through an interaction involving Asp-212. This deprotonation of Asp-57 causes the release of a proton into the extracellular medium. Reprotonation of Asp-57 occurs through the Schiff base reprotonation pathway, which consists of a hydrogen-bonded network of residues spanning from Asp-96 to Asp-212. The results also indicate that the transmembrane alpha-helical structural changes observed during N formation (Rothschild, K.J., Marti, T., Sonar, S., He, Y.W., Rath, P., Fischer, W., Bousche, O., and Khorana, H. G. (1993) J. Biol. Chem. 268, 27046-27052) do not require deprotonation of Asp-96 or of the Schiff base.

Site-directed isotope labelling and FTIR spectroscopy of bacteriorhodopsin.

Insight into integral membrane proteins function is presently limited by the difficulty of producing three-dimensional crystals. In addition, X-ray structures of proteins normally do not provide information about the protonation state and structural changes of individual residues. We report here the first use of site-directed isotope labelling and Fourier transform infrared (FTIR) difference spectroscopy to detect structural changes at the level of single residues in an integral membrane protein. Two site-directed isotope labeled (SDIL) tyrosine analogues of bacteriorhodopsin were produced which exhibit normal activity. FTIR spectroscopy shows that out of 11 tyrosines, only Tyr 185 is structurally active during the early photocycle and may be part of a proton wire.

Cell-free synthesis, functional refolding, and spectroscopic characterization of bacteriorhodopsin, an integral membrane protein.

Bacteriorhodopsin (bR) is an integral membrane protein which functions as a light-driven proton pump in Halobacterium halobium (also known as Halobacterium salinarium). The cell-free synthesis of bR in quantities sufficient for FTIR and NMR spectroscopy and the ability to selectively isotope label bR using aminoacylated suppressor tRNAs would provide a powerful approach for studying the role of specific amino acid residues. However, no integral membrane protein has yet been expressed in a cell-free system in quantities sufficient for such biophysical studies. We report the cell-free synthesis of bacterioopsin, its purification, its refolding in polar lipids from H. halobium, and its regeneration with all-trans-retinal to yield bacteriorhodopsin in a form functionally similar to bR in purple membrane. Importantly, the yields obtained from in vitro and in vivo expression are comparable. Functionality of the cell-free expressed bR is established using static and time-resolved absorption spectroscopy and FTIR difference spectroscopy.

Asp96 deprotonation and transmembrane alpha-helical structural changes in bacteriorhodopsin.

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.

Hydrogen bonding interactions with the Schiff base of bacteriorhodopsin. Resonance Raman spectroscopy of the mutants D85N and D85A.

The bacteriorhodopsin (bR) mutants Asp-85-->Asn (D85N) and Asp-85-->Ala (D85A) have a red-shifted chromophore absorption and exhibit no proton pumping (Otto, H., Marti, T., Holz, M., Mogi, T., Stern, L., Engel, F., Khorana, H. G., and Heyn, M. P. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1018-1022) consistent with the hypothesis that Asp-85 functions as a counterion and proton acceptor for the retinal Schiff base (Braiman, M. S., Mogi, T., Marti, T., Stern, L. J., Khorana, H. G., and Rothschild, K. J. (1988) Biochemistry 27, 8516-8520). Resonance Raman spectroscopy reveals that these mutants contain a mixture of all-trans and 13-cis/C = N syn chromophores, similar to dark-adapted purple membrane and acid-induced or deionized blue membrane. At high NaCl concentrations, both mutants adopt a predominantly all-trans chromophore structure similar to acid purple membrane. A comparison of the Schiff base C = NH+ stretch frequency (vC = N) and deuterium isotope shift for D85N, D85A as well as various forms of bR, including light-adapted bR, blue membrane, and acid purple membrane, provides information about hydrogen bonding interactions to the Schiff base. D85N has as strong a hydrogen bond as light-adapted bR despite the loss of the negative charge at residue 85. In contrast, D85A has a weaker hydrogen bond. These results can be explained if a direct interaction exists between the Schiff base and Asn-85 in D85N and between the Schiff base and a substituted water molecule in D85A. Many of the properties of wild type bR, D85N, D85A, blue membrane, and acid purple membrane can be explained on the basis of changes in the local hydrogen bonding near the Schiff base.

Static and time-resolved absorption spectroscopy of the bacteriorhodopsin mutant Tyr-185-->Phe: evidence for an equilibrium between bR570 and an O-like species.

The light-dark adaptation, photocycle kinetics, and acid-induced blue formation of the bacteriorhodopsin (bR) mutant Tyr-185-->Phe (Y185F) expressed in Halobacterium halobium have been investigated by both static and time-resolved visible absorption spectroscopy. Evidence is presented that a pH-dependent equilibrium exists between a bR570-like form (bRY185F570) and a red-shifted species in the light-adapted form of Y185F. In two related papers, we show that this species has vibrational features similar to the O intermediate. Key findings are that light adaptation causes formation of a purple species similar to bR570 and a second long-lived red-shifted species with a lambda max near 630 nm, well above the pH for the acid-induced blue transition. The concentration of the red-shifted species is pH- and salt-dependent, decreasing reversibly at high pH and high ionic strength. The dark-adapted state of Y185F also contains a small amount of the red-shifted species which is reversibly titratable. Dark adaptation is much slower than wild-type bR and causes a parallel decay of light-adapted bR and the red-shifted species. Time-resolved visible absorption spectroscopy reveals that the purple and the red-shifted species undergo separate photocycles. The purple species exhibits a relatively normal photocycle except for an increased rate of M formation kinetics. The red-shifted species has a photocycle involving a red-shifted K intermediate and a second longer lived intermediate possibly similar to N. The apparent absence of an O intermediate in the late photocycle of Y185F is attributed to cancellation by depletion bands due to the photoreacting red-shifted species.(ABSTRACT TRUNCATED AT 250 WORDS)

Time-resolved Fourier transform infrared spectroscopy of the bacteriorhodopsin mutant Tyr-185-->Phe: Asp-96 reprotonates during O formation; Asp-85 and Asp-212 deprotonate during O decay.

The protonation state of key aspartic acid residues in the O intermediate of bacteriorhodopsin (bR) has been investigated by time-resolved Fourier transform infrared (FTIR) difference spectroscopy and site-directed mutagenesis. In an earlier study (Bousché et al., J. Biol Chem. 266, 11063-11067, 1991) we found that Asp-96 undergoes a deprotonation during the M-->N transition, confirming its role as a proton donor in the reprotonation pathway leading from the cytoplasm to the Schiff base. In addition, both Asp-85 and Asp-212, which protonate upon formation of the M intermediate, remain protonated in the N intermediate. In this study, we have utilized the mutant Tyr-185-->Phe (Y185F), which at high pH and salt concentrations exhibits a photocycle similar to wild type bR but has a much slower decay of the O intermediate. Y185F was expressed in native Halobacterium halobium and isolated as intact purple membrane fragments. Time-resolved FTIR difference spectra and visible difference spectra of this mutant were measured from hydrated multilayer films. A normal N intermediate in the photocycle of Y185F was identified on the basis of characteristic chromophore and protein vibrational bands. As N decays, bands characteristic of the all-trans O chromophore appear in the time-resolved FTIR difference spectra in the same time range as the appearance of a red-shifted photocycle intermediate absorbing near 640 nm. Based on our previous assignment of the carboxyl stretch bands to the four membrane embedded Asp groups: Asp-85, Asp-96, Asp-115 and Asp-212, we conclude that during O formation: (i) Asp-96 undergoes reprotonation. (ii) Asp-85 may undergo a small change in environment but remains protonated. (iii) Asp-212 remains partially protonated. In addition, reisomerization of the chromophore during the N-->O transition is accompanied by a major reversal of protein conformational changes which occurred during the earlier steps in the photocycle. These results are discussed in terms of a proposed mechanism for proton transport.