研究目的
To understand the functional consequences of the allosteric activation of evolutionarily conserved signaling pathways in the rhodopsin structure by studying the binding of Ce6 to purified bovine rhodopsin.
研究成果
Ce6 binds weakly to bovine rhodopsin with higher affinity for Meta II and opsin states than the dark state. It acts as an allosteric modulator, perturbing the cytoplasmic domain and inhibiting G protein activation, without shifting the absorbance maximum or increasing dark noise. The results support Ce6's role in modulating rhodopsin structure and function, potentially generalizing to other GPCRs, but its physiological relevance in vision may be limited.
研究不足
The binding affinity of Ce6 is weak (μM range), and high concentrations used in NMR may lead to Ce6 aggregation, potentially affecting results. The study uses bovine rhodopsin as a model, and extrapolation to deep-sea fish rhodopsin, while supported by sequence conservation, may not fully capture in vivo conditions. The mechanism of red light sensitization is not fully elucidated, and energy transfer hypotheses were not experimentally validated.
1：Experimental Design and Method Selection:
The study used biophysical and biochemical approaches including absorbance spectroscopy, fluorescence spectroscopy, and NMR spectroscopy (1H, 19F, 1H-15N HSQC) to investigate Ce6 binding to bovine rhodopsin. Theoretical models included binding affinity calculations and conformational analysis.
2：Sample Selection and Data Sources:
Bovine rhodopsin was purified from bovine retinae or expressed in HEK293 cells. Ce6 was obtained commercially. Samples included dark-state, Meta II, and opsin conformations of rhodopsin.
3：List of Experimental Equipment and Materials:
Equipment included spectrophotometers (e.g., for absorbance and fluorescence), NMR spectrometers (Bruker 600 MHz, 800 MHz, 900 MHz), light sources (Fiber-Lite DC 950, Schott KL1500), and filters. Materials included sodium phosphate buffer, dodecyl maltoside (DM), DMSO, and specific reagents like 11-cis-retinal and 9-cis-14-fluororetinal.
4：Experimental Procedures and Operational Workflow:
Procedures involved sample preparation (e.g., labeling rhodopsin with 19F or 15N), titration with Ce6, illumination for light activation, and data acquisition at specified time points. NMR experiments were conducted in the dark to prevent unwanted activation.
5：Data Analysis Methods:
Data were analyzed using software like SigmaPlot for binding curves, TopSpin for NMR data, and statistical tests (e.g., student's t-test). Methods included spectral subtraction for absorbance, fluorescence correction, and chemical shift analysis.
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NMR Spectrometer
600 MHz
Bruker
Used for acquiring 1D and 2D NMR spectra to study protein-ligand interactions and conformational changes.
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NMR Spectrometer
800 MHz
Bruker
Used for selective excitation 1H NMR experiments.
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NMR Spectrometer
900 MHz
Bruker
Used for 1H-15N HSQC NMR spectroscopy.
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Retinal
9-cis-retinal
Sigma-Aldrich
Used in reconstitution experiments.
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CryoProbe
5 mm QCI CryoProbe
Bruker
Used for sensitive NMR detection.
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Software
TopSpin Version 2.0
Bruker
Used for NMR data acquisition and analysis.
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Light Source
Fiber-Lite DC 950
Dolan-Jenner industries
Used for illuminating samples in experiments.
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Light Source
Schott KL1500
Schott
Used for illumination with specific filters.
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Filter
630 nm long-pass filter
HV Skan
Used for selective red light illumination in absorbance spectroscopy.
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Filter
495 nm filter
Schott
Used for full bleaching of rhodopsin.
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Sepharose-4B
Sigma
Used for affinity purification of rhodopsin.
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Antibody
1D4
University of British Columbia
Used for immunoaffinity purification of rhodopsin.
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Chlorin e6
Frontier Scientific
The ligand studied for binding to rhodopsin.
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Fluororetinal
9-cis-14-fluororetinal
Synthesized and used for 19F NMR studies.
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Detergent
Dodecyl maltoside
Used for solubilizing rhodopsin in micelles.
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NMR Tube
Shigemi NMR tube
Shigemi
Used for NMR sample containment.
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Software
Sigmaplot 10.0
Sigmaplot
Used for scientific graphing and binding affinity calculations.
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Vacuum Manifold
Millipore
Used for filter binding assays.
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Scintillation Counter
LSC 6500
Beckman
Used for measuring radioactivity in GTPγS assays.
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Scintillation Cocktail
ScintiSafe LSC-cocktail
Fischer Scientific
Used in radioactivity measurements.
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