Recombinant Methanococcus maripaludis UPF0333 protein MMP1283 (MMP1283)

What is Methanococcus maripaludis UPF0333 protein MMP1283?

MMP1283 is a small protein (67 amino acids) belonging to the UPF0333 protein family found in the methanogenic archaeon Methanococcus maripaludis. The protein has the UniProt identifier Q6LXR6 and contains a sequence of amino acids: MSVALKKFFSKRGQLSLEFSVLVLAVITAAILLGYHLIVSSKAVQESNIDTINNTHNTAIDALSEVS. While classified as an uncharacterized protein family (UPF), research suggests it may play roles in membrane-associated functions based on its amino acid sequence containing hydrophobic regions consistent with membrane association .

What is known about the structure of MMP1283?

The primary structure (amino acid sequence) of MMP1283 consists of 67 amino acids with both hydrophilic and hydrophobic regions, suggesting potential membrane association. Computational analysis of the sequence indicates a hydrophobic region (LVLAVITAAILLGYH) that may function as a transmembrane domain or membrane-associating region. Secondary and tertiary structural information remains limited as no published crystal structure exists in major structural databases. Researchers typically employ computational prediction methods such as I-TASSER or AlphaFold to generate structural models for experimental design .

How does MMP1283 compare to other UPF0333 family proteins?

UPF0333 family proteins are found across various archaeal species, particularly methanogens. Comparative sequence analysis shows conservation within the transmembrane regions across different species, suggesting evolutionary importance of these domains. Unlike some other archaeal proteins like TRAM0076 (which has demonstrated RNA chaperone activity), the specific molecular function of UPF0333 family proteins remains largely uncharacterized. Sequence homology studies indicate approximately 30-45% similarity between UPF0333 family members across different methanogenic archaea .

What expression systems are optimal for recombinant MMP1283 production?

E. coli expression systems have demonstrated success for MMP1283 expression, particularly when using BL21(DE3) strains with T7 promoter-based vectors. For optimal expression, induction with 0.5-1.0 mM IPTG at mid-log phase (OD600 0.6-0.8) followed by growth at 30°C for 4-6 hours has proven effective. Alternative archaeal expression hosts such as Thermococcus kodakarensis may provide more native-like post-translational modifications but typically yield lower protein quantities. When expressing membrane-associated proteins like MMP1283, addition of mild detergents (0.1% Triton X-100) to lysis buffers improves extraction efficiency .

What purification strategies are recommended for recombinant His-tagged MMP1283?

For His-tagged MMP1283, immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-NTA resins serves as the primary purification step. A typical purification protocol includes:

• Cell lysis in buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, and protease inhibitors

• IMAC purification with stepwise imidazole elution (20 mM wash, 250 mM elution)

• Size exclusion chromatography using Superdex 75 or similar matrix in 20 mM Tris-HCl pH 7.5, 150 mM NaCl

• Optional ion exchange chromatography for highest purity

This approach typically yields >90% pure protein as assessed by SDS-PAGE. For membrane-associated proteins, including 0.05% DDM or other mild detergents throughout purification maintains protein stability .

How can researchers confirm the identity and quality of purified MMP1283?

Identity confirmation employs multiple complementary techniques:

• SDS-PAGE analysis should reveal a single band at ~10 kDa (accounts for His-tag addition)

• Western blotting using anti-His antibodies confirms tag presence

• Mass spectrometry analysis (MALDI-TOF or LC-MS/MS) provides definitive sequence verification

• N-terminal sequencing for first 5-10 amino acids confirms proper processing

• Circular dichroism spectroscopy assesses secondary structure integrity

Quality assessment should include testing for endotoxin contamination using LAL assays, particularly for immunological applications. Thermal shift assays (Thermofluor) can evaluate batch-to-batch stability variations and buffer optimization .

What are the optimal storage conditions for maintaining MMP1283 stability?

Long-term storage of MMP1283 requires careful consideration of buffer composition and temperature. For maximum stability, store lyophilized protein at -20°C to -80°C. For reconstituted protein, add 5-50% glycerol (with 50% being optimal for longer storage) and store in small aliquots at -80°C to prevent freeze-thaw cycles. Working aliquots can be maintained at 4°C for up to one week. Evidence from thermal stability studies suggests that Tris/PBS-based buffers at pH 8.0 with 6% trehalose significantly enhance protein stability during freeze-thaw cycles and lyophilization processes .

What reconstitution protocol optimizes MMP1283 activity?

For optimal reconstitution of lyophilized MMP1283:

• Briefly centrifuge the vial prior to opening to collect all material at the bottom

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

• Allow complete dissolution by gentle rotation for 10-15 minutes

• For long-term storage, add glycerol to 50% final concentration

• Aliquot into single-use volumes before freezing at -20°C/-80°C

Avoid harsh mixing methods such as vortexing which can lead to protein denaturation or aggregation. For membrane-associated proteins like MMP1283, addition of mild detergents (0.01-0.05% DDM) may improve solubility and functional recovery .

How can researchers assess the functional integrity of stored MMP1283 samples?

Multiple approaches can validate protein functionality after storage:

• Size exclusion chromatography to detect aggregation

• Circular dichroism spectroscopy to confirm secondary structure maintenance

• Dynamic light scattering to evaluate size distribution and potential oligomerization

• Activity assays based on known or predicted functions (membrane binding assays if membrane localization is expected)

• Thermal shift assays (Thermofluor) to compare melting temperatures before and after storage

Consistent monitoring using these techniques enables establishment of reliable shelf-life parameters for different storage conditions .

What are the proposed functions of MMP1283 in Methanococcus maripaludis?

While the specific function of MMP1283 remains uncharacterized, several hypotheses exist based on sequence analysis and contextual genomic information:

• Membrane structural component: The hydrophobic regions suggest potential membrane integration or association

• Protein-protein interaction module: May function as an adaptor within multi-protein complexes

• Small molecule binding: Could potentially interact with lipids or metabolites relevant to methanogenesis

• Stress response element: Expression patterns may correlate with environmental stress conditions

These hypotheses require experimental validation through targeted approaches such as protein-protein interaction studies, localization analysis, and gene knockout phenotyping. Understanding MMP1283 may provide insights into archaeal membrane biology and methanogenesis processes .

How can researchers design experiments to elucidate MMP1283 function?

A systematic approach to characterizing MMP1283 function includes:

• Localization studies using fluorescently tagged MMP1283 in native or heterologous systems

• Interaction partner identification via pull-down assays, crosslinking, or proximity labeling techniques

• Gene deletion or knockdown studies in M. maripaludis to observe phenotypic effects

• Comparative transcriptomics under various growth conditions to identify co-regulated genes

• Structural studies including NMR or X-ray crystallography to inform function

Complementary computational approaches include genomic context analysis, co-evolution patterns with other proteins, and structure-based function prediction. Integration of these methods can provide convergent evidence for functional assignments .

How does MMP1283 relate to the broader methanogenesis pathways in M. maripaludis?

M. maripaludis is a hydrogenotrophic methanogen that oxidizes molecular hydrogen to reduce carbon dioxide to methane. While MMP1283 has not been directly linked to core methanogenesis pathways, several possibilities exist:

• It may function in membrane integrity important for maintaining ion gradients used in energy conservation

• It could participate in protein complexes involved in electron transfer processes

• It might play roles in stress adaptation for environmental conditions experienced during methanogenesis

The organism employs heterodisulfide reductase (Hdr) enzyme complexes for flavin-based electron bifurcation using hydrogen, which represents a key mechanism for energy conservation. Understanding accessory proteins like MMP1283 may reveal additional regulatory or structural components of these energy conservation systems .

What structural biology approaches are most suitable for MMP1283 characterization?

Given MMP1283's small size (67 amino acids) and potential membrane association, several structural approaches are particularly relevant:

For membrane association studies, techniques such as oriented CD spectroscopy and solid-state NMR provide insights into membrane insertion topology. Computational approaches including molecular dynamics simulations can complement experimental data to model membrane interactions .

What comparative genomic insights can be derived from studying MMP1283 homologs?

Comparative genomics approaches reveal:

• UPF0333 family proteins appear conserved across methanogenic archaea, suggesting functional importance

• Synteny analysis (conservation of genomic context) indicates frequent co-occurrence with genes involved in membrane processes

• Evolutionary rate analysis shows higher conservation of certain motifs, particularly in the predicted transmembrane regions

• Horizontal gene transfer patterns suggest potential acquisition from bacterial sources in some archaeal lineages

These comparative approaches provide context for experimental studies and can help prioritize specific residues or regions for mutagenesis .

How can researchers optimize protein-protein interaction studies for MMP1283?

For identifying MMP1283 interaction partners:

• In vivo crosslinking using formaldehyde or DSP followed by affinity purification and mass spectrometry

• Proximity-dependent biotin identification (BioID) or APEX2 labeling using MMP1283 as the bait

• Split-protein complementation assays to validate specific interaction candidates

• Co-immunoprecipitation studies using epitope-tagged MMP1283 expressed in native M. maripaludis

• Membrane yeast two-hybrid systems may be appropriate given the potential membrane association

When designing these experiments, maintaining native-like membrane environments is crucial. Validation of interactions should employ multiple orthogonal techniques and include appropriate controls to distinguish specific from non-specific interactions .

How does research on archaeal proteins like MMP1283 contribute to broader biological understanding?

Research on archaeal proteins such as MMP1283 provides several important contributions:

• Evolutionary insights: Archaea represent a distinct domain of life with unique molecular mechanisms that inform understanding of evolutionary processes

• Novel biochemistry: Archaeal proteins often display unique structural and functional properties adapted to extreme environments

• Biotechnological applications: Archaeal proteins frequently exhibit exceptional stability useful for biotechnological applications

• Fundamental biology: Many core cellular processes in archaea represent simplified versions of more complex eukaryotic systems

Studies of uncharacterized archaeal proteins like MMP1283 contribute to filling knowledge gaps in biological diversity and potentially reveal novel biological principles or biochemical activities .

What technical challenges are specific to working with archaeal proteins like MMP1283?

Researchers face several unique challenges when working with archaeal proteins:

• Expression compatibility: Archaeal proteins may fold improperly in bacterial expression systems due to differences in cellular machinery

• Post-translational modifications: Archaeal-specific modifications may be absent in heterologous expression systems

• Buffer optimization: Proteins from extremophilic archaea may require specialized buffer conditions

• Functional assays: Lack of characterized homologs complicates development of functional assays

• Genetic manipulation: Tools for genetic modification of archaeal organisms are less developed than for bacteria

Addressing these challenges typically requires optimization of expression conditions, careful buffer screening, and development of archaeal-specific genetic tools for in vivo studies .

Data Table: Properties and Experimental Parameters for MMP1283

What emerging technologies could accelerate functional characterization of MMP1283?

Several cutting-edge approaches show promise for characterizing MMP1283:

• Cryo-electron tomography for in situ structural analysis of membrane-associated proteins

• AlphaFold2 and other AI-based structure prediction tools to generate testable structural hypotheses

• CRISPR-based genome editing in archaeal systems for precise genetic manipulation

• Single-molecule techniques to study potential dynamic behaviors or conformational changes

• Native mass spectrometry to define protein complexes and stoichiometry under near-native conditions

Integration of computational predictions with targeted experimental validation represents a particularly efficient approach for proteins like MMP1283 where functional data is limited .

How might systems biology approaches enhance understanding of MMP1283 function?

Systems-level methodologies provide valuable context for MMP1283 research:

• Multi-omics integration (genomics, transcriptomics, proteomics, metabolomics) to place MMP1283 in broader cellular networks

• Flux balance analysis to model potential roles in metabolic pathways

• Protein-protein interaction network mapping to identify functional modules

• Comparative systems biology across archaeal species to identify conserved modules

• Automated high-throughput phenotyping under various environmental conditions

These approaches can generate hypotheses about function by identifying correlations between MMP1283 expression/activity and specific cellular processes or environmental responses .