A13C NMR study on [3-13C]-, [1-13C]Ala-, or [1-13C]Val-labeled transmembrane peptides of bacteriorhodopsin in lipid bilayers: Insertion, rigid-body motions, and local conformational fluctuations at ambient temperature

Kimura, Shigeki; Naito, Akira; Tuzi, Satoru; Saitô, Hazime

doi:10.1002/1097-0282(200101)58:1<78::aid-bip80>3.0.co;2-c

Cited by 26 publications

(22 citation statements)

References 58 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…among the plots. This finding is consistent with our view that 13 C-NMR peaks of bR, as classified as a II helices, were caused by conformational fluctuation at ambient temperature [39]. Fig.…”

Section: R E S U L T Ssupporting

confidence: 93%

“…Such upfield displacements are most prominent for the C‐terminal α helix (0.43 p.p.m.) and is interpreted in terms of reduced thermal fluctuation at low temperature, because downfield displacement of such an α helix peak due to conformational fluctuation is present in lipid bilayer [39]. 13 C chemical shifts of the transmembrane α helices exhibited similar but less pronounced displacements as large as 0.21 and 0.27 p.p.m.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Cytoplasmic surface structure of bacteriorhodopsin consisting of interhelical loops and C‐terminal α helix, modified by a variety of environmental factors as studied by ¹³C‐NMR

Yamaguchi¹,

Yonebayashi²,

Konishi³

et al. 2001

European Journal of Biochemistry

Self Cite

View full text Add to dashboard Cite

We have examined the 13 C-NMR spectra of [3-13 C] Alalabeled bacteriorhodopsin and its mutants by varying a variety of environmental or intrinsic factors such as ionic strength, temperature, pH, truncation of the C-terminal a helix, and site-directed mutation at cytoplasmic loops, in order to gain insight into a plausible surface structure arising from the C-terminal a helix and loops. It is found that the surface structure can be characterized as a complex stabilized by salt bridges or metal-mediated linkages among charged side chains. The surface complex in bacteriorhodopsin is most pronounced under the conditions of 10 mm NaCl at neutral pH but is destabilized to yield relaxed states when environmental factors are changed to high ionic strength, low pH and higher temperature. These two states were readily distinguished by associated spectral changes, including suppressed (cross polarization-magic angle spinning NMR) or displaced (upfield) 13 C signals from the C-terminal a helix, or modified spectral features in the loop region. It is also noteworthy that such spectral changes, when going from the complexed to relaxed states, occur either when the C-terminal a helix is deleted or sitedirected mutations were introduced at a cytoplasmic loop. These observations clearly emphasize that organization of the cytoplasmic surface complex is important in the stabilization of the three-dimensional structure at ambient temperature, and subsequently plays an essential role in biological functions.

show abstract

Section: R E S U L T Ssupporting

confidence: 93%

Section: Resultsmentioning

confidence: 99%

Cytoplasmic surface structure of bacteriorhodopsin consisting of interhelical loops and C‐terminal α helix, modified by a variety of environmental factors as studied by ¹³C‐NMR

Yamaguchi¹,

Yonebayashi²,

Konishi³

et al. 2001

European Journal of Biochemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…The intense peak at 14.1 ppm was assigned to a lipid methyl peak from egg PC in view of its absence without ppR. Seven 13 C NMR signals were resolved for [3-13 C]Ala-ppR, which are ascribed to the transmembrane K-helices, corresponding to the peak positions of bR consisting of normal K I helices and K II helices with low frequency £uctuation [17], although 13 C NMR signals from the loop regions were completely suppressed in spite of the presence of three Ala residues. The spectral feature of the transmembrane helices in ppR is very similar to that of bR in spite of sequence homology of 27% [3], because similar amounts of Ala residues are present between the two types of proteins.…”

Section: Comparison Ofmentioning

confidence: 99%

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

et al. 2003

Self Cite

View full text Add to dashboard Cite

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 K K-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 K K-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 K K-helix protruding from the membrane surface is involved in the complex formation and subsequent £uctuation frequency is reduced by one order of magnitude. ß

show abstract

“…If conformational energy changes as the transition state forms, then one expects obligatory partitioning of transition state binding energy among multiple interactions (steric, electrostatic, hydrogen-bonding, or solvation forces) at highly delocalized positions throughout the protein fold. Since plasma membrane transport proteins consist mainly of bundled helices that exhibit rigid-body behaviour [16,17] it is unlikely that conformational remodelling of helix-helix interfaces could occur without changing conformational energy. Thus, localized control of translocation specificity (catalytic power) is also quite unlikely, and instead the determinants of specificity ought to be distributed rather broadly at dynamic interfaces throughout the helix-rich structure (a hypothesis that should be broadly testable by TSR scanning approaches [5,6]).…”

Section: Discussionmentioning

confidence: 99%

The "Transport Specificity Ratio": a structure-function tool to search the protein fold for loci that control transition state stability in membrane transport catalysis

King

2004

BMC Biochem

View full text Add to dashboard Cite

BackgroundIn establishing structure-function relationships for membrane transport proteins, the interpretation of phenotypic changes can be problematic, owing to uncertainties in protein expression levels, sub-cellular localization, and protein-folding fidelity. A dual-label competitive transport assay called "Transport Specificity Ratio" (TSR) analysis has been developed that is simple to perform, and circumvents the "expression problem," providing a reliable TSR phenotype (a constant) for comparison to other transporters.ResultsUsing the Escherichia coli GABA (4-aminobutyrate) permease (GabP) as a model carrier, it is demonstrated that the TSR phenotype is largely independent of assay conditions, exhibiting: (i) indifference to the particular substrate concentrations used, (ii) indifference to extreme changes (40-fold) in transporter expression level, and within broad limits (iii) indifference to assay duration. The theoretical underpinnings of TSR analysis predict all of the above observations, supporting that TSR has (i) applicability in the analysis of membrane transport, and (ii) particular utility in the face of incomplete information on protein expression levels and initial reaction rate intervals (e.g., in high-throughput screening situations). The TSR was used to identify gab permease (GabP) variants that exhibit relative changes in catalytic specificity (kcat/Km) for [14C]GABA (4-aminobutyrate) versus [3H]NA (nipecotic acid).ConclusionsThe TSR phenotype is an easily measured constant that reflects innate molecular properties of the transition state, and provides a reliable index of the difference in catalytic specificity that a carrier exhibits toward a particular pair of substrates. A change in the TSR phenotype, called a Δ(TSR), represents a specificity shift attributable to underlying changes in the intrinsic substrate binding energy (ΔGb) that translocation catalysts rely upon to decrease activation energy (). TSR analysis is therefore a structure-function tool that enables parsimonious scanning for positions in the protein fold that couple to the transition state, creating stability and thereby serving as functional determinants of catalytic power (efficiency, or specificity).

show abstract

A13C NMR study on [3-13C]-, [1-13C]Ala-, or [1-13C]Val-labeled transmembrane peptides of bacteriorhodopsin in lipid bilayers: Insertion, rigid-body motions, and local conformational fluctuations at ambient temperature

Cited by 26 publications

References 58 publications

Cytoplasmic surface structure of bacteriorhodopsin consisting of interhelical loops and C‐terminal α helix, modified by a variety of environmental factors as studied by ¹³C‐NMR

Cytoplasmic surface structure of bacteriorhodopsin consisting of interhelical loops and C‐terminal α helix, modified by a variety of environmental factors as studied by ¹³C‐NMR

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

The "Transport Specificity Ratio": a structure-function tool to search the protein fold for loci that control transition state stability in membrane transport catalysis

Contact Info

Product

Resources

About

A13C NMR study on [3-13C]-, [1-13C]Ala-, or [1-13C]Val-labeled transmembrane peptides of bacteriorhodopsin in lipid bilayers: Insertion, rigid-body motions, and local conformational fluctuations at ambient temperature

Cited by 26 publications

References 58 publications

Cytoplasmic surface structure of bacteriorhodopsin consisting of interhelical loops and C‐terminal α helix, modified by a variety of environmental factors as studied by 13C‐NMR

Cytoplasmic surface structure of bacteriorhodopsin consisting of interhelical loops and C‐terminal α helix, modified by a variety of environmental factors as studied by 13C‐NMR

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

The "Transport Specificity Ratio": a structure-function tool to search the protein fold for loci that control transition state stability in membrane transport catalysis

Contact Info

Product

Resources

About

Cytoplasmic surface structure of bacteriorhodopsin consisting of interhelical loops and C‐terminal α helix, modified by a variety of environmental factors as studied by ¹³C‐NMR

Cytoplasmic surface structure of bacteriorhodopsin consisting of interhelical loops and C‐terminal α helix, modified by a variety of environmental factors as studied by ¹³C‐NMR

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