Recombinant Serratia proteamaculans UPF0266 membrane protein Spro_2816 (Spro_2816)

What is Spro_2816 and what organism does it originate from?

Spro_2816 is a membrane protein belonging to the UPF0266 family (Uncharacterized Protein Family 0266), originating from the gram-negative bacterium Serratia proteamaculans (strain 568) . The protein consists of 152 amino acids with a molecular mass of approximately 17.7 kDa . As a member of the UPF0266 family, this protein belongs to a group of functionally uncharacterized proteins, indicating that its precise biological role remains to be fully elucidated. The classification within this family suggests potential structural or functional similarities with other UPF0266 members across bacterial species.

The complete amino acid sequence of Spro_2816 (MSLTDGVLLIFTALMLVYAIYDEFGMNLLKGKTLLKVQLKRRNRIDCLIFVGLITILLYRNVTTQGAVITTYLLISLALIAIYISYIRWPKMLFKAQGFFYANAFIEYNRIKAMNLSEDGILVIDLEQRRLLIQVTQLDDLEKIYHFFVENQ) reveals characteristics typical of membrane proteins, including hydrophobic regions that likely form transmembrane domains . Preliminary sequence analysis suggests the presence of multiple membrane-spanning regions, consistent with its classification as a membrane protein.

What structural features characterize Spro_2816 membrane protein?

Structural analysis of Spro_2816 indicates several key features consistent with its identification as a membrane protein. The 152-amino acid sequence contains multiple hydrophobic segments that likely form transmembrane domains, allowing it to span the bacterial cell membrane . These hydrophobic regions are interspersed with more hydrophilic segments that may be exposed to either the cytoplasmic or extracellular environment, creating a characteristic membrane protein topology.

Computational analysis suggests that Spro_2816 likely contains approximately 3-4 transmembrane helices, based on hydrophobicity patterns in the primary sequence. The N-terminal region appears to contain a signal sequence or initial transmembrane domain (approximately residues 5-25), followed by alternating hydrophobic and hydrophilic segments. The relatively small size (152 amino acids) indicates it likely has a compact structure with limited extramembrane domains.

When analyzing the sequence patterns further, several potential structural motifs can be identified:

• An N-terminal hydrophobic region likely forming the first transmembrane domain

• A positively charged region (residues 30-40) potentially involved in protein-protein interactions

• Additional hydrophobic segments constituting subsequent transmembrane domains

• C-terminal region potentially involved in signaling or interaction with other cellular components

What expression systems are most suitable for recombinant production of Spro_2816?

For successful recombinant production of Spro_2816, researchers should consider several expression systems, each offering distinct advantages depending on research objectives . The selection of an appropriate expression system is a critical decision that significantly impacts protein yield, folding, and functionality.

• Use specialized E. coli strains designed for membrane protein expression:

  • C41(DE3) and C43(DE3) strains that have adapted to tolerate membrane protein overexpression

  • Lemo21(DE3) for tunable expression through rhamnose-controlled lysozyme production

  • BL21(DE3) pLysS for tighter control of basal expression

• Optimize expression conditions:

  • Lower induction temperature (16-20°C) to slow protein synthesis and improve folding

  • Reduced inducer concentration to prevent overwhelming the membrane insertion machinery

  • Rich media formulations with proper aeration for robust cell growth

  • Induction at higher cell densities (OD600 ~0.8-1.0) for greater biomass

For structural studies requiring post-translational modifications or when E. coli expression yields poor results, eukaryotic expression systems should be considered:

• Yeast systems (Pichia pastoris, Saccharomyces cerevisiae) may provide better folding machinery for complex membrane proteins and allow for scale-up in bioreactors

• Insect cell systems using baculovirus vectors often yield higher quantities of properly folded membrane proteins

• Cell-free expression systems combined with supplied lipids or detergents can overcome toxicity issues while allowing direct incorporation into membrane mimetics

What experimental approaches are recommended for determining the function of Spro_2816?

As a member of the UPF0266 family of uncharacterized proteins, determining the function of Spro_2816 requires a systematic multi-faceted approach. The following methodological framework is recommended for researchers investigating this protein's function:

• Comparative genomics and bioinformatics analysis:

  • Examine the genomic context of Spro_2816 to identify co-regulated genes and potential operons

  • Perform phylogenetic analysis to identify orthologs across bacterial species, particularly those with functional annotations

  • Use advanced structure prediction tools (AlphaFold2, RoseTTAFold) to predict protein structure and identify potential binding sites

  • Apply protein-protein interaction prediction algorithms to identify potential binding partners

  • Analyze conserved residues across the UPF0266 family to identify functionally important regions

• Gene knockout and phenotypic characterization:

  • Generate Spro_2816 knockout strains in Serratia proteamaculans using CRISPR-Cas9 or traditional homologous recombination

  • Design comprehensive phenotypic screening protocols examining:

    • Growth rates under various conditions (temperature, pH, osmolarity)

    • Membrane integrity using fluorescent dyes (propidium iodide, FM4-64)

    • Stress responses (oxidative, antimicrobial, heavy metal)

    • Metabolic profiling using LC-MS/MS

  • Conduct comparative transcriptomics (RNA-Seq) between wild-type and knockout strains to identify affected pathways

• Protein localization and interaction studies:

  • Use fluorescent protein fusions to confirm membrane localization and observe dynamics

  • Perform co-immunoprecipitation coupled with mass spectrometry to identify interaction partners

  • Apply techniques like FRET (Förster Resonance Energy Transfer) or BiFC (Bimolecular Fluorescence Complementation) to validate protein-protein interactions in vivo

  • Conduct split-ubiquitin yeast two-hybrid assays specific for membrane protein interactions

How can researchers address the purification challenges specific to Spro_2816?

Purification of membrane proteins like Spro_2816 presents significant challenges due to their hydrophobic nature and requirement for a lipid environment. A methodical approach to purification should include:

• Optimization of solubilization conditions:

  • Test a panel of detergents including mild (DDM, LMNG), moderate (DM), and harsh (SDS, FC-12) options

  • Evaluate novel amphipathic polymers like SMA that extract proteins within native lipid nanodiscs

  • Determine optimal detergent concentration, temperature, and buffer composition through systematic screening

  • Consider lipid-detergent mixed micelles to stabilize the protein

• Chromatographic purification strategy:

  • Begin with affinity chromatography using engineered tags (His, Strep, FLAG)

  • Implement size exclusion chromatography to separate protein-detergent complexes from aggregates

  • Consider ion exchange chromatography as an additional purification step

  • Validate protein quality at each purification stage using SDS-PAGE and Western blotting

The table below compares the effectiveness of different detergents for Spro_2816-like membrane proteins:

• Assessment of protein homogeneity and stability:

  • Apply dynamic light scattering to monitor aggregation and size distribution

  • Perform thermal shift assays to identify stabilizing conditions

  • Monitor protein stability over time in different storage conditions

  • Use circular dichroism to assess secondary structure integrity

What approaches can be used to investigate potential protein-protein interactions involving Spro_2816?

Investigating protein-protein interactions (PPIs) involving membrane proteins like Spro_2816 requires specialized techniques that accommodate their hydrophobic nature. The following methodological approaches are recommended:

• Membrane-specific yeast two-hybrid systems:

  • Split-ubiquitin membrane yeast two-hybrid (MYTH) specifically designed for membrane proteins

  • DHFR protein-fragment complementation assay adapted for membrane protein interactions

  • Methodology should include appropriate controls and validation of membrane localization

  • Screen against genomic libraries from Serratia proteamaculans for comprehensive interactome mapping

• Co-immunoprecipitation approaches:

  • Cross-linking protocols optimized for membrane proteins prior to solubilization

  • Gentle detergent solubilization to maintain protein-protein interactions

  • Tandem affinity purification to reduce false positives

  • Mass spectrometry analysis with specialized membrane protein identification parameters

• Proximity-based labeling methods:

  • BioID or TurboID fusion constructs to identify proximal proteins in vivo

  • APEX2-based proximity labeling for temporal control of labeling reactions

  • Quantitative proteomics to distinguish specific from non-specific interactions

  • Comparison between active and control conditions to identify context-dependent interactions

These methods should be implemented with appropriate controls, including:

• Non-interacting membrane protein controls

• Cytoplasmic protein controls to identify nonspecific binding

• Reciprocal tagging approaches to confirm interactions

How should researchers design experiments to investigate the effect of environmental conditions on Spro_2816 expression and function?

Investigating how environmental conditions affect Spro_2816 expression and function requires a systematic experimental design that accounts for the protein's membrane localization and bacterial origin. The following methodological framework is recommended:

• Expression analysis under varying conditions:

  • Design a qRT-PCR assay targeting the Spro_2816 gene with appropriate reference genes

  • Develop reporter constructs (e.g., Spro_2816 promoter fused to GFP) to monitor expression in real-time

  • Create a comprehensive matrix of environmental conditions to test:

    • Temperature ranges (15-42°C)

    • pH gradients (5.0-9.0)

    • Osmotic stress conditions

    • Nutrient limitations

    • Exposure to antimicrobial compounds

    • Growth phase variations

  • Include time-course sampling to capture dynamic expression patterns

• Protein localization and abundance studies:

  • Generate antibodies specific to Spro_2816 or use epitope-tagged versions

  • Employ fractionation protocols to isolate membrane compartments

  • Use Western blotting with quantitative analysis to measure protein levels

  • Apply proteomics approaches to monitor changes in the membrane proteome

  • Implement fluorescence microscopy to observe changes in localization patterns

• Functional characterization across conditions:

  • Develop specific assays based on predicted functional properties

  • Measure membrane integrity and permeability under various conditions

  • Assess potential transport activity with fluorescent substrates

  • Monitor protein-protein interactions under different environmental stresses

  • Examine contribution to stress responses through phenotypic assays

What techniques are recommended for analyzing post-translational modifications of Spro_2816?

Despite being a bacterial protein with potentially fewer post-translational modifications (PTMs) than eukaryotic proteins, Spro_2816 may still undergo PTMs that affect its function, localization, or interactions. A comprehensive approach to PTM analysis would include:

• Mass spectrometry-based PTM identification:

  • Employ high-resolution tandem mass spectrometry (MS/MS) with multiple fragmentation methods

  • Use enrichment strategies specific to predicted modifications (phosphorylation, acetylation, methylation)

  • Apply both bottom-up (peptide-level) and top-down (intact protein) proteomics

  • Implement targeted multiple reaction monitoring for quantitative analysis of specific PTMs

  • Consider hydrogen-deuterium exchange mass spectrometry to detect PTM-induced conformational changes

• Site-specific PTM validation:

  • Generate site-specific antibodies against predicted modified residues

  • Employ site-directed mutagenesis to create non-modifiable variants

  • Use residue-specific chemical labeling approaches

  • Apply cross-linking mass spectrometry to identify PTM-dependent interactions

• Structural and functional impact assessment:

  • Compare structures of modified and unmodified proteins using cryo-EM or crystallography

  • Perform molecular dynamics simulations to predict PTM effects on protein behavior

  • Assess functional consequences through activity assays with modified and unmodified variants

  • Examine changes in protein-protein interaction profiles dependent on modification state

How can researchers resolve contradictory findings in studies of Spro_2816 function?

When faced with contradictory findings regarding Spro_2816 function, researchers should implement a structured approach to resolve discrepancies :

• Systematic context evaluation:

  • Categorize contradictions based on contextual factors as outlined in the literature :

    • Internal to the experimental system (species, cell type, protein isoform)

    • External factors (experimental conditions, reagents, methodologies)

    • Endogenous/exogenous variables (natural vs. induced protein expression)

    • Known controversies in the field

    • Contradictions arising from literature limitations

  • Create a comprehensive comparison table documenting all experimental variables across contradictory studies

  • Identify specific differences in experimental design that could explain divergent results

• Replication studies with controlled variables:

  • Design experiments that systematically test each variable identified in the context evaluation

  • Implement standardized protocols across different laboratory settings

  • Use identical reagents, cell lines, and experimental conditions where possible

  • Employ blinded analysis to minimize confirmation bias

  • Conduct power analysis to ensure adequate statistical strength

• Advanced meta-analysis approaches:

  • Perform quantitative synthesis of available data using meta-analysis techniques

  • Apply Bayesian methods to incorporate prior knowledge and uncertainty

  • Develop causal models to explain apparent contradictions

  • Use sensitivity analysis to determine which factors most strongly influence outcomes

  • Implement contradiction resolution frameworks developed for biomedical literature

The following table outlines common sources of contradictions in membrane protein studies and resolution strategies:

What bioinformatic tools and databases are most valuable for analyzing Spro_2816 and related UPF0266 family proteins?

For comprehensive analysis of Spro_2816 and the UPF0266 family, researchers should utilize a strategic combination of bioinformatic tools and databases:

• Sequence analysis and evolutionary tools:

  • BLAST/PSI-BLAST for identifying distant homologs across bacterial species

  • HMMER for building and searching with profile hidden Markov models

  • MUSCLE/CLUSTAL for multiple sequence alignment of UPF0266 family members

  • IQ-TREE/RAxML for phylogenetic tree construction with appropriate evolutionary models

  • ConSurf for identifying evolutionarily conserved residues

  • CLANS for visualization of sequence similarity networks

• Structural prediction and analysis:

  • AlphaFold2/RoseTTAFold for state-of-the-art protein structure prediction

  • SWISS-MODEL for homology modeling when templates are available

  • TMHMM/TOPCONS for transmembrane topology prediction

  • PyMOL/Chimera for structural visualization and analysis

  • CASTp for binding pocket prediction

  • MDWeb for preparing systems for molecular dynamics simulations

• Functional annotation resources:

  • InterPro for integrated protein domain analysis

  • STRING for protein-protein interaction network prediction

  • KEGG for pathway mapping and metabolic context

  • Gene Ontology for functional term enrichment analysis

  • UniProt for curated protein information

  • PredictProtein for comprehensive sequence-based feature prediction

What are the potential research applications of Spro_2816 beyond basic characterization?

While the specific function of Spro_2816 remains to be fully characterized, research on this membrane protein could lead to several promising research applications:

• Membrane protein research model:

  • Use Spro_2816 as a model system for developing improved membrane protein expression methods

  • Explore as a test case for membrane protein reconstitution techniques

  • Develop as a standard for evaluating membrane protein structural prediction algorithms

  • Employ as a benchmark for optimizing membrane protein purification protocols

  • Utilize for validating new membrane mimetic systems

• Comparative bacterial membrane biology:

  • Investigate as a representative of bacterial membrane organization

  • Study evolutionary conservation of UPF0266 family across bacterial species

  • Examine potential roles in bacterial adaption to environmental stresses

  • Explore function in bacterial membrane maintenance or remodeling

  • Investigate potential roles in bacterial communication or sensing

• Novel functional discovery platforms:

  • Develop high-throughput screening methods to identify interacting partners

  • Create reporter systems to monitor protein activity in response to stimuli

  • Design biosensors based on conformational changes if identified

  • Establish conditional expression systems to study phenotypic effects

  • Implement CRISPR interference approaches for temporal control of expression

• Structural biology methodology advancement:

  • Use as a test case for developing improved membrane protein structure determination techniques

  • Apply as model system for validating computational structure prediction algorithms

  • Develop as standard for membrane protein reconstitution protocols

  • Employ for optimizing electron microscopy sample preparation methods

  • Utilize for advancing native mass spectrometry approaches for membrane proteins

What emerging technologies are likely to accelerate research on uncharacterized membrane proteins like Spro_2816?

Several cutting-edge technologies are poised to transform research on uncharacterized membrane proteins like Spro_2816:

• Advanced structural biology approaches:

  • Cryo-electron tomography for visualizing membrane proteins in their native cellular context

  • Micro-electron diffraction (MicroED) for structure determination from nanocrystals

  • Integrative structural biology combining multiple data types for comprehensive models

  • Serial femtosecond crystallography using X-ray free electron lasers

  • Correlative light and electron microscopy for linking structure to function

• Single-molecule techniques:

  • Single-molecule FRET for dynamic structural analysis

  • Nanopore-based single-molecule sensing for functional characterization

  • Atomic force microscopy with chemical recognition for topological mapping

  • Single-molecule tracking in living cells to monitor dynamics and interactions

  • Optical tweezers to measure mechanical properties and conformational changes

• AI and computational advances:

  • Deep learning for improved structure and function prediction

  • Molecular dynamics simulations with enhanced sampling techniques

  • Machine learning classification of membrane protein structures and functions

  • Automated literature mining to connect disparate findings

  • In silico screening for ligands and interaction partners

• Next-generation membrane mimetics:

  • Advanced nanodisc technologies with controlled lipid composition

  • Cell-free expression systems coupled with membrane scaffolds

  • 3D printed artificial membranes with precise control of properties

  • Droplet interface bilayers for electrical measurements

  • DNA-origami scaffolds for precise positioning of membrane proteins

What are common experimental pitfalls when working with recombinant Spro_2816 and how can they be addressed?

Researchers working with recombinant Spro_2816 may encounter several challenges specific to membrane proteins. Here are common pitfalls and methodological solutions:

• Expression and toxicity issues:

  • Pitfall: Overexpression causing toxicity to host cells

  • Solution: Implement tightly regulated expression systems with lower induction levels

  • Pitfall: Formation of inclusion bodies

  • Solution: Lower expression temperature (16-20°C), use specialized strains (C41/C43), or explore fusion partners

  • Pitfall: Poor expression yield

  • Solution: Optimize codon usage, consider alternative expression systems, or use cell-free expression

• Membrane extraction and solubilization problems:

  • Pitfall: Inefficient extraction from membranes

  • Solution: Screen multiple detergents, optimize detergent:protein ratios, and consider extraction time

  • Pitfall: Protein aggregation during solubilization

  • Solution: Add stabilizing agents (glycerol, specific lipids), maintain low temperature throughout

  • Pitfall: Loss of function during extraction

  • Solution: Use milder extraction methods (SMA polymers, native nanodiscs) that preserve lipid environment

• Purification challenges:

  • Pitfall: Co-purification of contaminants

  • Solution: Implement tandem purification strategies using orthogonal tags, optimize wash conditions

  • Pitfall: Protein degradation during purification

  • Solution: Add protease inhibitors, minimize processing time, keep samples cold, consider on-column techniques

  • Pitfall: Poor yield after multiple purification steps

  • Solution: Minimize purification steps, optimize each step individually, validate recovery

How can researchers optimize protocols for Spro_2816 reconstitution into membrane mimetic systems?

Reconstitution of Spro_2816 into membrane mimetic systems requires careful optimization to maintain protein structure and function. The following methodological framework addresses key considerations:

• Selection of appropriate membrane mimetic system:

  • Evaluate protein requirements based on size, stability, and functional assays

  • Consider liposomes for functionality studies and transport assays

  • Use nanodiscs for structural studies requiring a native-like bilayer

  • Explore bicelles for NMR applications

  • Test amphipols for single-particle cryo-EM

  • Consider SMALPs for maintaining native lipid interactions

• Optimization of lipid composition:

  • Analyze lipid composition of Serratia proteamaculans membranes as starting point

  • Test lipid mixtures systematically varying:

    • Headgroup composition (PE, PG, cardiolipin ratios)

    • Acyl chain length and saturation

    • Inclusion of bacterial-specific lipids

    • Cholesterol or ergosterol content if applicable

  • Implement lipid screen assays using thermal stability as readout

  • Consider native mass spectrometry to identify co-purifying lipids

• Reconstitution method selection and optimization:

  • For liposomes: Compare detergent removal methods

    • Dialysis (gentle but time-consuming)

    • Bio-Beads adsorption (faster but potential for protein adsorption)

    • Cyclodextrin complexation (rapid but expensive)

    • Dilution method (simple but may result in larger vesicles)

  • For nanodiscs: Optimize scaffold protein:lipid:target ratios

  • For bicelles: Determine optimal q-value (long-chain:short-chain lipid ratio)

  • Monitor reconstitution kinetics to identify optimal endpoints