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1.
Solid State Nucl Magn Reson ; 84: 242-248, 2017.
Article in English | MEDLINE | ID: mdl-28781142

ABSTRACT

Chloride ions play important roles in many chemical and biological processes. This paper investigates the possibility of localizing 35Cl nuclei using solid-state NMR. It demonstrates that distances shorter than 3.8Å, between 13C atoms and 35Cl atoms in 10% uniformly labeled 13C L-tyrosine·HCl and natural abundance Glycine·HCl can be measured using rotational-echo (adiabatic passage) double-resonance (RE(AP)DOR). Furthermore the effect of quadrupolar interaction on the REDOR/REAPDOR experiment is quantified. The dephasing curve is plotted in a three dimensional chart as a function of the dephasing time and of the strength of quadrupolar interaction felt by each orientation. During spinning each orientation feels a quadrupolar interaction that varies in time, and therefore at each moment in time we reorder the crystallite orientations as a function of their contribution to the dephasing curve. In this way the effect of quadrupolar interaction on the dipolar dephasing curve can be fitted with a polynomial function. The numerical investigation performed allows us to generate REDOR/REAPDOR curves which are then used to simulate the experimental data.

2.
Solid State Nucl Magn Reson ; 82-83: 35-41, 2017.
Article in English | MEDLINE | ID: mdl-28187333

ABSTRACT

Chloride ions play important roles in many chemical and biological processes. This paper investigates the possibility of localizing 35Cl nuclei using solid-state NMR. It demonstrates that distances shorter than 3.8Å, between 13C atoms and 35Cl atoms in 10% uniformly labeled 13C L-tyrosine·HCl and natural abundance Glycine·HCl can be measured using rotational-echo (adiabatic passage) double-resonance (RE(AP)DOR). Furthermore the effect of quadrupolar interaction on the REDOR/REAPDOR experiment is quantified. The dephasing curve is plotted in a three dimensional chart as a function of the dephasing time and of the strength of quadrupolar interaction felt by each orientation. During spinning each orientation feels a quadrupolar interaction that varies in time, and therefore at each moment in time we reorder the crystallite orientations as a function of their contribution to the dephasing curve. In this way the effect of quadrupolar interaction on the dipolar dephasing curve can be fitted with a polynomial function. The numerical investigation performed allows us to generate REDOR/REAPDOR curves which are then used to simulate the experimental data.


Subject(s)
Glycine/chemistry , Magnetic Resonance Spectroscopy/methods , Rotation , Electrons
3.
Appl Magn Reson ; 34(3-4): 237-263, 2008 Aug.
Article in English | MEDLINE | ID: mdl-19194532

ABSTRACT

Dynamic nuclear polarization (DNP) results in a substantial nuclear polarization enhancement through a transfer of the magnetization from electrons to nuclei. Recent years have seen considerable progress in the development of DNP experiments directed towards enhancing sensitivity in biological nuclear magnetic resonance (NMR). This review covers the applications, hardware, polarizing agents, and theoretical descriptions that were developed at the Francis Bitter Magnet Laboratory at Massachusetts Institute of Technology for high-field DNP experiments. In frozen dielectrics, the enhanced nuclear polarization developed in the vicinity of the polarizing agent can be efficiently dispersed to the bulk of the sample via (1)H spin diffusion. This strategy has been proven effective in polarizing biologically interesting systems, such as nanocrystalline peptides and membrane proteins, without leading to paramagnetic broadening of the NMR signals. Gyrotrons have been used as a source of high-power (5-10 W) microwaves up to 460 GHz as required for the DNP experiments. Other hardware has also been developed allowing in situ microwave irradiation integrated with cryogenic magic-angle-spinning solid-state NMR. Advances in the quantum mechanical treatment are successful in describing the mechanism by which new biradical polarizing agents yield larger enhancements at higher magnetic fields. Finally, pulsed methods and solution experiments should play a prominent role in the future of DNP.

4.
Minerva Cardioangiol ; 54(5): 687-93, 2006 Oct.
Article in English | MEDLINE | ID: mdl-17019403

ABSTRACT

AIM: Dual antiplatelet treatment with aspirin and a thienopyridine is the antithrombotic treatment recommended after percutaneous coronary intervention with stent implantation (PCI-S). Optimal treatment in patients with an indication for long-term oral anticoagulation (OAC) undergoing PCI-S is currently undefined. The aim of this study was to evaluate the contemporary management of these patients, and determine the safety and the efficacy of the various regimens. METHODS: A systematic review of the literature reporting on this issue was carried out. RESULTS: The adopted strategies showed substantial variability, and the regimens used included: substitution of OAC for dual antiplatelet therapy in 25-54% of cases, addition to OAC of a single antiplatelet agent in 12-25% and institution of triple therapy with OAC (or low-molecular-weight heparin), aspirin and a thienopyridine in about 60%. OAC was systematically aimed at a lower intensity in 33% of cases, whereas in another 29% this was pursued only when a high hemorrhagic risk was perceived. Both safety and efficacy of the various regimens appeared suboptimal, with a 30-day occurrence of major bleeding and thrombotic complications of 3-7% and 4%, respectively. CONCLUSIONS: Due to the suboptimal safety and/or efficacy of the various regimens adopted, the optimal antithrombotic treatment in patients with an indication for OAC undergoing PCI-S remains to be defined. Since the number of this patient subgroup is foreseen to progressively increase over the next years, large scale registries and clinical trials are warranted.


Subject(s)
Angioplasty, Balloon, Coronary , Anticoagulants/therapeutic use , Platelet Aggregation Inhibitors/therapeutic use , Stents , Drug Therapy, Combination , Humans , Time Factors
7.
J Am Chem Soc ; 123(15): 3507-19, 2001 Apr 18.
Article in English | MEDLINE | ID: mdl-11472123

ABSTRACT

We describe a magic-angle spinning NMR experiment for selective (13)C-(15)N distance measurements in uniformly (13)C,(15)N-labeled solids, where multiple (13)C-(15)N and (13)C-(13)C interactions complicate the accurate measurement of structurally interesting, weak (13)C-(15)N dipolar couplings. The new experiment, termed FSR (frequency selective REDOR), combines the REDOR pulse sequence with a frequency selective spin-echo to recouple a single (13)C-(15)N dipolar interaction in a multiple spin system. Concurrently the remaining (13)C-(15)N dipolar couplings and all (13)C-(13)C scalar couplings to the selected (13)C are suppressed. The (13)C-(15)N coupling of interest is extracted by a least-squares fit of the experimentally observed modulation of the (13)C spin-echo intensity to the analytical expression describing the dipolar dephasing in an isolated heteronuclear spin pair under conventional REDOR. The experiment is demonstrated in three uniformly (13)C,(15)N-labeled model systems: asparagine, N-acetyl-L-Val-L-Leu and N-formyl-L-Met-L-Leu-L-Phe; in N-formyl-[U-(13)C,(15)N]L-Met-L-Leu-L-Phe we have determined a total of 16 internuclear distances in the 2.5-6 A range.


Subject(s)
Asparagine/chemistry , Peptides/chemistry , Carbon Radioisotopes , Magnetic Resonance Spectroscopy , Models, Chemical , Molecular Conformation , Nitrogen Radioisotopes
8.
Proc Natl Acad Sci U S A ; 98(4): 1571-6, 2001 Feb 13.
Article in English | MEDLINE | ID: mdl-11171992

ABSTRACT

Unidirectional proton transport in bacteriorhodopsin is enforced by the switching machinery of the active site. Threonine 89 is located in this region, with its O--H group forming a hydrogen bond with Asp-85, the acceptor for proton transfer from the Schiff base of the retinal chromophore. Previous IR spectroscopy of [3-(18)O]threonine-labeled bacteriorhodopsin showed that the hydrogen bond of the O--D group of Thr-89 in D(2)O is strengthened in the K photocycle intermediate. Here, we show that the strength and orientation of this hydrogen bond remains unchanged in the L intermediate and through the M intermediate. Furthermore, a strong interaction between Asp-85 and the O--H (O--D) group of Thr-89 in M is indicated by a shift in the C==O stretching vibration of the former because of (18)O substitution in the latter. Thus, the strong hydrogen bond between Asp-85 and Thr-89 in K persists through M, contrary to structural models based on x-ray crystallography of the photocycle intermediates. We propose that, upon photoisomerization of the chromophore, Thr-89 forms a tight, persistent complex with one of the side-chain oxygens of Asp-85 and is thereby precluded from participating in the switching process. On the other hand, the loss of hydrogen bonding at the other oxygen of Asp-85 in M may be related to the switching event.


Subject(s)
Aspartic Acid/chemistry , Bacteriorhodopsins/chemistry , Membrane Proteins/chemistry , Proton Pumps/chemistry , Threonine/chemistry , Isotope Labeling , Oxygen Isotopes
9.
Article in English | MEDLINE | ID: mdl-11088380

ABSTRACT

The phase diagram of parallel, charged spherocylinders is computed. The topology of the diagram is found to be similar to the uncharged one, but there are several qualitative changes. Regions of phase coexistence are significantly narrower and positional ordering is stabilized by the electrostatic repulsions. The nematic phase occupies a very narrow zone. We suggest that soft repulsions between surfactant micelles may be responsible for the absence of a nematic phase in most surfactant systems. We also present comparisons with the observed nematic-smectic phase transition for fd and tobacco mosaic virus particles.


Subject(s)
Inovirus/chemistry , Models, Chemical , Tobacco Mosaic Virus/chemistry , Entropy , Mathematical Computing , Micelles , Osmolar Concentration , Particle Size , Surface-Active Agents/chemistry
10.
Biochim Biophys Acta ; 1460(1): 95-105, 2000 Aug 30.
Article in English | MEDLINE | ID: mdl-10984593

ABSTRACT

In recent years, significant progress has been made in elucidating the structure of bacteriorhodopsin. However, the molecular mechanism by which vectorial proton motion is enforced remains unknown. Given the advantages of a protonated Schiff base for both photoisomerization and thermal reisomerization of the chromophore, a five-state proton pump can be rationalized in which the switch in the connectivity of the Schiff base between the two sides of the membrane is decoupled from double bond isomerization. This decoupling requires tight control of the Schiff base until it is deprotonated and decisive release after it is deprotonated. NMR evidence has been obtained for both the tight control and the decisive release: strain develops in the chromophore in the first half of the photocycle and disappears after deprotonation. The strain is associated with a strong interaction between the Schiff base and its counterion, an interaction that is broken when the Schiff base deprotonates. Thus the counterion appears to play a critical role in energy transduction, controlling the Schiff base in the first half of the photocycle by 'electrostatic steering'. NMR also detects other events during the photocycle, but it is argued that these are secondary to the central mechanism.


Subject(s)
Bacteriorhodopsins/chemistry , Magnetic Resonance Spectroscopy/methods , Isomerism , Light , Photochemistry , Proton-Motive Force , Retinaldehyde/chemistry , Schiff Bases/chemistry , Static Electricity
11.
J Magn Reson ; 146(1): 132-9, 2000 Sep.
Article in English | MEDLINE | ID: mdl-10968966

ABSTRACT

Heteronuclear dipolar recoupling with rotational-echo double-resonance (REDOR) is investigated in the rapid magic-angle spinning regime, where radiofrequency irradiation occupies a significant fraction of the rotor period (10-60%). We demonstrate, in two model (13)C-(15)N spin systems, [1-(13)C, (15)N] and [2-(13)C, (15)N]glycine, that REDOR DeltaS/S(0) curves acquired at high MAS rates and relatively low recoupling fields are nearly identical to the DeltaS/S(0) curve expected for REDOR with ideal delta-function pulses. The only noticeable effect of the finite pi pulse length on the recoupling is a minor scaling of the dipolar oscillation frequency. Experimental results are explained using both numerical calculations and average Hamiltonian theory, which is used to derive analytical expressions for evolution under REDOR recoupling sequences with different pi pulse phasing schemes. For xy-4 and extensions thereof, finite pulses scale only the dipolar oscillation frequency by a well-defined factor. For other phasing schemes (e.g., xx-4 and xx-4) both the frequency and amplitude of the oscillation are expected to change.


Subject(s)
Magnetic Resonance Spectroscopy/methods , Algorithms , Glycine/chemistry , Molecular Conformation
13.
Proc Natl Acad Sci U S A ; 97(9): 4643-8, 2000 Apr 25.
Article in English | MEDLINE | ID: mdl-10758159

ABSTRACT

The photoisomerization of the retinal in bacteriorhodopsin is selective and efficient and yields perturbation of the protein structure within femtoseconds. The stored light energy in the primary intermediate is then used for the net translocation of a proton across the membrane in the microsecond to millisecond regime. This study is aimed at identifying how the protein changes on photoisomerization by using the O-H groups of threonines as internal probes. Polarized Fourier-transform IR spectroscopy of [3-(18)O]threonine-labeled and unlabeled bacteriorhodopsin indicates that 3 of the threonines (of a total of 18) change their hydrogen bonding. One is exchangeable in D(2)O, but two are not. A comprehensive mutation study indicates that the residues involved are Thr-89, Thr-17, and Thr-121 (or Thr-90). The perturbation of only three threonine side chains suggests that the structural alteration at this stage of the photocycle is local and specific. Furthermore, the structural change of Thr-17, which is located >11 A from the retinal chromophore, implicates a specific perturbation channel in the protein that accompanies the retinal motion.


Subject(s)
Bacteriorhodopsins/chemistry , Retinaldehyde/chemistry , Amino Acid Sequence , Amino Acid Substitution , Bacteriorhodopsins/metabolism , Halobacterium salinarum/metabolism , Isomerism , Kinetics , Light , Models, Molecular , Mutagenesis, Site-Directed , Photochemistry , Protein Conformation , Recombinant Proteins/chemistry , Retinaldehyde/metabolism , Spectroscopy, Fourier Transform Infrared , Threonine
14.
Biochim Biophys Acta ; 1495(2): 140-9, 2000 Feb 02.
Article in English | MEDLINE | ID: mdl-10656971

ABSTRACT

Listeria monocytogenes and some other infectious bacteria polymerize their host cell's actin into tails that propel the bacteria through the cytoplasm. Here we show that reconstitution of this behavior in simpler media resolves two aspects of the mechanism of force transduction. First, since dilute reconstitution media have no cytoskeleton, we consider what keeps the tail from being pushed backward rather than the bacterium being propelled forward. The dependence of the partitioning of motion on the friction coefficient of the tail is derived. Consistent with experiments, we find that the resistance of the tail to motion is sensitive to its length. That even small tails are stationary in intact cells is attributed to anchoring to the cytoskeleton. Second, the comparatively low viscosity of some reconstitution media magnifies the effects of diffusion, such that a large gap will develop between the bacterium and its tail if they are unattached. At the viscosities of diluted platelet extracts, steady-state gaps of several bacterium lengths are predicted. Since such gaps are not observed, we conclude that Listeria must be attached to their tails. We consider what purposes such attachments might serve under physiological conditions. The implications for related pathogens and amoeboid locomotion are also discussed.


Subject(s)
Listeria monocytogenes/physiology , Actins , Culture Media , Diffusion , Friction , Listeria monocytogenes/pathogenicity , Locomotion , Mathematics , Movement
15.
Biochemistry ; 39(2): 362-71, 2000 Jan 18.
Article in English | MEDLINE | ID: mdl-10630997

ABSTRACT

Constraints on the proximity of the carboxyl carbons of the Asp-85 and Asp-212 side chains to the 14-carbon of the retinal chromophore have been established for the bR(555), bR(568), and M(412) states of bacteriorhodopsin (bR) using solid-state NMR spectroscopy. These distances were examined via (13)C-(13)C magnetization exchange, which was observed in two-dimensional RF-driven recoupling (RFDR) and spin diffusion experiments. A comparison of relative RFDR cross-peak intensities with simulations of the NMR experiments yields distance measurements of 4.4 +/- 0.6 and 4.8 +/- 1.0 A for the [4-(13)C]Asp-212 to [14-(13)C]retinal distances in bR(568) and M(412), respectively. The spin diffusion data are consistent with these results and indicate that the Asp-212 to 14-C-retinal distance increases by 16 +/- 10% upon conversion to the M-state. The absence of cross-peaks from [14-(13)C]retinal to [4-(13)C]Asp-85 in all states and between any [4-(13)C]Asp residue and [14-(13)C]retinal in bR(555) indicates that these distances exceed 6.0 A. For bR(568), the NMR distance constraints are in agreement with the results from recent diffraction studies on intact membranes, while for the M state the NMR results agree with theoretical simulations employing two bound waters in the region of the Asp-85 and Asp-212 residues. The structural information provided by NMR should prove useful for refining the current understanding of the role of aspartic acid residues in the proton-pumping mechanism of bR.


Subject(s)
Bacteriorhodopsins/chemistry , Anisotropy , Aspartic Acid/chemistry , Binding Sites , Magnetic Resonance Spectroscopy/methods , Retinaldehyde/chemistry , Spectroscopy, Fourier Transform Infrared
16.
Biophys J ; 77(6): 3407-23, 1999 Dec.
Article in English | MEDLINE | ID: mdl-10585963

ABSTRACT

The acrosomal process of the sea cucumber Thyone briareus can extend 90 microm in 10 s, but an epithelial goldfish keratocyte can only glide a few microns in the same time. Both speeds reflect the rate of extension of an actin network. The difference is in the delivery of actin monomers to the polymerization region. Diffusion supplies monomers fast enough to support the observed speed of goldfish keratocytes, but previous models have indicated that the acrosomal process of Thyone extends too rapidly for diffusion to keep up. Here we reexamine the assumptions made in earlier models and present a new model, the Actin Reconcentration Model, that includes more biological detail. Salt and water fluxes during the acrosomal reaction and the nonideality of the cytoplasm are particularly significant for actin delivery. We find that the variability of the acrosomal growth curve can be explained by the salt and water fluxes, and that nonideality magnifies the effect of actin concentration changes. We calculate the speed of process growth using biologically relevant parameters from the literature and find that the predictions of the model fall among the experimental data.


Subject(s)
Acrosome/metabolism , Actins/metabolism , Sea Cucumbers/metabolism , Acrosome/physiology , Actins/physiology , Animals , Biological Transport, Active , Biophysical Phenomena , Biophysics , Male , Models, Biological , Osmotic Pressure , Sea Cucumbers/physiology , Sperm Motility/physiology , Water/metabolism
17.
Biochemistry ; 38(30): 9676-83, 1999 Jul 27.
Article in English | MEDLINE | ID: mdl-10423246

ABSTRACT

The all-trans to 13-cis photoisomerization of the retinal chromophore of bacteriorhodopsin occurs selectively, efficiently, and on an ultrafast time scale. The reaction is facilitated by the surrounding protein matrix which undergoes further structural changes during the proton-transporting reaction cycle. Low-temperature polarized Fourier transform infrared difference spectra between bacteriorhodopsin and the K intermediate provide the possibility to investigate such structural changes, by probing O-H and N-H stretching vibrations [Kandori, Kinoshita, Shichida, and Maeda (1998) J. Phys. Chem. B 102, 7899-7905]. The measurements of [3-18O]threonine-labeled bacteriorhodopsin revealed that one of the D2O-sensitive bands (2506 cm(-1) in bacteriorhodopsin and 2466 cm(-1) in the K intermediate, in D2O exhibited 18(O)-induced isotope shift. The O-H stretching vibrations of the threonine side chain correspond to 3378 cm(-1) in bacteriorhodopsin and to 3317 cm(-1) in the K intermediate, indicating that hydrogen bonding becomes stronger after the photoisomerization. The O-H stretch frequency of neat secondary alcohol is 3340-3355 cm(-1). The O-H stretch bands are preserved in the T46V, T90V, T142N, T178N, and T205V mutant proteins, but diminished in T89A and T89C, and slightly shifted in T89S. Thus, the observed O-H stretching vibration originates from Thr89. This is consistent with the atomic structure of this region, and the change of the S-H stretching vibration of the T89C mutant in the K intermediate [Kandori, Kinoshita, Shichida, Maeda, Needleman, and Lanyi (1998) J. Am. Chem. Soc. 120, 5828-5829]. We conclude that all-trans to 13-cis isomerization causes shortening of the hydrogen bond between the OH group of Thr89 and a carboxyl oxygen atom of Asp85.


Subject(s)
Bacteriorhodopsins/chemistry , Threonine/chemistry , Aspartic Acid/chemistry , Bacteriorhodopsins/genetics , Deuterium Oxide/chemistry , Halobacterium salinarum/chemistry , Hydrogen Bonding , Isomerism , Mutagenesis, Site-Directed , Photochemistry , Protein Structure, Secondary , Schiff Bases , Spectroscopy, Fourier Transform Infrared/methods , Structure-Activity Relationship , Threonine/genetics
18.
Biochemistry ; 38(5): 1562-72, 1999 Feb 02.
Article in English | MEDLINE | ID: mdl-9931023

ABSTRACT

15N solid-state NMR (SSNMR) spectra of guanidyl-15N-labeled bacteriorhodopsin (bR) show perturbation of an arginine residue upon deprotonation of the retinal Schiff base during the photocycle. At the epsilon position, an upfield shift of 4 ppm is observed while the eta nitrogens develop a pair of 'wing' peaks separated by 24 ppm. Proton-driven spin diffusion between the two 'wing' peaks indicates that they arise from a single Arg residue. An unusually asymmetric environment for this residue is indicated by comparison with guanidyl-15N chemical shifts in a series of arginine model compounds. The 'wing' peaks are tentatively assigned to Arg-82 on the basis of the SSNMR investigations of the alkaline and neutral dark-adapted forms of the D85N bacteriorhodopsin mutant. Another, less asymmetric pair of eta signals, that is not affected by Schiff base deprotonation or D85 mutation, is tentatively assigned to Arg-134. The results are discussed in relation to existing models of bR structure and function.


Subject(s)
Arginine/metabolism , Asparagine/genetics , Aspartic Acid/genetics , Bacteriorhodopsins/metabolism , Proton-Motive Force , Amino Acid Substitution/genetics , Arginine/chemistry , Bacteriorhodopsins/chemistry , Bacteriorhodopsins/genetics , Carbon Isotopes , Crystallography, X-Ray , Halobacterium salinarum , Magnetic Resonance Spectroscopy , Photochemistry
19.
Biochim Biophys Acta ; 1365(3): 363-72, 1998 Jul 20.
Article in English | MEDLINE | ID: mdl-9711293

ABSTRACT

The behavior of threonine residues in the bacteriorhodopsin (bR) photocycle has been investigated by Fourier transform infrared difference spectroscopy. L-Threonine labeled at the hydroxyl group with 18O (L-[3-(18)O]threonine) was incorporated into bR and the bR-->M FTIR difference spectra measured. Bands are assigned to threonine vibrational modes on the basis of 18O induced isotope frequency shifts and normal mode calculations. In the 3500 cm-1 region, a negative band is assigned to the OH stretch of threonine. In the 1125 cm-1 region, a negative band is assigned to a mixed CH3 rock/CO stretch mode. The frequency of both these bands indicates the presence of at least one hydrogen bonded threonine hydroxyl group in light adapted bR which undergoes a change in structure by formation of the M intermediate. Spectral changes induced by the substitution Thr-89-->Asn but not Thr-46-->Asn or Asp-96-->Asn are consistent with the assignment of these bands to Thr-89. These results along with another related study on the mutant Thr-89-->Asn indicate that the active site of bR includes Thr-89 and that its interaction with the retinylidene Schiff base and Asp-85 may play an important role in regulating the color of bacteriorhodopsin and the transfer of a proton to the Schiff base.


Subject(s)
Bacteriorhodopsins/chemistry , Threonine/chemistry , Light , Models, Molecular , Mutagenesis, Site-Directed , Spectroscopy, Fourier Transform Infrared/methods
20.
Biochemistry ; 37(22): 8088-96, 1998 Jun 02.
Article in English | MEDLINE | ID: mdl-9609703

ABSTRACT

To enforce vectorial proton transport in bacteriorhodopsin (bR), it is necessary that there be a change in molecular structure between deprotonation and reprotonation of the chromophore-i.e., there must be at least two different M intermediates in the functional photocycle. We present here the first detection of multiple M intermediates in native wild-type bacteriorhodopsin by solid-state NMR. Illumination of light-adapted [zeta-15N-Lys]-bR at low temperatures shifts the 15N signal of the retinal Schiff base (SB) downfield by about 150 ppm, indicating a deprotonated chromophore. In 0.3 M Gdn-HCl at pH 10.0, two different M states are obtained, depending on the temperature during illumination. The M state routinely prepared at the lower temperature, Mo, decays to the newly observed M state, Mn, and the N intermediate, as the temperature is increased. Both relax to bR568 at 0 degreesC. A unique reaction sequence is derived: bR568-->Mo-->(Mn+N)-->bR568. Mo and Mn have similar chemical shifts at [12-13C]ret, [14-13C]ret, and [epsilon-13C]Lys216, indicating that Mn, like Mo, has a 13-cis and C=N anti chromophore. However, a small splitting in the [14-13C]ret signal of Mo reveals that it has at least two substates. The 7 ppm greater shielding of the SB nitrogen in Mn compared to Mo suggests an increase in basicity and/or hydrogen bonding. Probing the peptide backbone of the protein, via [1-13C]Val labeling, reveals a substantial structural change between Mo and Mn including the relaxation of perturbations at some sites and the development of new perturbations at other sites. The combination of the change in the protein structure and the increase in the pKa of the SB suggests that the demonstrated Mo-->Mn transition may function as the "reprotonation switch" required for vectorial proton transport.


Subject(s)
Bacteriorhodopsins/chemistry , Carbon Isotopes , Guanidine , Halobacterium salinarum , Light , Lysine/chemistry , Magnetic Resonance Spectroscopy , Nitrogen Isotopes , Photochemistry , Proline/chemistry , Protein Conformation , Protons , Schiff Bases , Spectrophotometry
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