Thr-90 Plays a Vital Role in the Structure and Function of Bacteriorhodopsin
2004; Elsevier BV; Volume: 279; Issue: 16 Linguagem: Inglês
10.1074/jbc.m313988200
ISSN1083-351X
AutoresAlex Perálvarez‐Marín, M. Márquez, José‐Luis Bourdelande, Enric Querol, Esteve Padrós,
Tópico(s)Nicotinic Acetylcholine Receptors Study
ResumoThe role of Thr-90 in the bacteriorhodopsin structure and function was investigated by its replacement with Ala and Val. The mutant D115A was also studied because Asp-115 in helix D forms a hydrogen bond with Thr-90 in helix C. Differential scanning calorimetry showed a decreased thermal stability of all three mutants, with T90A being the least stable. Light-dark adaptation of T90A was found to be abnormal and salt-dependent. Proton transport monitored using pyranine signals was ∼10% of wild type for T90A, 20% for T90V, and 50% for D115A. At neutral or alkaline pH, the M rise of these mutants was faster than that of wild type, whereas M decay was slower in T90A. Overall, Fourier transform infrared (FTIR) difference spectra of T90A were strongly pH-dependent. Spectra recorded on films adjusted at the same pH at 243 or 277 K, dry or wet, showed similar features. The D115A and T90V FTIR spectra were closer to WT, showing minor structural differences. The band at 1734 cm-1 of the deconvoluted FTIR spectrum, corresponding to the carboxylate of Asp-115, was absent in all mutants. In conclusion, Thr-90 plays a critical role in maintaining the operative location and structure of helix C through three complementary interactions, namely an interhelical hydrogen bond with Asp-115, an intrahelical hydrogen bond with the peptide carbonyl oxygen of Trp-86, and a steric contact with the retinal. The interactions established by Thr-90 emerge as a general feature of archaeal rhodopsin proteins. The role of Thr-90 in the bacteriorhodopsin structure and function was investigated by its replacement with Ala and Val. The mutant D115A was also studied because Asp-115 in helix D forms a hydrogen bond with Thr-90 in helix C. Differential scanning calorimetry showed a decreased thermal stability of all three mutants, with T90A being the least stable. Light-dark adaptation of T90A was found to be abnormal and salt-dependent. Proton transport monitored using pyranine signals was ∼10% of wild type for T90A, 20% for T90V, and 50% for D115A. At neutral or alkaline pH, the M rise of these mutants was faster than that of wild type, whereas M decay was slower in T90A. Overall, Fourier transform infrared (FTIR) difference spectra of T90A were strongly pH-dependent. Spectra recorded on films adjusted at the same pH at 243 or 277 K, dry or wet, showed similar features. The D115A and T90V FTIR spectra were closer to WT, showing minor structural differences. The band at 1734 cm-1 of the deconvoluted FTIR spectrum, corresponding to the carboxylate of Asp-115, was absent in all mutants. In conclusion, Thr-90 plays a critical role in maintaining the operative location and structure of helix C through three complementary interactions, namely an interhelical hydrogen bond with Asp-115, an intrahelical hydrogen bond with the peptide carbonyl oxygen of Trp-86, and a steric contact with the retinal. The interactions established by Thr-90 emerge as a general feature of archaeal rhodopsin proteins. The purple membrane patches of Halobacterium salinarum cells contain a single protein, bacteriorhodopsin (BR), 1The abbreviations used are: BR, bacteriorhodopsin; DSC, differential scanning calorimetry; FTIR, Fourier transform infrared; WT, wild type. 1The abbreviations used are: BR, bacteriorhodopsin; DSC, differential scanning calorimetry; FTIR, Fourier transform infrared; WT, wild type. which translocates protons from the interior to the exterior of the cell upon photon absorption by a retinal molecule (1Oesterhelt D. Stoeckenius W. Proc. Natl. Acad. Sci. U. S. A. 1973; 70: 2853-2857Crossref PubMed Scopus (944) Google Scholar). The native structure of bacteriorhodopsin consists of a bundle of seven densely packed α-helices forming a para-crystalline arrangement of BR trimers (2Henderson R. Unwin P.N. Nature. 1975; 257: 28-32Crossref PubMed Scopus (1589) Google Scholar, 3Grigorieff N. 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Protein-protein and lipid-protein interactions as well as the lateral pressure exerted by the lipid chains on the protein molecules have important roles in this arrangement (9Curran A.R. Templer R.H. Booth P.J. Biochemistry. 1999; 38: 9328-9336Crossref PubMed Scopus (111) Google Scholar, 10Weik M. Patzelt H. Zaccai G. Oesterhelt D. Mol. Cell. 1998; 1: 411-419Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 11Heyes C.D. El-Sayed M.A. J. Biol. Chem. 2002; 277: 29437-29443Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Because of the high purple membrane density, the protein, in turn, should comply with some characteristics to allow the formation of the hexameric arrangement. Helix-helix interactions as well as retinal protein interactions are important elements involved in the BR compactness. When a relaxation of the protein structure occurs, the hexagonal arrangement appears impaired. One example refers to the bleached membrane, where the retinal absence compels the helices to lose several interactions (12Cladera J. Galisteo M.L. Sabés M. Mateo P.L. Padrós E. Eur. J. Biochem. 1992; 207: 581-585Crossref PubMed Scopus (48) Google Scholar, 13Moller C. Buldt G. Dencher N.A. Engel A. Muller D.J. J. Mol. Biol. 2000; 301: 869-879Crossref PubMed Scopus (39) Google Scholar). Another case corresponds to the triple or quadruple mutants E9Q/E194Q/E204Q and E9Q/E74Q/E194Q/E204Q, which exhibit a more relaxed conformation as compared with WT (14Sanz C. Lazarova T. Sepulcre F. González-Moreno R. Bourdelande J.L. Querol E. Padrós E. FEBS Lett. 1999; 456: 191-195Crossref PubMed Scopus (28) Google Scholar, 15Padrós E. Sanz C. Lazarova T. Márquez M. Sepulcre F. Trapote X. Muñoz F.-X. González-Moreno R. Bourdelande J.L. Querol E. Dér A. Keszthelyi L. Bioelectronic Applications of Photochromic Pigments. 335. IOS Press, Amsterdam2001: 120-136Google Scholar).The mutant T90A shows some characteristics indicative of a certain degree of softening of both the para-crystalline arrangement and the interactions within the seven-helix bundle. This is demonstrated by the decreased cooperativity and temperature of the differential scanning calorimetry pre-transition, as well as by a decreased temperature of the main transition (16Perálvarez A. Barnadas R. Sabés M. Querol E. Padrós E. FEBS Lett. 2001; 508: 399-402Crossref PubMed Scopus (14) Google Scholar). Because Thr-90 most probably does not form part of the proton transport chain (6Luecke H. Schobert B. Richter H.T. Cartailler J.P. Lanyi J.K. J. Mol. Biol. 1999; 291: 899-911Crossref PubMed Scopus (1295) Google Scholar, 7Facciotti M.T. Rouhani S. Burkard F.T. Betancourt F.M. Downing K.H. Rose R.B. McDermott G. Glaeser R.M. Biophys. J. 2001; 81: 3442-3455Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), it is likely that its importance relies on structural aspects. An earlier work already demonstrated the inability of the iodoacetic acid-derivatized T90C to fold normally in detergent/phospholipid micelles (17Flitsch S.L. Khorana H.G. Biochemistry. 1989; 28: 7800-7805Crossref PubMed Scopus (40) Google Scholar), indicating that the -OH function and/or the side chain bulkiness is an important structural feature. According to the high-resolution BR structures, Thr-90 (helix C) and Asp-115 (helix D) form a hydrogen bond (Refs. 6Luecke H. Schobert B. Richter H.T. Cartailler J.P. Lanyi J.K. J. Mol. Biol. 1999; 291: 899-911Crossref PubMed Scopus (1295) Google Scholar and 7Facciotti M.T. Rouhani S. Burkard F.T. Betancourt F.M. Downing K.H. Rose R.B. McDermott G. Glaeser R.M. Biophys. J. 2001; 81: 3442-3455Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, and see Fig. 1). These two amino acids, which are conserved among various archaebacterial strains (18Ihara K. Umemura T. Katagiri I. Kitajima-Ihara T. Sugiyama Y. Kimura Y. Mukohata Y. J. Mol. Biol. 1999; 285: 163-174Crossref PubMed Scopus (138) Google Scholar, 19Zhai Y. Heijne W.H. Smith D.W. Saier Jr., M.H. Biochim. Biophys. Acta. 2001; 1511: 206-223Crossref PubMed Scopus (58) Google Scholar), may be important in structural terms. Another significant structural interaction may be the hydrogen bond formed by the -OH group of Thr-90 with the peptide carbonyl oxygen of Trp-86, as bends and twists in transmembrane helices are induced by Thr through formation of an hydrogen bond with the backbone (20Gray T.M. Matthews B.W. J. Mol. Biol. 1984; 175: 75-81Crossref PubMed Scopus (223) Google Scholar, 21Ballesteros J.A. Shi L. Javitch J.A. Mol. Pharmacol. 2001; 60: 1-19Crossref PubMed Scopus (399) Google Scholar). The BR structural models effectively show a kink in helix C at this level (Fig. 1). On the other hand, Pro residues are known as helix-breaking residues, and, thus, the adjacent Pro-91 may contribute to bending the helix. Again, sequence comparison shows that Pro-91 is conserved (18Ihara K. Umemura T. Katagiri I. Kitajima-Ihara T. Sugiyama Y. Kimura Y. Mukohata Y. J. Mol. Biol. 1999; 285: 163-174Crossref PubMed Scopus (138) Google Scholar). Finally, there are van der Waals contacts between the methyl group of Thr-90 and the retinal chain that may also be important for the correct location of retinal.In this work, we analyze in detail the behavior of the T90A, T90V, and D115A mutants to determine the structural and functional consequences of the disruption of the interactions involving the Thr-90 side chain. In the T90A mutant, we describe a chromoprotein with very different properties as compared with wild type BR, including a decrease in the efficiency of the proton transport and some critical alterations in the photocycle. In addition, the D115A and T90V mutations provide further knowledge about the role of the set of interactions involving Thr-90.EXPERIMENTAL PROCEDURESThe construction and expression of T90A and D115A mutants in Halobacterium salinarum was carried out as described (14Sanz C. Lazarova T. Sepulcre F. González-Moreno R. Bourdelande J.L. Querol E. Padrós E. FEBS Lett. 1999; 456: 191-195Crossref PubMed Scopus (28) Google Scholar). The mutant T90V was a generous gift from Dr. J. K. Lanyi.UV-visible spectra of dark- or light-adapted purple membrane suspensions (1.5·10-5m, and 3.5·10-5m in the case of T90A) were recorded with a Cary Bio3 spectrophotometer using an integrating sphere when necessary. The difference spectra were obtained by subtracting light-adapted minus dark-adapted samples.Flash-induced transient absorbance changes were monitored using a LKS50 instrument from Applied Photophysics. A Q-switched neodymium:yttrium-aluminum-garnet (Nd-YAG) laser (Spectron Laser Systems; pulse width ∼9 ns; E = 5 mJ/pulse/cm2; repetition frequency 0.5 Hz) at 532 nm was used for light excitation. Transient pH changes in the bulk medium were followed by measuring the absorbance changes of 50 μm pyranine at 460 nm in a purple membrane suspension in 1 m KCl, pH 7 (22Grzesiek S. Dencher N.A. FEBS Lett. 1986; 208: 337-342Crossref Scopus (91) Google Scholar). To obtain the net absorbance changes of pyranine, the traces of samples in the absence of the dye were subtracted from those in its presence. The negative signal of ΔΔA indicates the release of protons by BR (pyranine protonation), whereas the positive signal indicates BR proton uptake. Photocycle reactions of purple membrane suspensions of T90A and D115A mutants in 1 m KCl were followed by the acquisition of absorbance spectra at 410, 555, and 660 nm as a function of time at pH 6.5 and pH 10.Infrared experiments were performed at 277 and 243 K with wet and dry samples. The temperature was controlled and maintained using a homemade cell holder and a cryostat. Membrane samples were suspended in 150 mm KCl and either 3 mm carbonate-bicarbonate for pH 10 or3mm sodium phosphate for pH 7. Preparation of membrane films and spectra acquisition were done as described (23Lazarova T. Padrós E. Biochemistry. 1996; 35: 8354-8358Crossref PubMed Scopus (8) Google Scholar) with a Bio-Rad FTS6000 spectrometer at 2 cm-1 resolution. At least three cycles of 350 scans were averaged (i.e. at least 1050 interferograms were accumulated per spectrum). Difference spectra were calculated by subtracting unphotolyzed BR from the corresponding photointermediate. Absorption spectra were Fourier self-deconvoluted using the Kauppinnen algorithm (24Kauppinnen J.K. Moffatt D.J. Mantsch H.H. Cameron D.G. Anal. Chem. 1981; 53: 1454-1457Crossref Scopus (300) Google Scholar), with a Lorentzian band shape, a full width at a half-height of 10 cm-1, and a band narrowing factor k of 2. When necessary, films were illuminated with blue light to drive any remaining intermediate back to BR. Differential scanning calorimetry (DSC) experiments were performed as described previously (16Perálvarez A. Barnadas R. Sabés M. Querol E. Padrós E. FEBS Lett. 2001; 508: 399-402Crossref PubMed Scopus (14) Google Scholar).RESULTSLight-Dark Adaptation—Light-dark adaptation was found to be abnormal in T90A. Fig. 2 shows the absorbance and difference spectra (light-adapted minus dark-adapted) in water, 150 mm KCl, and 1 m KCl (pH 7, room temperature). In H2O, a decrease of the extinction coefficient and virtually no shift of the maximum upon illumination are observed. When the ionic strength is increased, a more normal behavior is observed (increase of the extinction coefficient and red shift upon light adaptation), due probably to the partial recovery of the normal isomer ratio in dark-adapted form. Under the conditions presented in Fig. 2, dark adaptation is very slow, taking at least 1 month to complete. In contrast, T90V and D115A show a normal dark adaptation, although all mutants present a shifted absorbance maxima as compared with WT (see Table I).Fig. 2Light-dark absorption spectra of T90A.Left column, absorption spectra of dark-adapted (dash-dot line) and light-adapted (solid line) membrane suspensions of T90A at pH 7 in water (A), 150 mm KCl (B), or 1 m KCl (C). Right column, difference spectra obtained by subtracting dark-adapted spectra from light-adapted spectra.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table ITime constants of M rise and decay obtained by fitting the absorption changes at 410 nm fromFig. 3(1 m KCl, pH 6.5 or 10.0, 293 K) to a biexponential function The corresponding fraction amplitudes are shown in parentheses. Maximum absorbance (Max. Abs.) values at 150 mm KCl, pH 7.0 are also shown. DA, dark-adapted; LA, light-adapted.WTD115AT90VT90AMax. Abs. DA (nm)558551545548Max. Abs. LA (nm)568557551553M rise pH 6.5 (μs)4.1 (0.10)8.2 (0.30)4.5 (0.50)1.5 (0.70)70 (0.90)50 (0.70)40 (0.50)60 (0.30)M rise pH 10 (μs)3 (0.80)2.1 (0.75)1.5 (0.65)2.1 (0.90)20 (0.20)80 (0.25)20 (0.35)50 (0.10)M decay pH 6.5 (ms)3 (1.0)7.2 (0.90)4.5 (0.80)9 (0.70)70 (0.10)70 (0.20)93 (0.30)M decay pH 10 (ms)3 (0.50)5 (0.40)5.7 (0.45)21 (0.40)80 (0.50)51 (0.60)155 (0.55)235 (0.60) Open table in a new tab Photocycle Reactions and Proton Uptake and Release—Fig. 3A shows kinetic traces of the light-induced absorption changes for T90A, T90V, D115A, and WT in 1 m KCl, pH 6.5 (room temperature) normalized to the M amplitude. All mutants present a faster M rise and a slower M decay than WT (see Table I). Fig. 3A also shows that D115A presents a higher amount of O intermediate than T90A, T90V, or wild type and that T90A and T90V exhibit a longer-living O intermediate. At pH 10, like at neutral pH, the M rise of T90A and of T90V is faster and the M decay slower than those of the WT, whereas D115A has a faster M decay (Fig. 3B and Table I). On the other hand, at neutral or at alkaline pH, both the amplitude of the M intermediate (maximal signal at 410 nm) and the signal at the λmax of 555 nm (disappearance of the BR form) of T90A are ∼20% of the WT signal, whereas they are ∼40% for T90V and ∼50% for D115A. At pH 4, no signal was detected for T90A at any of the wavelengths analyzed, indicating the absence of photocycle intermediates under these conditions, whereas both T90V and D115A showed small signals (data not shown).Fig. 3Kinetics of photocycle intermediates. The traces show the absorption changes corresponding to the M intermediate (410 nm; top), BR depletion and recovery (555 nm for T90A, T90V and D115A and 570 nm for WT; middle), and the O intermediate (660 nm; bottom). Column A, pH 6.5, 1 m KCl, 293 K. Column B, pH 10, 1 m KCl, 293 K. The absorption changes at 410, 660, and 570/555 nm, were normalized to the amplitude of the M intermediate for each pigment at the respective pH.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4 shows the pyranine signal for T90A, T90V, D115A, and WT at pH 7. As is apparent, a weak signal is obtained for T90A and T90V, amounting to ∼10% of the WT signal for T90A and ∼20% for T90V. This is in keeping with the low accumulation of the M intermediate obtained at neutral pH and with the decreased pumping efficiency of T90A incorporated into liposomes, which was found to be <20% (16Perálvarez A. Barnadas R. Sabés M. Querol E. Padrós E. FEBS Lett. 2001; 508: 399-402Crossref PubMed Scopus (14) Google Scholar). The signal of D115A also appears decreased, to ∼50% of the WT signal, again in accordance with the amplitude of the M intermediate for this mutant.Fig. 4Light-induced pyranine signals. Pyranine absorption changes were measured at 460 nm in suspensions of T90A, T90V, D115A, and WT in 150 mm KCl (pH 7) at 293 K. Proton uptake from BR causes an increase of absorbance, whereas proton release causes a decrease. Protein concentration, 1·5·10-5m.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fourier Transform Infrared Spectra—A general characteristic of the infrared difference spectra of T90A films, obtained by continuous illumination, is their low intensity in accordance with the small signal obtained in flash photolysis experiments. On the other hand, we have found that the difference spectra depend mainly on the pH. As shown in Fig. 5A, the spectra taken at pH 10, at either 243 or 277 K, dry or wet, are similar to each other and relatively similar to the WT M1 intermediate spectrum (25Braiman M.S. Ahl P.L. Rothschild K.J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5221-5225Crossref PubMed Scopus (107) Google Scholar). Asp-85 appears protonated (band at 1762–1763 cm-1), and a small negative band appears at 1747 cm-1 that is not present in the WT difference spectrum. The amide I is also somewhat changed in comparison to the WT M1 intermediate. The pair of bands at 1640 cm-1 (negative) and 1624 cm-1 (positive) indicate the presence of a protonated Schiff base in the unphotolyzed pigment and a deprotonated Schiff base in the M1-like intermediate. In the amide II, the C = C stretching band appears at 1530 cm-1, in keeping with the known inverse relationship with the λmax of the visible absorption spectrum, which is 550 nm at pH 10. On the other hand, the retinal negative peaks at 1201 and 1167 cm-1 indicate that the retinal is in the all-trans configuration in the unphotolyzed state (26Maeda A. Sasaki J. Pfefferle J.M. Shichida Y. Yoshizawa T. Photochem. Photobiol. 1991; 54: 911-921Crossref Scopus (84) Google Scholar). It is interesting to note that the small negative peak at 1276 cm-1 is not present, indicating that the conformational changes of the T90A photocycle do not affect Tyr-185 (27He Y. Krebs M.P. Fischer W.B. Khorana H.G. Rothschild K.J. Biochemistry. 1993; 32: 2282-2290Crossref PubMed Scopus (30) Google Scholar).Fig. 5FTIR difference spectra of T90A. The films were prepared from suspensions in 150 mm KCl at pH 10 (A) and pH 7 (B). Conditions were as follows: 1, dry sample at 277 K; 2, wet sample at 277 K; 3, dry sample at 243 K; and 4, wet sample at 243 K. The spectra are scaled so that all of them have the same intensity.View Large Image Figure ViewerDownload Hi-res image Download (PPT)At pH 7 (Fig. 5B), the difference spectra depend more on the particular conditions, especially on the water content. The carboxylate band of Asp-85 is less evident, and it shifted to 1764–1768 cm-1, depending on the temperature and the state of the sample. The negative band at 1698 cm-1, which is particularly evident in the wet samples, probably corresponds to the band at 1692 cm-1 in WT and T90A at pH 10. This shift may indicate that the corresponding reverse turns of the unphotolyzed protein have a different structure in wet samples at pH 7 from those at pH 10. The positive band at 1509 cm-1 is also mainly seen in wet samples (Fig. 5B). It may be reminiscent of the band at 1506 cm-1, seen in the O difference spectra (28Zscherp C. Heberle J. J. Phys. Chem. 1997; 101: 10542-10547Crossref Scopus (93) Google Scholar). The most striking feature observed at pH 7 is the positive 1222 cm-1 peak, which again is more intense in wet samples and is accompanied by a low intensity of the 1200 cm-1 band. These features indicate that at neutral pH, the retinal adopts a distorted configuration in T90A as compared with WT. Difference spectra for D115A and T90V were more similar to WT, with only some deviation appearing in the amide I and in the fingerprinting region of the retinal (data not shown).Fig. 6A presents FTIR difference spectra collected under conditions of the N intermediate. D115A shows an N-like intermediate, whereas the main feature of T90A and T90V is an M-like intermediate (compare with the spectrum of WT). In D115A, the peak of protonated Asp-85 is shifted to a more M-like position (1761 cm-1), and the 1650 cm-1 peak corresponding to helical conformational changes is absent, as in T90A. The T90V mutant shows an M-like spectrum, although, as in D115A, a small positive band at 1186 cm-1 (characteristic of N) is present. Fig. 6B shows the absorbance FTIR spectra of WT, T90A, T90V, and D115A films at pH 7 in the carboxylate region after band narrowing by deconvolution. Two bands can be seen in the WT sample at 1734 and 1740 cm-1, which are assigned to Asp-115 and Asp-96, respectively (29Sasaki J. Lanyi J.K. Needleman R. Yoshizawa T. Maeda A. Biochemistry. 1994; 33: 3178-3184Crossref PubMed Scopus (91) Google Scholar). In the mutants, the band at 1734 cm-1 is missing, whereas the band at 1740 cm-1 keeps its position in T90A and shifts to 1741 cm-1 in T90V and D115A. Similar spectra were found at pH 10 (data not shown). This indicates that, in T90A and T90V, the -COOH group of Asp-115 does not contribute to the carboxylate region and, thus, is most likely deprotonated at neutral or alkaline pH.Fig. 6N-like FTIR difference spectra and carboxylic region of the FTIR deconvoluted spectra of T90A, T90V, D115A, and WT.A, comparison of T90A, T90V, D115A, and WT FTIR difference spectra under conditions for WT to yield the N intermediate (pH 10, wet sample, at 277 K) 1, WT; 2, T90A; 3, T90V; and 4, D115A. B, carboxylic region of the deconvoluted spectra of purple membrane dry films at 293 K obtained from membrane suspensions in 150 mm KCl, pH 7 1, WT; 2, T90A; 3, T90V; and 4, D115A. The parameters used for deconvolution are a full bandwidth at a half-height of 10 cm-1 and a narrowing factor k of 2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Thermal Stability—DSC experiments were performed on wild type and mutant BR to determine the influence of the mutations on the structural stability. Fig. 7 shows the DSC scans after baseline subtraction. As is known, the WT purple membrane presents two transitions, namely the pre-transition, which is assigned to the reversible disorganization of the paracrystalline arrangement, and the main transition, which is caused by the irreversible (partial) protein denaturation. Fig. 7 shows that the mutations induce a clear destabilization. As described previously (16Perálvarez A. Barnadas R. Sabés M. Querol E. Padrós E. FEBS Lett. 2001; 508: 399-402Crossref PubMed Scopus (14) Google Scholar), T90A has the main transition at 83 °C and a decreased area of the transition curve, thus presenting a dramatic decrease in conformational stability as compared with WT (main transition at 98 °C). T90V and D115A show an intermediate behavior. They have the same main transition temperature (92 °C), but T90V shows a lower cooperativity. The mutations affect the pre-transition even more. As compared with WT, there is a decrease in the temperature of the pre-transition of 12 °C for D115A, 20 °C for T90V, and ∼30 °C for T90A. Additionally, an important decrease in cooperativity is observed, especially for T90V and T90A. Overall, these data indicate a more relaxed structure of the BR mutants, giving rise, in turn, to a decreased stability of the para-crystalline arrangement.Fig. 7DSC thermograms of wild type and mutants D115A, T90V, and T90A. Purple membrane patches were suspended in H2Oat a concentration of 2.0 mg/ml, pH 7. The curves were corrected with the instrumental and chemical baselines. Scans were taken at 1.5 K/min. Scale bar represents an apparent heat capacity of 5.10-4 cal/° C.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONThe study of the BR mutants T90A, T90V, and D115A presented in this paper provides the means to evaluate the scope of the three interactions held by the Thr-90 side chain, namely the steric interaction with the retinal at the level of C11-C13 and the hydrogen bonds established with Asp-115 and the carbonyl oxygen of Trp-86. In accordance with our previous conclusions (16Perálvarez A. Barnadas R. Sabés M. Querol E. Padrós E. FEBS Lett. 2001; 508: 399-402Crossref PubMed Scopus (14) Google Scholar), the results reveal Thr-90 as a key element in the structure of BR. Focusing on proton pumping, mutagenesis on Thr-90 yields a protein with a clear decrease in the proton-pumping ability. Thus, substitution of Thr-90 with Ala, which avoids all the interactions of residue 90, decreases the pumping efficiency to only ∼10% of the pyranine signal in comparison with WT. Similarly, substitution of Thr-90 with Val, which keeps the steric interaction with the retinal but loses the hydrogen bonds, shows a proton pumping ∼20% of that of WT. In accordance with this trend, the D115A mutant, which only loses the hydrogen bonding of Thr-90 with Asp-115, shows a proton pumping of ∼50% as compared with WT. Previous data on T90V expressed in Escherichia coli and reconstituted into liposomes also showed a somewhat decreased proton-pumping activity, although not as important as in our case (∼70%; Ref. 30Marti T. Otto H. Mogi T. Roesselet S.J. Heyn M.P. Khorana H.G. J. Biol. Chem. 1991; 266: 6919-6927Abstract Full Text PDF PubMed Google Scholar), whereas T90C showed similar proton pumping as WT (17Flitsch S.L. Khorana H.G. Biochemistry. 1989; 28: 7800-7805Crossref PubMed Scopus (40) Google Scholar). This discrepancy may be due to a more relaxed conformation of the protein in the monomeric state that is obtained by reconstitution, as compared with the crystalline lattice of purple membrane.The kinetics and yield of the photocycle intermediates constitute a valuable appraisal of the disruption of the function of BR produced by the mutations. First of all, the amount of M intermediate is much decreased in T90A compared with WT and is decreased to half of the value of WT in T90V and D115A, suggesting a back-reaction to the purple form from one of the intermediates preceding M. On the other hand, the M rise is faster than WT in all mutants at neutral or alkaline pH, whereas the M decay is slower in all mutants at both pH conditions, except for D115A, which shows faster M decay kinetics at alkaline pH. This indicates that the deprotonated state of the Schiff base, i.e. the M intermediate, is favored in T90A and T90V, in agreement with FTIR data (see Fig. 6A). On the other hand, the absorbance maximum and dark-light adaptation properties give information on the environment of the retinal chromophore. As described under "Results," all three mutants show shifted absorbance maxima, but only T90A has abnormal dark-light adaptation kinetics. This means that the steric interaction between the methyl group of Thr-90 and the retinal participates in the
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