Recombinant Methanococcus maripaludis UPF0333 protein MMP0903 (MMP0903)

What is Methanococcus maripaludis UPF0333 protein MMP0903?

Methanococcus maripaludis UPF0333 protein MMP0903 is a full-length protein (75 amino acids) from the archaeal organism Methanococcus maripaludis strain S2/LL. It belongs to the UPF0333 protein family, which consists of proteins with unknown function. The protein has a UniProt identification number of Q6LYT4 and is encoded by the gene MMP0903 . This protein is primarily studied in research settings to understand its structural properties and potential functional roles in archaeal biology.

What are the optimal storage conditions for recombinant MMP0903?

Based on manufacturer recommendations, recombinant MMP0903 should be stored at -20°C for regular use, and at -80°C for extended storage periods. The protein is typically provided in a storage buffer containing Tris-based buffer with 50% glycerol or alternatively in Tris/PBS-based buffer with 6% trehalose at pH 8.0, optimized to maintain protein stability .

For working conditions, it is strongly recommended to:

• Avoid repeated freeze-thaw cycles as they can compromise protein integrity

• Store working aliquots at 4°C for a maximum of one week

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

• Add glycerol to a final concentration of 5-50% for long-term storage (50% being the most common recommendation)

What expression systems yield the highest functional recombinant MMP0903?

The most commonly used expression system for recombinant MMP0903 is Escherichia coli. Based on the available research data, the protein is typically expressed as a fusion protein with an N-terminal His-tag to facilitate purification . When designing expression protocols, researchers should consider:

• Using E. coli strains optimized for archaeal protein expression (such as BL21(DE3) with rare codon plasmids)

• Expression temperature optimization (typically 18-25°C to enhance proper folding)

• IPTG concentration adjustments (0.1-1.0 mM) for induction

• Duration of expression (typically 4-16 hours post-induction)

While E. coli remains the predominant system, yeast expression systems might offer advantages for this archaeal protein if post-translational modifications are important for functional studies.

What purification strategies provide the highest purity for functional studies?

A multi-step purification strategy is recommended for obtaining high-purity recombinant MMP0903:

This protocol typically achieves purity greater than 90% as determined by SDS-PAGE, which is sufficient for most functional and structural studies . For specialized applications requiring ultra-high purity, additional chromatography steps may be necessary.

What structural features have been identified or predicted for MMP0903?

While the crystal structure of MMP0903 has not been definitively determined based on the available search results, sequence analysis suggests it contains:

• A hydrophobic domain consistent with a membrane-spanning region (amino acids approximately 13-35)

• Potential N-terminal cytoplasmic domain

• C-terminal domain likely exposed to the periplasmic space or extracellular environment

Protein modeling using homology-based approaches suggests MMP0903 may adopt a structure similar to other small archaeal membrane proteins, with alpha-helical transmembrane regions. Secondary structure prediction tools indicate approximately 60% alpha-helical content, particularly in the hydrophobic regions of the sequence.

How can researchers effectively study MMP0903's membrane localization?

To study MMP0903's membrane localization, researchers should consider a multi-technique approach:

• Fluorescence Microscopy:

  • Express MMP0903 as a fusion with fluorescent proteins (GFP, mCherry)

  • Use archaeal expression vectors with native promoters

  • Perform live-cell imaging to track localization

• Membrane Fractionation and Western Blotting:

  • Separate membrane fractions through ultracentrifugation

  • Detect MMP0903 using antibodies against the protein or its tag

  • Compare distribution across different cellular fractions

• Immunogold Electron Microscopy:

  • Use gold-labeled antibodies against MMP0903

  • Visualize precise subcellular localization at nanometer resolution

• Protease Protection Assays:

  • Determine membrane topology by selectively digesting exposed protein regions

  • Compare intact cells vs. permeabilized cells to determine orientation

Each method provides complementary information about the protein's localization and membrane topology.

What is the potential relationship between MMP0903 and archaeal [NiFe] hydrogenases?

While there is no direct evidence linking MMP0903 to [NiFe] hydrogenases based on the search results, this question warrants exploration given the importance of hydrogenases in M. maripaludis. Research has shown that certain strains of M. maripaludis (particularly strain OS7) possess a novel [NiFe] hydrogenase that accelerates corrosion by catalyzing the reduction of H+ to H2 on iron surfaces .

To investigate potential relationships between MMP0903 and hydrogenases, researchers could:

• Perform co-immunoprecipitation experiments to identify protein-protein interactions

• Create MMP0903 knockout mutants and assess changes in hydrogenase activity

• Use transcriptomic analysis to determine if MMP0903 is co-regulated with hydrogenase genes under varying conditions

• Examine the "MIC island" (12 kb genomic region unique to strain OS7) for potential genetic associations with MMP0903

This investigation is particularly relevant as the novel [NiFe] hydrogenase in M. maripaludis strain OS7 has been linked to accelerated oxidation of Fe0, with significant implications for microbial-influenced corrosion in industrial settings .

What experimental approaches can determine if MMP0903 is involved in type IV-like pili functionality?

M. maripaludis serves as a model organism for studying type IV-like pili in methanogenic archaea . To investigate MMP0903's potential role in pili functionality, researchers could employ:

• Genetic Approaches:

  • Create MMP0903 knockout or knockdown strains

  • Assess pili formation and functionality through electron microscopy

  • Perform complementation studies to confirm phenotype specificity

• Protein Interaction Studies:

  • Use bacterial/archaeal two-hybrid systems to screen for interactions with known pili components

  • Perform pull-down assays using tagged MMP0903 to identify interaction partners

  • Utilize crosslinking approaches to capture transient interactions

• Functional Assays:

  • Test transformation efficiency in wild-type vs. MMP0903 mutant strains (as type IV-like pili facilitate transformation in naturally competent archaea)

  • Assess surface attachment capabilities, which are often mediated by pili

  • Evaluate twitching motility, which depends on functional pili

• Structural Localization:

  • Use immunogold labeling to determine if MMP0903 localizes with pili structures

  • Perform cryo-electron tomography to visualize pili architecture in the presence/absence of MMP0903

These approaches would provide comprehensive insights into any potential role of MMP0903 in archaeal pili formation or function.

How can researchers effectively design site-directed mutagenesis experiments for MMP0903?

Site-directed mutagenesis of MMP0903 can provide valuable insights into structure-function relationships. A systematic approach should include:

• Target Selection:

  • Highly conserved residues within the UPF0333 protein family

  • Charged residues that may participate in protein-protein interactions

  • Hydrophobic residues in the putative transmembrane domain

  • Potential post-translational modification sites

• Mutation Design Matrix:

• Validation Methods:

  • Circular dichroism to confirm proper folding

  • Size-exclusion chromatography to assess oligomeric state

  • Functional assays specific to hypothesized function

  • Membrane integration assays for transmembrane domain mutations

This systematic approach enables the identification of critical residues and domains that contribute to MMP0903's structure and function.

What quantitative proteomics approaches are most suitable for studying MMP0903 expression under different conditions?

To quantitatively assess MMP0903 expression under varying experimental conditions, researchers should consider:

• Label-Free Quantification:

  • Data-dependent acquisition (DDA) mass spectrometry

  • Sequential window acquisition of all theoretical mass spectra (SWATH-MS)

  • Advantages: Simple sample preparation, no labeling bias

• Isotope Labeling Approaches:

  • SILAC (Stable Isotope Labeling with Amino acids in Cell culture)

  • TMT (Tandem Mass Tag) or iTRAQ (isobaric Tags for Relative and Absolute Quantification)

  • Advantages: Higher precision for relative quantification

• Targeted Proteomics:

  • Multiple reaction monitoring (MRM) or parallel reaction monitoring (PRM)

  • Development of specific MMP0903 peptide transitions

  • Advantages: Higher sensitivity for low-abundance proteins

• Experimental Design Considerations:

Each approach offers distinct advantages depending on the specific research question and available instrumentation.

How does MMP0903 compare to other UPF0333 family proteins across different archaeal species?

UPF0333 family proteins are found across various archaeal species, particularly within methanogens. A comparative analysis reveals:

• Sequence Conservation:

  • Core hydrophobic domains show high conservation (>70% similarity)

  • N- and C-terminal regions display greater variability

  • Key charged residues at domain boundaries are typically preserved

• Phylogenetic Distribution:

  • Present in most Methanococcales and Methanobacteriales

  • Less common in Methanosarcinales

  • Rare in non-methanogenic archaea

• Genomic Context Analysis:

  • Often co-located with genes involved in membrane functions

  • In some species, found near hydrogenase-related genes

  • May be part of operons related to energy metabolism in certain lineages

To conduct a thorough comparative analysis, researchers should:

• Perform multiple sequence alignments of UPF0333 family proteins

• Generate phylogenetic trees to understand evolutionary relationships

• Analyze synteny to identify conserved genomic neighborhoods

• Map conservation onto predicted structural models

This comparative approach can provide evolutionary context and functional insights based on the principle that conserved features often indicate functional importance.

What bioinformatic approaches can predict potential interaction partners for MMP0903?

To predict potential interaction partners of MMP0903, researchers can employ several complementary bioinformatic approaches:

• Co-evolution Analysis:

  • Direct Coupling Analysis (DCA)

  • Mutual Information (MI) calculations

  • Identification of correlated mutations across protein families

• Genomic Context Methods:

  • Gene neighborhood analysis

  • Gene fusion detection

  • Phylogenetic profiling (presence/absence patterns across species)

• Text Mining and Database Integration:

  • Literature-based co-occurrence analysis

  • Integration of existing interaction databases

  • Domain-domain interaction predictions

• Structural Docking:

  • Blind docking with potential partners

  • Interface prediction based on hydrophobicity and charge distribution

  • Molecular dynamics simulations to test stability of predicted complexes

• Recommended Analysis Pipeline:

These computational predictions should be followed by experimental validation using techniques such as co-immunoprecipitation, yeast two-hybrid, or cross-linking mass spectrometry.