Structure and protein environment of the retinal chromophore in light- and dark-adapted bacteriorhodopsin studied by solid-state NMR

Our previous solid-state 13C NMR studies on bR have been directed at characterizing the structure and protein environment of the retinal chromophore in bR568 and bR548, the two components of the dark-adapted protein. In this paper, we extend these studies by presenting solid-state NMR spectra of light-adapted bR (bR568) and examining in more detail the chemical shift anisotropy of the retinal resonances near the ionone ring and Schiff base. Magic angle spinning (MAS) 13C NMR spectra were obtained of bR568, regenerated with retinal specifically 13C labeled at positions 12-15, which allowed assignment of the resonances observed in the dark-adapted bR spectrum. Of particular interest are the assignments of the 13C-13 and 13C-15 resonances. The 13C-15 chemical resonance for bR568 (160.0 ppm) is upfield of the 13C-15 resonance for bR548 (163.3 ppm). This difference is attributed to a weaker interaction between the Schiff base and its associated counterion in bR568. The 13C-13 chemical shift for bR568 (164.8 ppm) is close to that of the all-trans-retinal protonated Schiff base (PSB) model compound (approximately 162 ppm), while the 13C-13 resonance for bR548 (168.7 ppm) is approximately 7 ppm downfield of that of the 13-cis PSB model compound. The difference in the 13C-13 chemical shift between bR568 and bR548 is opposite that expected from the corresponding 15N chemical shifts of the Schiff base nitrogen and may be due to conformational distortion of the chromophore in the C13 = C14-C15 bonds.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of unsaturated fatty acids during bacteriorhodopsin preparation from Halobacterium halobium

AIMS: Isolation and characterization of unsaturated fatty acids during bacteriorhodopsin preparation from Halobacterium halobium. METHODS AND RESULTS: Halobacterium halobium was cultivated in a composite medium. Cells were collected by centrifugation followed by ultrasonic disruption, and the resulting suspension was subject to centrifugation for preparation of both pellet and supernatant. The pellet was saved in order to prepare bacteriorhodopsin, while the supernatant was used for the isolation of crude fatty acids by saponification and extraction. Crystallization then took place in acetone at -16 degrees C to remove fatty acids in which the carbon chain length was shorter than 13. The sample was obtained after purification and analysed by gas chromatography. The results demonstrated that Halobacterium halobium could synthesize multiple unsaturated fatty acids, particularly the three important polyunsaturated fatty acids arachidonic acid (1.12%), eicosapentaenoic acid (16.76%) and docosahexaenoic acid (9.38%). CONCLUSION: Important unsaturated fatty acids were isolated and characterized from the waste, which was produced during the preparation of bacteriorhodopsin from Halobacterium halobium. SIGNIFICANCE AND IMPACT OF THE STUDY: Halobacterium halobium has already been used for decades to prepare bacteriorhodopsin. We found that several important unsaturated fatty acids could be extracted from the bacterial waste, which extends its application scope and might bring additional benefits to humanity.     

Long-distance effects of site-directed mutations on backbone conformation in bacteriorhodopsin from solid state NMR of [1-13C]Val-labeled proteins.

We have recorded 13C cross-polarization-magic angle spinning and dipolar decoupled-magic angle spinning NMR spectra of [1-13C]Val-labeled wild-type bacteriorhodopsin (bR), and the V49A, V199A, T46V, T46V/V49A, D96N, and D85N mutants, in order to study conformational changes of the backbone caused by site-directed mutations along the extracellular surface and the cytoplasmic half channel. On the basis of spectral changes in the V49A and V199A mutants, and upon specific cleavage by chymotrypsin, we assigned the three well-resolved 13C signals observed at 172.93, 172.00, and 171. 11 ppm to [1-13C]Val 69, Val 49, and Val 199, respectively. The local conformations of the backbone at these residues are revealed by the conformation-dependent 13C chemical shifts. We find that at the ambient temperature of these measurements Val 69 is not in a beta-sheet, in spite of previous observations by electron microscopy and x-ray diffraction at cryogenic temperatures, but in a flexible turn structure that undergoes conformational fluctuation. Results with the T46V mutant suggest that there is a long-distance effect on backbone conformation between Thr 46 and Val 49. From the spectra of the D85N and E204Q mutants there also appears to be coupling between Val 49 and Asp 85 and between Asp 85 and Glu 204, respectively. In addition, the T2 measurement indicates conformational interaction between Asp 96 and extracellular surface. The protonation of Asp 85 in the photocycle therefore might induce changes in conformation or dynamics, or both, throughout the protein, from the extracellular surface to the side chain of Asp 96.     

NMR crystallography: the effect of deuteration on high resolution 13C solid state NMR spectra of a 7-TM protein

The effect of deuteration on the 13C linewidths of U-13C, 15N 2D crystalline bacteriorhodopsin (bR) from Halobacterium salinarium, a 248-amino acid protein with seven-transmembrane (7TM) spanning regions, has been studied in purple membranes as a prelude to potential structural studies. Spectral doubling of resonances was observed for receptor expressed in 2H medium (for both 50:50% 1H:2H, and a more highly deuterated form) with the resonances being of similar intensities and separated by <0.3 ppm in the methyl spectral regions in which they were readily distinguished. Line-widths of the methyl side chains were not significantly altered when the protein was expressed in highly deuterated medium compared to growth in fully protonated medium (spectral line widths were about 0.5 ppm on average for receptor expressed both in the fully protonated and highly deuterated media from the C delta, C gamma 1, and C gamma 2 Ile 13C signals observed in the direct, 21-39 ppm, and indirect, 9-17 ppm, dimensions). The measured 13C NMR line-widths observed for both protonated and deuterated form of the receptor are sufficiently narrow, indicating that this crystalline protein morphology is suitable for structural studies. 1) decoupling comparison of the protonated and deuterated bR imply that deuteration may be advantageous for samples in which low power 1H decoupling is required.     

Mechanism of proton pumping in bacteriorhodopsin by solid-state NMR: the protonation state of tyrosine in the light-adapted and M states.

Solid-state 13C NMR spectra were employed to characterize the protonation state of tyrosine in the light-adapted (bR568) and M states of bacteriorhodopsin (bR). Difference spectra (isotopically labeled bR minus natural-abundance bR) were obtained for [4'-13C]Tyr-labeled bR, regenerated with [14-13C]retinal as an internal marker to identify the photocycle states. The [14-13C]retinal has distinct chemical shifts for bR555, for bR568, and for the M intermediate generated and thermally trapped at pH 10 in the presence of 0.3 M KCl or 0.5 M guanidine. Previous work has demonstrated that tyrosine and tyrosinate are easily distinguished on the basis of the chemical shift of the 4'-13C label and that both NMR signals are detectable in dark-adapted bR, although the tyrosinate signal is only present at pH values greater than 12. In the present work, we show that neither the light-adapted form of bR prepared at pH 7 or 10 nor the M state thermally trapped at -80 degrees C in 0.3 M KCl pH 10, or in 0.5 M guanidine pH 10, shows any detectable tyrosinate. In addition, after the M samples were briefly warmed (approximately 30 s), no tyrosinate was observed. However, small (1-2 ppm) changes in the structure or dispersion in the Tyr peak were observed in the M state phototrapped by either method. These changes were reversible when the sample was warmed, although on a time scale slower than the relaxation of the retinal back to the bR568 conformer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Surface dynamics of bacteriorhodopsin as revealed by (13)C NMR studies on [(13)C]Ala-labeled proteins: detection of millisecond or microsecond motions in interhelical loops and C-terminal alpha-helix

We have recorded (13)C NMR spectra of [2-(13)C]-, [1-(13)C]-, [3-(13)C],- and [1,2,3-(13)C(3)]Ala-labeled bacteriorhodopsin (bR), and its mutants, A196G, A160G, and A103C, by means of cross polarization-magic angle spinning (CP-MAS) and dipolar decoupled-magic angle spinning (DD-MAS) techniques, to reveal the conformation and dynamics of bR, with emphasis on the loop and C-terminus structures. The (13)C NMR signals of the loop (C-D, E-F, and F-G) regions were almost completely suppressed from [2-(13)C]-, [1-(13)C]Ala-, and [1-(13)C]Gly-labeled bR, due to the presence of conformational fluctuation with correlation times of 10(-4) s that interfered with the peak-narrowing by magic angle spinning. The observation of such suppressed peaks for specific residues provides a unique means of detecting intermediate frequency motions on the time scale of ms or micros in the surface loops of membrane proteins. Instead, the three well-resolved (13)C CP-MAS NMR signals of [2-(13)C]Ala-bR, at 50.38, 49.90, and 47.96 ppm, were ascribed to the C-terminal alpha-helix previously proposed from the data for [3-(13)C]Ala-bR: the former two peaks were assigned to Ala 232 and 238, in view of the results of successive proteolysis experiments, while the highest-field peak was ascribed to Ala 235 prior to Pro 236. Even such (13)C NMR signals were substantially broadened when (13)C NMR spectra of fully labeled [1,2,3-(13)C]Ala-bR were recorded, because the broadening and splitting of peaks due to the accelerated transverse relaxation rate caused by the increased number of relaxation pathways through a number of (13)C-(13)C homo-nuclear dipolar interactions and scalar J couplings, respectively, are dominant among (13)C-labeled nuclei. In addition, approximate correlation times for local conformational fluctuations of different domains, including the C-terminal tail, C-terminal alpha-helix, loops, and transmembrane alpha-helices, were estimated by measurement of the spin-lattice relaxation times in the laboratory frame and spin-spin relaxation times under the conditions of cross-polarization-magic angle spinning, and comparative study of suppressed specific peaks between the CP-MAS and DD-MAS experiments.     

High resolution 13C-solid state NMR of bacteriorhodopsin: assignment of specific aspartic acids and structural implications of single site mutations

Three mutant strains of Halobacterium sp. GRB with the site of mutation in the bacterioopsin gene (PM 326: Asp96 Asn; PM 374: Asp96 Gly; PM 384: Asp85 Glu) were grown in a synthetic medium containing (4-¹³C)-Asp. The mutant bacteriorhodopsins labeled with (4-¹³C)-Asp (37%–45%), and owing to the metabolism of Halobacteria also with (11-¹³C)-Trp (50%–100%), were isolated as purple membranes and ¹³C Solid State Magic Angle Sample Spinning (MASS) Nuclear Magnetic Resonance (NMR) spectra of the samples were taken. The Asp96 mutants lacked the signal at 171.3 ppm which was previously assigned to a protonated internal Asp (Engelhard et al. 1989 a). This observation supports the conclusion that Asp96 is protonated in the ground state. PM 384 (Asp85 Glu) has an absorption maximum at 610 run. It can be converted into a purple form (_max = 5.40 nm) by treatment with a detergent (CHAPSO). The NMR-spectra of these two species differ from each other and from the wild type. The intensity of the resonance at 173 ppm in the wild type spectrum is reduced in both forms of the mutant protein. It is probable that this signal is caused by Asp85. The amino acid changes result not only in a perturbation of their direct environment but also effects on Trp residues and the chromophore protein interaction can be observed.Abbreviations CHAPSO 3-(3-cholamidopropyl)-dimethylammonio2-hydroxy-1-propane sulfonate - CP crosspolarization - bR bacteriorhodopsin - FTIR Fourier-transform-infrared - FID free induction decay - IR Infrared - MASS Magic angle sample spinning - NMR Nuclear magnetic resonance - TMS tetramethylsilane This research was supported by the Deutsche Forschungsgemeinschaft #SFB 60G9 Offprint requests to: M. Engelhard     

Dynamic aspects of extracellular loop region as a proton release pathway of bacteriorhodopsin studied by relaxation time measurements by solid state NMR

Local dynamics of interhelical loops in bacteriorhodopsin (bR), the extracellular BC, DE and FG, and cytoplasmic AB and CD loops, and helix B were determined on the basis of a variety of relaxation parameters for the resolved 13C and 15N signals of [1-13C]Tyr-, [15N]Pro- and [1-13C]Val-, [15N]Pro-labeled bR. Rotational echo double resonance (REDOR) filter experiments were used to assign [1-13C]Val-, [15N]Pro signals to the specific residues in bR. The previous assignments of [1-13C]Val-labeled peaks, 172.9 or 171.1 ppm, to Val69 were revised: the assignment of peak, 172.1 ppm, to Val69 was made in view of the additional information of conformation-dependent 15N chemical shifts of Pro bonded to Val in the presence of 13C-15N correlation, although no assignment of peak is feasible for 13C nuclei not bonded to Pro. 13C or 15N spin-lattice relaxation times (T1), spin-spin relaxation times under the condition of CP-MAS (T2), and cross relaxation times (TCH and TNH) for 13C and 15N nuclei and carbon or nitrogen-resolved, 1H spin-lattice relaxation times in the rotating flame (1H T1 rho) for the assigned signals were measured in [1-13C]Val-, [15N]Pro-bR. It turned out that V69-P70 in the BC loop in the extracellular side has a rigid beta-sheet in spite of longer loop and possesses large amplitude motions as revealed from 13C and 15N conformation-dependent chemical shifts and T1, T2, 1H T1 rho and cross relaxation times. In addition, breakage of the beta-sheet structure in the BC loop was seen in bacterio-opsin (bO) in the absence of retinal.     

Effect of acid pH on the absorption spectra and photoreactions of bacteriorhodopsin.

Purple membranes (PM) from Halobacterium halobium were incorporated into 7.5% polyacrylamide gels to prevent aggregation which occurs in suspensions at low pH. At pH 7.0, the circular dichroism (CD) spectra and visible absorption spectra of light- and dark-adapted bacteriorhodopsin (bR558, respectively) and the flash photolysis cycle of bR568 in gels were essentially the same as those in PM suspensions. Titration of the gels with hydrochloric acid showed the transition to a form absorbing at 605 nm (bR605 acid) with pK = 2.9 and to a second form absorbing at 565 nm (bR565 acid) with pK = 0.5. Isosbestic points were seen for each transition in both light- and dark-adapted gels. In addition, a third isosbestic point was evident between pH 3.5 and 7. Visible CD spectra of bR568, bR605 acid, and bR565 acid all showed the bilobed pattern typical of bR568 in suspensions of PM. Flash kinetic spectrophotometry (with 40-microseconds time resolution) of bR605 acid and bR565 acid showed transient absorbance changes with at least one transiently blue-shifted form for each and an early red-shifted intermediate for bR565 acid. Chromophore extraction from membrane suspensions yielded all-trans-retinal for bR565 acid and a mixture of 13-cis and trans isomers for bR605 acid.     

Solid-state 13C NMR of the retinal chromophore in photointermediates of bacteriorhodopsin: characterization of two forms of M

Solid-state 13C NMR spectra of the M photocycle intermediate of bacteriorhodopsin (bR) have been obtained from purple membrane regenerated with retinal specifically 13C labeled at positions 5, 12, 13, 14, and 15. The M intermediate was trapped at -40 degrees C and pH = 9.5-10.0 in either 100 mM NaCl [M (NaCl)] or 500 mM guanidine hydrochloride [M (Gdn-HCl)]. The 13C-12 chemical shift at 125.8 ppm in M (NaCl) and 128.1 ppm in M (Gdn-HCl) indicates that the C13 = C14 double bond has a cis configuration, while the 13C-13 chemical shift at 146.7 ppm in M (NaCl) and 145.7 ppm in M (Gdn-HCl) demonstrates that the Schiff base is unprotonated. The principal values of the chemical shift tensor of the 13C-5 resonance in both M (NaCl) and M (Gdn-HCl) are consistent with a 6-s-trans structure and a negative protein charge localized near C-5 as was observed in dark-adapted bR. The approximately 5 ppm upfield shift of the 13C-5 M resonance (approximately 140 ppm) relative to 13C-5 bR568 and bR548 (approximately 145 ppm) is attributed to an unprotonated Schiff base in the M chromophore. Of particular interest in this study were the results obtained from 13C-14 M. In M (NaCl), a dramatic upfield shift was observed for the 13C-14 resonance (115.2 ppm) relative to unprotonated Schiff base model compounds (approximately 128 ppm). In contrast, in M (Gdn-HCl) the 13C-14 resonance was observed at 125.7 ppm. The different 13C-14 chemical shifts in these two M preparations may be explained by different C = N configurations of the retinal-lysine Schiff base linkage, namely, syn in NaCl and anti in guanidine hydrochloride.     

Protein, lipid and water organization in bacteriorhodopsin crystals: a molecular view of the purple membrane at 1.9 A resolution.

BACKGROUND: Bacteriorhodopsin (bR) from Halobacterium salinarum is a proton pump that converts the energy of light into a proton gradient that drives ATP synthesis. The protein comprises seven transmembrane helices and in vivo is organized into purple patches, in which bR and lipids form a crystalline two-dimensional array. Upon absorption of a photon, retinal, which is covalently bound to Lys216 via a Schiff base, is isomerized to a 13-cis,15-anti configuration. This initiates a sequence of events - the photocycle - during which a proton is transferred from the Schiff base to Asp85, followed by proton release into the extracellular medium and reprotonation from the cytoplasmic side. RESULTS: The structure of bR in the ground state was solved to 1.9 A resolution from non-twinned crystals grown in a lipidic cubic phase. The structure reveals eight well-ordered water molecules in the extracellular half of the putative proton translocation pathway. The water molecules form a continuous hydrogen-bond network from the Schiff-base nitrogen (Lys216) to Glu194 and Glu204 and includes residues Asp85, Asp212 and Arg82. This network is involved both in proton translocation occurring during the photocycle, as well as in stabilizing the structure of the ground state. Nine lipid phytanyl moieties could be modeled into the electron-density maps. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of single crystals demonstrated the presence of four different charged lipid species. CONCLUSIONS: The structure of protein, lipid and water molecules in the crystals represents the functional entity of bR in the purple membrane of the bacteria at atomic resolution. Proton translocation from the Schiff base to the extracellular medium is mediated by a hydrogen-bond network that involves charged residues and water molecules.     

NMR crystallography: The effect of deuteration on high resolution C solid state NMR spectra of a 7-TM protein

The effect of deuteration on the ¹³C linewidths of U-¹³C, ¹⁵N 2D crystalline bacteriorhodopsin (bR) from Halobacterium salinarium, a 248-amino acid protein with seven-transmembrane (7TM) spanning regions, has been studied in purple membranes as a prelude to potential structural studies. Spectral doubling of resonances was observed for receptor expressed in ²H medium (for both 50:50% 1H:2H, and a more highly deuterated form) with the resonances being of similar intensities and separated by < 0.3 ppm in the methyl spectral regions in which they were readily distinguished. Line-widths of the methyl side chains were not significantly altered when the protein was expressed in highly deuterated medium compared to growth in fully protonated medium (spectral line widths were about 0.5 ppm on average for receptor expressed both in the fully protonated and highly deuterated media from the Cδ, Cγ₁, and Cγ₂ Ile ¹³C signals observed in the direct, 21-39 ppm, and indirect, 9-17 ppm, dimensions). The measured ¹³C NMR line-widths observed for both protonated and deuterated form of the receptor are sufficiently narrow, indicating that this crystalline protein morphology is suitable for structural studies. ¹H decoupling comparison of the protonated and deuterated bR imply that deuteration may be advantageous for samples in which low power ¹H decoupling is required.     

Solid state NMR study of [epsilon-13C]Lys-bacteriorhodopsin: Schiff base photoisomerization.

Previous solid state 13C-NMR studies of bacteriorhodopsin (bR) have inferred the C = N configuration of the retinal-lysine Schiff base linkage from the [14-13C]retinal chemical shift (1-3). Here we verify the interpretation of the [14-13C]-retinal data using the [epsilon-13C]lysine 216 resonance. The epsilon-Lys-216 chemical shifts in bR555 (48 ppm) and bR568 (53 ppm) are consistent with a C = N isomerization from syn in bR555 to anti in bR568. The M photointermediate was trapped at pH 10.0 and low temperatures by illumination of samples containing either 0.5 M guanidine-HCl or 0.1 M NaCl. In both preparations, the [epsilon-13C]Lys-216 resonance of M is 6 ppm downfield from that of bR568. This shift is attributed to deprotonation of the Schiff base nitrogen and is consistent with the idea that the M intermediate contains a C = N anti chromophore. M is the only intermediate trapped in the presence of 0.5 M guanidine-HCl, whereas a second species, X, is trapped in the presence of 0.1 M NaCl. The [epsilon-13C]Lys-216 resonance of X is coincident with the signal for bR568, indicating that X is either C = N anti and protonated or C = N syn and deprotonated.     

Solid-state 13C NMR study of tyrosine protonation in dark-adapted bacteriorhodopsin

Solid-state 13C MAS NMR spectra were obtained for dark-adapted bacteriorhodopsin (bR) labeled with [4'-13C]Tyr. Difference spectra (labeled minus natural abundance) taken at pH values between 2 and 12, and temperatures between 20 and -90 degrees C, exhibit a single signal centered at 156 ppm, indicating that the 11 tyrosines are protonated over a wide pH range. However, at pH 13, a second line appears in the spectrum with an isotropic shift of 165 ppm. Comparisons with solution and solid-state spectra of model compounds suggest that this second line is due to the formation of tyrosinate. Integrated intensities indicate that about half of the tyrosines are deprotonated at pH 13. This result demonstrates that deprotonated tyrosines in a membrane protein are detectable with solid-state NMR and that neither the bR568 nor the bR555 form of bR present in the dark-adapted state contains a tyrosinate at pH values between 2 and 12. Deprotonation of a single tyrosine in bR568 would account for 3.6% of the total tyrosine signal, which would be detectable with the current signal-to-noise ratio. We observe a slight heterogeneity and subtle line-width changes in the tyrosine signal between pH 7 and pH 12, which we interpret to be due to protein environmental effects (such as changes in hydrogen bonding) rather than complete deprotonation of tyrosine residue(s).     

13C NMR of cyanylated flavodoxin from Megasphaera elsdenii and of thiocyanate model compounds.

Both of the thiol groups of Megasphaera elsdenii flavodoxin have been cyanylated using 13C-enriched cyanide. This chemical modification increases the dissociation constant of the apoflavodoxin-flavin mononucleotide (FMN) complex from 0.4 nM to 2 microM. The thiocyanate carbons of the cyanylated cysteine residues in apoflavodoxin had 13C chemical shifts of 109.4 ppm and 112.2 ppm, which were replaced by signals at 115.5 ppm and 109.6 ppm when FMN was bound. The signals at 109.4 ppm and 112.2 ppm due to the cyanylated apoflavodoxin were unstable at 28 degrees C, and they were slowly replaced signals at 114.5 ppm and 115.3 ppm which are attributed to an inactive form of the apoprotein, which does not bind FMN. At alkaline pH values or after prolonged incubation at neutral pH, the signals at 114.5 ppm and 115.3 ppm were replaced by signals at approximately 171 ppm. On the basis of results obtained with model compounds, the signals at 171 ppm are assigned to the 2-imino carbon of the 2-iminothiazolidine ring formed by the cyclization of the appropriate thiocyanate group. After determining the chemical shift of the thiocyanate carbon of model compounds in a range of solvents, we conclude that the thiocyanate carbons will have a minimal chemical shift of approximately 109 ppm in apolar solvents which do not contain hydrogen bond donors. In water, a more polar hydrogen-bonding solvent, the chemical shift increases to approximately 115 ppm. We also conclude that the chemical shift of a thiocyanate carbon can be used as a probe of its molecular environment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phase transitions of the purple membranes of Halobacterium halobium

Purple membranes of Halobacterium halobium were studied by differential scanning calorimetry. No transition was detected at temperatures below 70 degrees C. A small endothermic transition was seen at about 80 degrees C and a larger one at 100 degrees C. The larger transition is the irreversible denaturation of bacteriorhodopsin. The smaller transition is accompanied by a change in the visible absorption spectrum and is believed to be reversible, involving a cooperative change in crystalline structure of the membrane.     

Solid state 13C NMR study of collagen molecular dynamics in hard and soft tissues

The molecular dynamics of the collagen backbone in intact connective tissues has been elucidated using 13C line shape analysis. Since one-third of the amino acid residues in collagen are glycines, we have labeled: (a) reconstituted lathrytic (uncross-linked) chick calvaria collagen fibrils; (b) rat tail tendon (cross-linked); and (c) rat calvaria (cross-linked and mineralized) collagen with [1-13C]glycine. The proton-enhanced and normal 90 degrees - t proton-decoupled spectrum of each collagen sample shows an asymmetric chemical shift powder pattern for the glycine carbonyl carbon. The powder line width, delta, (delta = sigma zz - sigma xx) at 22 degrees C for the uncross-linked reconstituted collagen fibril is 108 ppm, whereas the maximum value of delta (140 ppm) is observed for the cross-linked and mineralized collagen fibrils in rat calvaria. The powder line widths for the cross-linked fibrils in tail tendons and demineralized calvaria are 124 and 120 ppm, respectively. However, since the same line shape and line width (145 ppm) are observed for all samples at -35 degrees C, the difference in delta values observed at room temperature is attributed to differences in molecular mobility of collagen in various samples. The line shapes are analyzed using a dynamic model in which azimuthal orientation of the collagen backbone is assumed to fluctuate as a consequence of reorientation about the helix axis. The observed line shapes are sensitive to motions having correlation times less than approximately 10(-4) s and the analysis provides the values of the root mean square fluctuation in azimuthal angle, gamma rms, due to such motions. It is found that gamma rms equals 41 degrees, 33 degrees, and 14 degrees for the uncross-linked, cross-linked, and mineralized collagens, respectively. These results provide the first information about the extent that cross-linking and mineralization restrict molecular motion in collagen.     

Fourier transform infrared study of the halorhodopsin chloride pump

Halorhodopsin (hR) is a light-driven chloride pump located in the cell membrane of Halobacterium halobium. Fourier transform infrared difference spectroscopy has been used to study structural alterations occurring during the hR photocycle. The frequencies of peaks attributed to the retinylidene chromophore are similar to those observed in the spectra of the related protein bacteriorhodopsin (bR), indicating that in hR as in bR an all-trans----13-cis isomerization occurs during formation of the early bathoproduct. Spectral features due to protein structural alterations are also similar for the bR and hR photocycles. For example, formation of the red-shifted primary photoproducts of both hR and bR results in similar carboxyl peaks in the 1730-1745-cm-1 region. However, in contrast to bR, no further changes are observed in the carboxyl region during subsequent steps in the hR photocycle, indicating that additional carboxyl groups are not directly involved in chloride translocation. Overall, the close similarity of vibrations in hR and bR photoproduct difference spectra supports the existence of some common elements in the molecular mechanisms of energy transduction and active transport by these two proteins.     

Dynamic structure of pharaonis phoborhodopsin (sensory rhodopsin II) and complex with a cognate truncated transducer as revealed by site-directed 13C solid-state NMR

We have recorded (13)C nuclear magnetic resonance (NMR) spectra of [3-(13)C]Ala, [1-(13)C]Val-labeled pharaonis phoborhodopsin (ppR or sensory rhodopsin II) incorporated into egg PC (phosphatidylcholine) bilayer, by means of site-directed high-resolution solid-state NMR techniques. Seven (13)C NMR signals from transmembrane alpha-helices were resolved for [3-(13)C]Ala-ppR at almost the same positions as those of bacteriorhodopsin (bR), except for the suppressed peaks in the loop regions in spite of the presence of at least three Ala residues. In contrast, (13)C NMR signals from the loops were visible from [1-(13)C]Val-ppR but their peak positions of the transmembrane alpha-helices are not always the same between ppR and bR. The motional frequency of the loop regions in ppR was estimated as 10(5) Hz in view of the suppressed peaks from [3-(13)C]Ala-ppR due to interference with proton decoupling frequency. We found that conformation and dynamics of ppR were appreciably altered by complex formation with a cognate truncated transducer pHtr II (1-159). In particular, the C-terminal alpha-helix protruding from the membrane surface is involved in the complex formation and subsequent fluctuation frequency is reduced by one order of magnitude.