Recombinant Rhodocyclus tenuis Light-harvesting polypeptide B-885 alpha-2 chain, partial

What is the Recombinant Rhodocyclus tenuis Light-harvesting polypeptide B-885 alpha-2 chain?

The Recombinant Rhodocyclus tenuis Light-harvesting polypeptide B-885 alpha-2 chain is a protein component of the photosynthetic apparatus in the purple non-sulfur bacterium Rhodocyclus tenuis. It functions as part of the light-harvesting complex, specifically as an antenna pigment polypeptide that captures light energy and transfers it to the photosynthetic reaction center. According to product information, this protein is also known as "Antenna pigment polypeptide alpha-2 chain LH-1" .

Methodology for identification:

• UV-visible spectroscopy shows characteristic absorption profiles when complexed with bacteriochlorophyll

• Mass spectrometry can confirm molecular weight (similar to the approach used in high potential iron-sulfur protein from the same organism)

• SDS-PAGE with expected purity of >85% as indicated in product specifications

What are the optimal storage conditions for this recombinant protein?

Storage conditions significantly impact protein stability and functionality. Based on product specifications:

For reconstitution:

• Briefly centrifuge the vial before opening to bring contents to the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is the default recommendation)

• Aliquot for long-term storage at -20°C/-80°C

What expression systems are suitable for this protein?

Based on the available data, the baculovirus expression system is employed for commercial production of this recombinant protein . This system is advantageous for membrane proteins that may require post-translational modifications.

Alternative methodological approaches include:

• E. coli expression systems, which have been successfully used for other Rhodocyclus tenuis proteins such as the High potential iron-sulfur protein (HiPIP)

• For E. coli expression, researchers have demonstrated successful protocols involving:

  • Cloning the structural gene

  • Overexpression under optimized induction conditions

  • Purification to apparent homogeneity

  • Verification of proper folding through spectroscopic methods

How can I verify the identity and integrity of the expressed protein?

Multiple complementary techniques should be used to verify both identity and structural integrity:

The approach used for HiPIP from R. tenuis demonstrated that "All the observed properties of the recombinant protein parallel those of the native protein...indicating correct folding and incorporation of the iron-sulfur cluster" . Similar verification strategies can be applied to the light-harvesting polypeptide.

How do protein-pigment interactions influence spectroscopic properties?

Protein-pigment interactions are fundamental to the function of light-harvesting complexes. Research methodologies to investigate these interactions include:

• Site-directed mutagenesis of amino acids involved in pigment binding

• Spectroscopic analysis using:

  • Absorption spectroscopy to detect shifts in maxima

  • Circular dichroism to evaluate exciton coupling

  • Fluorescence spectroscopy to assess energy transfer efficiency

• Reconstitution experiments with:

  • Different bacteriochlorophyll analogs

  • Varying protein:pigment ratios

  • Modified lipid environments

Previous research on light-harvesting complexes has shown that "a light-harvesting antenna protein retains its folded conformation in the absence of protein-lipid and protein-pigment interactions" , but these interactions are critical for full functionality of the complex.

What approaches can be used to study quaternary structure?

The quaternary structure of light-harvesting complexes can be investigated using several complementary approaches:

Electron microscopy analysis has been successfully applied to light-harvesting complexes , providing valuable information about their quaternary organization. For pigment-protein complexes, it's essential to assess the structure in both pigment-bound and pigment-free states to understand the role of cofactors in assembly.

How can energy transfer efficiency be investigated in reconstituted complexes?

Energy transfer in light-harvesting complexes requires sophisticated time-resolved spectroscopic methods:

• Femtosecond transient absorption spectroscopy:

  • Uses pump-probe setup with ultrafast laser pulses

  • Measures energy migration through absorption changes

  • Provides kinetic components for transfer rate determination

• Time-resolved fluorescence spectroscopy:

  • Employs streak cameras or time-correlated single-photon counting

  • Measures fluorescence decay profiles at different wavelengths

  • Extraction of energy transfer times through multi-exponential fitting

• Steady-state methods for comparative studies:

  • Fluorescence quantum yield measurements

  • Excitation spectrum analysis compared with absorption profiles

Research protocol considerations:

• Temperature control is crucial (measurements at both room and cryogenic temperatures)

• Sample concentration optimization to minimize self-absorption effects

• Detergent or lipid environment selection to maintain native-like conformation

What experimental challenges exist in studying protein-lipid interactions?

Investigating protein-lipid interactions in membrane proteins like light-harvesting complexes presents several methodological challenges:

• Extraction and purification considerations:

  • Detergent selection is critical for maintaining native-like structure

  • Delipidation can cause conformational changes

  • Some boundary lipids may be tightly bound and co-purify with the protein

• Reconstitution approaches:

  • Incorporation into liposomes or nanodiscs for a membrane-like environment

  • Control of protein:lipid ratios and lipid composition

  • Verification of correct orientation and integration

• Analytical techniques:

  • Thin layer chromatography for phospholipid extraction and analysis

  • ESI-MS for identifying bound lipids

  • Fluorescence-based assays using labeled lipids

  • Molecular dynamics simulations to predict lipid binding sites

Dissertation research has specifically addressed "protein-lipid interactions of natural and model light-harvesting complex 2 in purple bacterium Rhodobacter sphaeroides" , providing methodological guidance applicable to Rhodocyclus tenuis proteins.

How do mutations affect pigment binding and spectroscopic properties?

Site-directed mutagenesis provides powerful insights into structure-function relationships:

• Mutation design strategy:

  • Target conserved residues potentially involved in pigment coordination

  • Focus on histidine residues (potential Mg2+ ligands)

  • Examine aromatic residues that may participate in π-stacking

  • Investigate hydrogen-bonding residues affecting electronic structure

• Experimental workflow:

  • Generate point mutations using PCR-based methods

  • Express and purify under identical conditions to wild-type

  • Perform spectroscopic characterization to assess changes

• Analytical methods:

  • Absorption spectroscopy to detect shifts in bacteriochlorophyll absorption maxima

  • Circular dichroism to evaluate altered pigment-protein interactions

  • Time-resolved spectroscopy to measure modified energy transfer rates

  • Thermal stability analysis to assess structural impact

What techniques reveal conformational dynamics in membrane environments?

Studying dynamics requires specialized techniques sensitive to motion on different timescales:

• Spectroscopic methods:

  • EPR spectroscopy with site-directed spin labeling

  • Solid-state NMR for proteins in lipid bilayers

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS)

  • Fluorescence techniques:

    • Fluorescence anisotropy for rotational freedom

    • FRET measurements for distance changes

    • Fluorescence correlation spectroscopy for diffusion properties

• Computational approaches:

  • Molecular dynamics simulations in explicit membrane environments

  • Analysis of protein flexibility and lipid interactions

  • Normal mode analysis to identify collective motions relevant to function

• Structural biology in native-like conditions:

  • Cryo-electron microscopy in nanodiscs or liposomes

  • Time-resolved X-ray techniques at X-ray free electron lasers

Research on "protein-lipid interactions of natural and model light-harvesting complex 2" provides methodological frameworks applicable to studying the B-885 alpha-2 chain in membrane environments.

How can isotopic labeling enhance structural studies?

Isotopic labeling enables sophisticated structural and dynamic studies:

• NMR spectroscopy applications:

  • Uniform 15N/13C labeling for backbone and side-chain assignments

  • Selective amino acid labeling to reduce spectral complexity

  • Deuteration to improve spectral quality for larger proteins

  • Methyl-group labeling for studying dynamics in large complexes

• Mass spectrometry applications:

  • Hydrogen-deuterium exchange (HDX-MS) to probe solvent accessibility

  • Cross-linking with mass spectrometry to identify interacting regions

  • Pulse-chase experiments to study assembly pathways

• Neutron scattering:

  • Small-angle neutron scattering with contrast matching

  • Allows visualization of specific components in complex assemblies

Protein expression protocols must be adapted for isotopic labeling:

• Growth in minimal media with isotope-enriched nitrogen and carbon sources

• Optimization of expression conditions that may differ from rich media

• Verification of proper folding after expression in isotope-enriched media

What methods assess thermal stability and unfolding pathways?

Thermal stability and unfolding studies provide insights into protein structure and dynamics:

• Differential scanning calorimetry (DSC):

  • Measures heat capacity changes during unfolding

  • Determination of melting temperature (Tm)

  • Identification of cooperative unfolding units

• Spectroscopic methods:

  • Temperature-dependent circular dichroism to monitor secondary structure changes

  • UV-visible spectroscopy to track bacteriochlorophyll spectral shifts

  • Fluorescence spectroscopy to monitor tertiary structure changes

  • FTIR spectroscopy for secondary structure analysis

• Hydrodynamic methods:

  • Dynamic light scattering to detect size changes and aggregation

  • Size-exclusion chromatography at varying temperatures

• Data analysis approaches:

  • Two-state versus multi-state unfolding models

  • Van't Hoff analysis to determine thermodynamic parameters

  • Comparison between pigment-bound and pigment-free states

These methodologies allow researchers to investigate how pigment binding affects protein stability, which is crucial for understanding structure-function relationships in light-harvesting complexes.