Recombinant Rhodobacter sphaeroides UPF0060 membrane protein RSKD131_0092 (RSKD131_0092)

What is the basic structure and characteristics of Rhodobacter sphaeroides UPF0060 membrane protein RSKD131_0092?

The RSKD131_0092 protein is a UPF0060 family membrane protein from Rhodobacter sphaeroides with 108 amino acids. The full amino acid sequence is: MGLSLAAYAGAALAEIAGCFAVWAWWRLGASALWLVPGALSLGTFAWLLALTPVEAAGRSYAVYGGVYVAASLLWLWAVEGVRPDRWDMGGAALVLAGAAVILWAPRG . This protein is a full-length membrane protein (1-108 aa) that is typically expressed with an N-terminal His tag to facilitate purification procedures. The hydrophobic nature of this sequence suggests multiple transmembrane domains, consistent with its classification as a membrane protein .

When investigating this protein's structural characteristics, researchers should note that membrane proteins often require specialized techniques for structural determination compared to soluble proteins. Computational predictions suggest this protein contains multiple transmembrane helices, though detailed 3D structural information remains limited.

How should researchers prepare and store recombinant RSKD131_0092 protein samples for optimal stability?

Recombinant RSKD131_0092 protein is typically supplied as a lyophilized powder, requiring careful handling for reconstitution and storage . For optimal stability and experimental consistency, follow these recommended procedures:

Reconstitution Protocol:

• Centrifuge the vial briefly before opening to collect all material at 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% (recommended optimum: 50%)

• Aliquot into smaller volumes to avoid repeated freeze-thaw cycles

Storage Recommendations:

• Store reconstituted aliquots at -20°C/-80°C for long-term storage

• Working aliquots can be stored at 4°C for up to one week

• Repeated freeze-thaw cycles should be strictly avoided as they compromise protein integrity

The protein is typically stored in Tris/PBS-based buffer with 6% trehalose at pH 8.0, which helps maintain structural integrity during freeze-thaw transitions . Researchers should verify protein stability after reconstitution through activity assays or structural analysis before proceeding with experiments.

What expression systems are recommended for recombinant RSKD131_0092 protein production?

Advantages of R. sphaeroides as an expression platform:

• Reduced toxic effects on the host, which often limit membrane protein expression

• Superior functional folding compared to traditional E. coli systems

• Lower rates of inclusion body formation and proteolytic degradation

• Higher biomass yield (3-fold increase) using anaerobic photoheterotrophic growth conditions

• Strong native puc promoter that enhances expression

Expression Optimization Table:

For challenging membrane proteins, the photoheterotrophic growth regime in R. sphaeroides has demonstrated substantial improvements in protein yield and quality, making it particularly valuable for functional studies of membrane proteins like RSKD131_0092 .

What experimental design approaches are most effective for studying RSKD131_0092 protein function?

When designing experiments to investigate RSKD131_0092 protein function, researchers should consider implementing a randomized block design to control for variables that might influence membrane protein behavior . This approach is particularly valuable when working with membrane proteins, which can be sensitive to experimental conditions.

Recommended Experimental Design Framework:

• Define clear research questions: Establish specific hypotheses about RSKD131_0092 function based on its sequence characteristics and predicted membrane topology

• Identify variables:

  • Independent variables: Expression conditions, detergent types, buffer compositions

  • Dependent variables: Protein activity, stability, interaction partners

  • Control variables: Temperature, pH, ionic strength

• Implement randomized block design:

  • Block based on known sources of variability (e.g., protein preparation batches)

  • Randomly assign treatments within blocks

  • This design reduces experimental error by accounting for systematic differences between blocks

• Statistical considerations:

  • Conduct power analysis to determine appropriate sample sizes

  • Implement appropriate statistical tests based on data distribution

  • Consider within-subjects design for experiments involving multiple conditions with the same protein preparation

When investigating protein-protein interactions or membrane integration, researchers should implement controls that account for the hydrophobic nature of membrane proteins and potential non-specific interactions with detergents or lipids.

How can researchers optimize solubilization and purification protocols for RSKD131_0092?

Membrane protein solubilization and purification represent significant challenges due to their hydrophobic nature. For RSKD131_0092, a strategic approach is necessary:

Solubilization Optimization:

• Detergent screening: Test a panel of detergents including:

  • Mild detergents: DDM, LMNG, Digitonin

  • Zwitterionic detergents: LDAO, FC-12

  • Start with 1% concentration and optimize based on extraction efficiency

• Lipid supplementation: Adding phospholipids (0.01-0.1%) during solubilization can stabilize the native conformation

Purification Protocol:

• Immobilized metal affinity chromatography (IMAC):

  • Utilize the N-terminal His-tag for efficient capture

  • Wash extensively to remove non-specifically bound proteins

  • Use a shallow imidazole gradient (20-300 mM) for elution

• Size exclusion chromatography:

  • Secondary purification to isolate monodisperse protein

  • Assess oligomeric state and sample homogeneity

Optimization Considerations:

The final purification protocol should be validated by assessing protein purity (>90% by SDS-PAGE), structural integrity, and functional activity . Researchers should note that repeated freeze-thaw cycles significantly impact membrane protein stability, so experimental planning should minimize such cycles.

What approaches can be used to investigate the membrane topology and interactions of RSKD131_0092?

Understanding membrane topology and protein interactions is crucial for characterizing RSKD131_0092 function. Several complementary approaches provide valuable insights:

Membrane Topology Analysis:

• In silico prediction:

  • Hydropathy analysis using tools like TMHMM, Phobius, or TOPCONS

  • Secondary structure prediction using PsiPred or JPred

  • Compare multiple algorithms to identify consensus predictions

• Experimental verification:

  • Cysteine scanning mutagenesis with membrane-impermeable labeling reagents

  • Protease protection assays to identify accessible regions

  • Fluorescence-based approaches with GFP fusions at different positions

Protein Interaction Studies:

• Co-immunoprecipitation:

  • Using mild detergents to preserve interactions

  • Coupled with mass spectrometry to identify interacting partners

• Crosslinking approaches:

  • Chemical crosslinkers of different lengths and specificities

  • Photoactivatable amino acid incorporation for zero-length crosslinking

• Reconstitution systems:

  • Proteoliposome preparation with defined lipid compositions

  • Allows functional studies in controlled membrane environments

When planning these experiments, researchers should consider that membrane proteins often exist in complexes, and disrupting the native membrane environment may alter their interactions and functions. Therefore, validation across multiple experimental approaches is strongly recommended.

What protocols are recommended for functional characterization of RSKD131_0092?

Functional characterization of membrane proteins like RSKD131_0092 requires specialized approaches that maintain the protein in a native-like environment. Based on research with similar membrane proteins, the following methodological approaches are recommended:

Functional Reconstitution:

• Proteoliposome preparation:

  • Solubilize purified protein in detergent (typically 0.1% DDM)

  • Mix with lipids (POPC:POPE:POPG at 7:2:1 ratio)

  • Remove detergent using Bio-Beads or dialysis

  • Verify incorporation by freeze-fracture electron microscopy

• Activity assays based on predicted function:

  • For transport proteins: liposome-based flux assays with appropriate substrates

  • For enzymatic activity: coupled assays with detectable products

  • For structural roles: membrane integrity and fluidity measurements

Advanced Biophysical Characterization:

• Circular dichroism (CD) spectroscopy:

  • Secondary structure assessment (far-UV CD)

  • Tertiary structure fingerprinting (near-UV CD)

  • Thermal stability measurements

• Fluorescence-based techniques:

  • Intrinsic tryptophan fluorescence for conformational studies

  • FRET analyses for interaction studies and distance measurements

When assessing functionality, researchers should implement both positive and negative controls, including denatured protein samples and liposomes without incorporated protein. Additionally, comparisons with known membrane proteins of similar structure can provide valuable benchmarking.

How can researchers effectively design mutagenesis studies to probe RSKD131_0092 structure-function relationships?

Strategic mutagenesis is a powerful approach for investigating structure-function relationships in membrane proteins like RSKD131_0092. A systematic and hypothesis-driven approach will yield the most valuable insights:

Mutagenesis Strategy Development:

• Target selection based on:

  • Sequence conservation analysis across UPF0060 family proteins

  • Predicted transmembrane regions and functional domains

  • Charged residues within transmembrane segments (often functionally critical)

  • Aromatic residues at membrane interfaces

• Types of mutations to consider:

  • Alanine scanning: Replace target residues with alanine to remove side chain interactions

  • Conservative substitutions: Maintain chemical properties (e.g., Leu→Ile, Asp→Glu)

  • Charge alterations: Reverse charge (Lys→Glu) or neutralize (Lys→Gln)

  • Cysteine substitutions: For subsequent labeling or crosslinking studies

Experimental Design Considerations:

When designing mutagenesis studies, researchers should implement a pre-experimental computational analysis to prioritize residues likely to be functionally significant. Additionally, all mutants should be validated for proper expression, folding, and membrane integration before functional assessments are conducted.

What analytical techniques should be employed for structural characterization of RSKD131_0092?

Structural characterization of membrane proteins presents unique challenges. For RSKD131_0092, a multi-technique approach will provide the most comprehensive structural insights:

Solution-State Techniques:

• Small-angle X-ray scattering (SAXS):

  • Provides low-resolution envelope of protein-detergent complexes

  • Sample requirements: 1-5 mg/mL protein in monodisperse state

  • Analysis yields radius of gyration, maximum dimension, and molecular envelope

• Nuclear magnetic resonance (NMR):

  • For specific domain characterization or dynamics studies

  • Requires isotopic labeling (¹⁵N, ¹³C) during protein expression

  • Can provide residue-level information on protein-ligand interactions

Solid-State Techniques:

• X-ray crystallography:

  • Requires formation of well-diffracting 3D crystals

  • Lipidic cubic phase crystallization often successful for membrane proteins

  • Provides atomic-resolution structures when successful

• Cryo-electron microscopy:

  • Single-particle analysis for larger membrane proteins or complexes

  • Sample requirements: 50-100 μg of highly pure, homogeneous protein

  • Recent advances allow near-atomic resolution for membrane proteins >150 kDa

Computational Approaches:

• Homology modeling:

  • Based on structurally characterized members of the UPF0060 family

  • Validate models using experimental constraints

  • Molecular dynamics simulations in explicit membrane environments

For RSKD131_0092, researchers should begin with biophysical characterization (CD spectroscopy, SEC-MALS) to assess sample quality before advancing to more resource-intensive structural studies. The complementary information from multiple techniques will provide the most reliable structural model.

How should researchers analyze expression optimization data for RSKD131_0092?

Optimizing expression conditions for membrane proteins like RSKD131_0092 requires systematic data collection and analysis. The following analytical framework will help researchers interpret their optimization experiments:

Data Collection Parameters:

• Expression variables to monitor:

  • Total protein yield (mg/L culture)

  • Proportion of correctly folded protein

  • Functional activity per unit protein

  • Expression timeline (optimal induction and harvest points)

• Statistical analysis approach:

  • Implement multifactorial design of experiments (DOE) approach

  • Analyze main effects and interaction effects between variables

  • Use response surface methodology to identify optimal conditions

Analytical Framework:

When interpreting expression data, researchers should prioritize conditions that produce functionally active protein rather than simply maximizing total yield. For R. sphaeroides expression systems, analyzing the impact of light intensity and oxygen levels on expression can reveal important optimization opportunities that aren't relevant in traditional expression systems .

What are the key considerations when interpreting structural data for membrane proteins like RSKD131_0092?

Structural data for membrane proteins requires specialized interpretation due to the presence of detergents, lipids, or nanodiscs that maintain protein solubility. Consider these key aspects when analyzing structural data for RSKD131_0092:

Interpreting Different Data Types:

Data Integration Approach:

Researchers should integrate multiple data types to build a comprehensive structural model. When inconsistencies arise between different techniques, consider the following hierarchy of reliability:

• High-resolution experimental structures (X-ray, cryo-EM)

• Direct spectroscopic measurements (NMR, EPR)

• Biophysical characterization (CD, fluorescence)

• Computational predictions and homology models

The amphipathic nature of membrane proteins requires careful attention to the membrane-water interface regions, which often contain functionally important residues that interact with both hydrophobic and hydrophilic environments.

How can comparative analysis with other UPF0060 family proteins inform research on RSKD131_0092?

Comparative analysis with related proteins provides valuable context for understanding RSKD131_0092 function and significance. The UPF0060 family contains members across various bacterial species with potentially conserved functions:

Comparative Analysis Framework:

• Sequence-based comparisons:

  • Multiple sequence alignment of UPF0060 family members

  • Identification of absolutely conserved residues (likely functionally critical)

  • Analysis of conservation patterns in predicted transmembrane segments

  • Evaluation of species-specific variations that might reflect functional adaptations

• Structural comparisons (when structures are available):

  • Superposition of experimentally determined structures

  • Comparison of electrostatic surface potentials

  • Analysis of conserved binding pockets or interaction interfaces

• Functional implications:

  • Cross-species complementation studies

  • Comparison of phenotypic effects when genes are disrupted

  • Analysis of genomic context and potential operon structures

Analysis of Evolutionary Conservation:

When interpreting comparative data, researchers should consider that sequence conservation may not directly correlate with structural conservation in membrane proteins, as similar 3D folds can be achieved with different amino acid compositions while maintaining hydrophobicity patterns.