FTIR difference spectroscopy of bacteriorhodopsin: Toward a molecular model

Rothschild, Kenneth J.

doi:10.1007/bf00762674

Cited by 295 publications

(283 citation statements)

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“…Ϫ1 (data not shown). Such spectral evolution has previously been attributed to the N3 O transitions of the proton pumping cycle (27).…”

Section: Resultsmentioning

confidence: 69%

A Reaction-induced Fourier Transform-Infrared Spectroscopic Study of the Lactose Permease

Patzlaff¹,

Brooker²,

Barry³

2000

Journal of Biological Chemistry

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In chemiosmotic coupling, a transmembrane ion gradient is used as the source of energy to drive reactions. This process occurs in all cells, but the microscopic mechanism is not understood. Here, Escherichia coli lactose permease was used in a novel spectroscopic method to investigate the mechanism of chemiosmotic coupling in secondary active transporters. To provide a light-triggered electrochemical gradient, bacteriorhodopsin was co-reconstituted with the permease, and reaction-induced Fourier transform-infrared spectra were obtained from the co-reconstituted samples. The bacteriorhodopsin contributions were subtracted from these data to give spectra reflecting permease conformational changes that are induced by an electrochemical gradient. Positive bands in the 1765-1730 cm ؊1 region are attributable to carboxylic acid residues in the permease and are consistent with changes of pK a , protonation state, or environment. This is the first direct information concerning gradient-induced structural changes in the permease at the single amino acid level. Ultimately, these structural changes facilitate galactoside binding and may be involved in the storage of free energy.In secondary active transport, proteins employ a transmembrane electrochemical potential to translocate solutes across the membrane. This process plays a central metabolic role in all living cells. The molecular mechanism by which a transporter transduces a protonmotive force into a solute gradient is the focus of our study. The lactose permease is a transmembrane protein that is responsible for the uptake of lactose into the bacterial cytoplasm (1). The permease is a member of the major facilitator superfamily; members of the major facilitator superfamily catalyze uniport, symport, or antiport (reviewed in Ref.2). The lactose permease is a symporter that can harness the free energy stored in a proton electrochemical gradient to drive the accumulation of lactose against a concentration gradient (1). The permease transports protons and lactose with a one-to-one stoichiometry. A number of mutagenesis studies have led to models suggesting that a few ionizable residues along transmembrane segments play key roles in the mechanism of H ϩ /lactose symport (3, 4). However, direct evidence regarding the mechanism of chemiosmotic coupling in the lactose permease and related transporters has not been available. The goal of this study was to obtain dynamic information about structural changes in the permease associated with the imposition of an electrochemical gradient. To carry out this experiment, we have employed co-reconstitution (5-7) of the permease with a light-driven proton pump, bacteriorhodopsin, and reaction-induced FT-IR 1 spectroscopy (8). Reaction-induced FT-IR spectroscopy requires that a single sample be modulated between two structural states of interest. Co-reconstituted samples provide such a light-activatable and reversible experimental system. When a laser flash is given, bacteriorhodopsin pumps protons across the membrane and creates a H ϩ...

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“…Ϫ1 (data not shown). Such spectral evolution has previously been attributed to the N3 O transitions of the proton pumping cycle (27).…”

Section: Resultsmentioning

confidence: 69%

A Reaction-induced Fourier Transform-Infrared Spectroscopic Study of the Lactose Permease

Patzlaff¹,

Brooker²,

Barry³

2000

Journal of Biological Chemistry

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“…30 33,40 e.g., the occurrence of α II -helix structures that have other dihedral angles as well as longer hydrogen bonds. 41 None of these hypotheses have, however, yet been conclusively confirmed.…”

Section: Introductionmentioning

confidence: 99%

Vibrational Coupling between Helices Influences the Amide I Infrared Absorption of Proteins: Application to Bacteriorhodopsin and Rhodopsin

Karjalainen

Barth

2012

J. Phys. Chem. B

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The amide I spectrum of multimers of helical protein segments was simulated using transition dipole coupling (TDC) for long-range interactions between individual amide oscillators and DFT data from dipeptides (la Cour Jansen et al. 2006, J. Chem. Phys. 125, 44312) for nearest neighbor interactions. Vibrational coupling between amide groups on different helices shift the helix absorption to higher wavenumbers. This effect is small for helix dimers (1 cm −1 ) at 10Å distance and only moderately affected by changes in the relative orientation between the helices. However, the effect becomes considerable when several helices are bundled in membrane proteins. Particular examples are the 7-helix membrane proteins bacteriorhodopsin (BR) and rhodopsin, where the upshift is 4.3 and 5.3 cm −1 , respectively, due to inter-helical coupling within a BR monomer. A further upshift of 4.0 cm −1 occurs when BR monomers associate to trimers. We propose that inter-helical vibrational coupling explains the experimentally observed unusually high wavenumber of the amide I band of BR.

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“…The photolabile substrate analogs (Kaplan et al, 1978), ATP[Et(PhNO,)], ADP[Et (PhNO,)] and PO, [Et(PhNO,)], in combination with infrared spectroscopy, allowed us to measure the effects of nucleotides in CK and to identify the amino acid residues implicated in the active site. The light-induced release of photolabile substrate permitted us to measure very accurately infrared difference spectra before and after illumination, reflecting changes that are related solely to substrate binding to CK (Mantele, 1993;Rotschild, 1992;Siebert, 1995). The overall infrared spectra corresponding to the parts of CK that are not affected by substrate binding are cancelled by simple subtraction.…”

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confidence: 99%

ADP‐Binding and ATP‐Binding Sites in Native and Proteinase‐K‐Digested Creative Kinase, Probed by Reaction‐Induced Difference Infrared Spectroscopy

Raimbault

Clottes

Leydier

et al. 1997

European Journal of Biochemistry

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Conformational changes induced by nucleotide binding to native creatine kinase (CK) from rabbit muscle and to proteinase-K-digested (nicked) CK, were investigated by infrared spectroscopy. Photochemical release of ATP from ATP[Et (PhNO,)] in the presence of creatine and native CK produced reaction-induced difference infrared spectra (RIDS) of CK related to structural changes of the enzyme that paralleled the reversible phosphoryl transfer from ATP to creatine. Similarly the photochemical release of ADP from ADP[Et (PhNO,)] in the presence of phosphocreatine and native CK allowed us to follow the backward reaction and its corresponding RIDS. Infrared spectra of native CK indicated that carboxylate groups of Asp or Glu, and some carbonyl groups of the peptide backbone are involved in the enzymatic reaction. Native and proteinase nicked CK have similar Stokes' radii, tryptophan fluorescence, fluorescence fraction accessible to iodide, and far-ultraviolet CD spectra, indicating that native and modified enzymes have the same quaternary structures. However, infrared data showed that the binding site of the y-phosphate group of the nucleotide was affected in nicked CK compared with that of the native CK.Furthermore, the infrared absorptions associated with ionized carboxylate groups of Asp or Glu amino acid residues were different in nicked CK and in native CK.

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FTIR difference spectroscopy of bacteriorhodopsin: Toward a molecular model

Cited by 295 publications

References 132 publications

A Reaction-induced Fourier Transform-Infrared Spectroscopic Study of the Lactose Permease

A Reaction-induced Fourier Transform-Infrared Spectroscopic Study of the Lactose Permease

Vibrational Coupling between Helices Influences the Amide I Infrared Absorption of Proteins: Application to Bacteriorhodopsin and Rhodopsin

ADP‐Binding and ATP‐Binding Sites in Native and Proteinase‐K‐Digested Creative Kinase, Probed by Reaction‐Induced Difference Infrared Spectroscopy

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