Recombinant Methanococcus maripaludis UPF0333 protein MMP1685 (MMP1685)

What is Methanococcus maripaludis UPF0333 protein MMP1685?

MMP1685 is a 74-amino acid protein from the archaeon Methanococcus maripaludis (strain S2/LL), classified in the UPF0333 protein family. Mass spectrometry analysis has identified MMP1685 as the major structural pilin in M. maripaludis type IV pili structures. The protein includes an N-terminal 12-amino-acid type IV pilin-like signal peptide that undergoes post-translational processing. The mature protein's complete amino acid sequence is MKFLEKLTSKKGQIAMELGILVMAAVAVAAIAAYFYATNVSNTGKQITNSTNQTTQALADAISDATSQMSNITD, with position 1-74 expressed in the recombinant form .

What are the key structural features of MMP1685?

MMP1685 exhibits several notable structural features:

• Signal peptide: A 12-amino-acid N-terminal sequence cleaved during maturation

• Post-cleavage modification: The mature N-terminal residue is a pyroglutamic acid

• Glycosylation sites: Four consensus sites for N-glycosylation

• Limited basic residues: Only a single lysine residue and no arginine residues

• Molecular weight discrepancy: Predicted mass of mature protein (6,398 Da) differs significantly from observed mass (9,728 Da) due to post-translational modifications

How does MMP1685 relate to other type IV pilin proteins in M. maripaludis?

MMP1685 shares structural similarities with other type IV pilin-like proteins in M. maripaludis, including MMP0233, MMP0236, and MMP0237. All contain type IV pilin-like signal peptides identified by FlaFind algorithm. MMP1685 contains a glutamine (Q) at the +1 position (similar to MMP0233, MMP0236, and MMP0237) and a glutamic acid (E) at the +5 position (similar to MMP0233 and MMP0236). These shared features suggest common processing pathways and potentially related functions in pili assembly and structure .

What specialized cultivation techniques are required for working with M. maripaludis?

Working with M. maripaludis requires anaerobic cultivation techniques due to its strict anaerobic nature. The recommended approach involves:

• Using formate as a growth substrate

• Maintaining completely oxygen-free conditions throughout cultivation

• Employing specialized anaerobic chambers or techniques for all manipulations

• Following strict protocols for media preparation to ensure proper nutrient availability

• Monitoring growth conditions carefully to maintain culture viability

How can researchers effectively express and purify recombinant MMP1685?

For effective expression and purification of recombinant MMP1685, researchers should:

• Employ anoxic affinity purification techniques to maintain protein integrity

• Use liposome-mediated transformation for introducing expression constructs

• Consider expressing the protein with appropriate tags for purification (determined during production process)

• Store purified protein in Tris-based buffer with 50% glycerol

• Maintain strict anaerobic conditions throughout the purification process to prevent protein oxidation

• Avoid repeated freeze-thaw cycles; store working aliquots at 4°C for up to one week

What analytical methods are most effective for characterizing MMP1685?

Based on research findings, the most effective analytical methods for characterizing MMP1685 include:

These methods have successfully revealed critical features of MMP1685, including its pyroglutamic acid N-terminus and extensive glycosylation patterns that account for the observed molecular weight discrepancy .

How do the glycosylation patterns of MMP1685 affect its structural and functional properties?

The glycosylation of MMP1685 presents a complex research question. MS/MS analysis has shown that MMP1685 contains multiple N-linked glycans similar to those observed in M. maripaludis flagellin. The observed mass difference between predicted (6,398 Da) and actual (9,728 Da) molecular weights suggests extensive glycosylation accounting for approximately 3,330 Da of additional mass.

To investigate this question methodologically:

• Perform site-directed mutagenesis of the four consensus N-glycosylation sites

• Compare glycosylation patterns using mass spectrometry before and after treatment with specific glycosidases

• Assess structural impacts through comparative circular dichroism analysis

• Evaluate functional impacts by testing pili assembly efficiency with differentially glycosylated variants

• Map glycosylation sites to predicted structural domains to infer potential functional roles

What are the mechanisms of MMP1685 incorporation into type IV pili structures?

Understanding MMP1685 incorporation into pili structures requires investigation of:

• Signal peptide processing: The 12-amino-acid N-terminal signal peptide is cleaved, with Q at the +1 position being critical for proper processing

• Pyroglutamic acid formation: The mature N-terminal residue undergoes cyclization to form pyroglutamic acid, which likely affects pili assembly

• Interaction domains: Analysis of the mature protein sequence suggests potential protein-protein interaction domains that facilitate incorporation into pili

• Assembly pathway: Research should examine the chronological order of pilin recruitment and incorporation

• Structural requirements: Comparison with other pilins can reveal conserved motifs necessary for assembly

Methodologically, researchers should consider complementation studies with mutant variants, proximity labeling techniques, and in vitro assembly assays to elucidate these mechanisms.

How does the expression of MMP1685 relate to Fe-S cluster metabolism in M. maripaludis?

While direct evidence linking MMP1685 to Fe-S cluster metabolism is limited in the provided literature, this represents an intriguing research direction. M. maripaludis contains numerous Fe-S proteins essential for methanogenesis and other metabolic processes. To investigate potential relationships:

• Perform co-expression analysis of MMP1685 with known Fe-S cluster proteins

• Analyze transcriptional responses to iron limitation or oxidative stress

• Investigate potential Fe-S binding motifs in the MMP1685 sequence

• Use techniques for Fe-S cluster reconstitution to test possible Fe-S binding capacity

• Employ UV-visible absorption spectroscopy to detect characteristic Fe-S cluster signatures

This investigation could reveal unexpected functional roles for MMP1685 beyond its structural role in pili formation.

What are the key considerations for designing experiments involving MMP1685?

When designing experiments involving MMP1685, researchers should consider:

• Anaerobic requirements: All experimental procedures must maintain strict anaerobic conditions

• Protein stability: MMP1685 should be stored appropriately (Tris-based buffer, 50% glycerol) at -20°C or -80°C for extended storage

• Post-translational modifications: Experimental design must account for the significant impact of glycosylation on protein properties

• Purification challenges: The limited number of basic residues (only one lysine, no arginine) makes trypsin-based analyses difficult

• N-terminal blockage: The presence of pyroglutamic acid at the N-terminus prevents standard N-terminal sequencing

How can researchers effectively study protein-protein interactions involving MMP1685?

To study protein-protein interactions involving MMP1685, researchers should employ:

• Crosslinking approaches: Chemical crosslinking followed by mass spectrometry analysis

• Pull-down assays: Using tagged versions of MMP1685 under anaerobic conditions

• Bacterial/archaeal two-hybrid systems: Adapted for use in anaerobic organisms

• Co-immunoprecipitation: With antibodies specific to MMP1685 or its interaction partners

• Surface plasmon resonance: For quantitative analysis of binding kinetics under anaerobic conditions

When designing these experiments, researchers must consider the potential impact of MMP1685's extensive glycosylation on interaction surfaces and binding properties .

What experimental controls are essential when working with recombinant MMP1685?

Essential experimental controls when working with recombinant MMP1685 include:

These controls help distinguish specific effects related to MMP1685 from artifacts of the experimental system .

How should researchers resolve discrepancies between predicted and observed molecular weights for MMP1685?

The significant difference between predicted (6,398 Da) and observed (9,728 Da) molecular weights of MMP1685 requires systematic analysis:

• Post-translational modification mapping: Use mass spectrometry to identify all modifications

• Glycan analysis: Characterize glycan structures using specialized MS techniques

• Deglycosylation experiments: Compare molecular weights before and after enzymatic deglycosylation

• Site-directed mutagenesis: Modify predicted glycosylation sites and assess impact on molecular weight

• Comparative analysis: Compare with other archaeal pilins to identify common modification patterns

This methodological approach can resolve the apparent discrepancy and provide valuable insights into archaeal protein processing.

What approaches can address the challenges of MMP1685 structural analysis?

Structural analysis of MMP1685 presents several challenges, including its small size, extensive glycosylation, and sensitivity to oxidation. Researchers should consider:

• Integrated structural biology approaches combining:

  • X-ray crystallography of deglycosylated variants

  • NMR spectroscopy for solution structure determination

  • Cryo-electron microscopy of assembled pili structures

  • Molecular dynamics simulations to model flexible regions

• Specialized sample preparation:

  • Anaerobic purification to maintain native conformation

  • Controlled partial deglycosylation to improve crystallization

  • Nanobody-assisted crystallization to stabilize flexible regions

• Analysis of the protein in context:

  • In situ structural studies of MMP1685 within assembled pili

  • Cross-linking mass spectrometry to capture interaction interfaces

How can researchers accurately interpret mass spectrometry data for MMP1685?

Mass spectrometry data for MMP1685 requires careful interpretation due to several complicating factors:

• Glycan heterogeneity: The presence of multiple glycoforms creates complex spectra that require deconvolution

• N-terminal modification: The pyroglutamic acid modification affects fragmentation patterns

• Limited tryptic sites: The single lysine residue limits traditional tryptic digest approaches

For accurate interpretation:

• Use multiple complementary proteases (e.g., AspN, GluC) to generate overlapping peptide maps

• Apply specialized glycoproteomics approaches to characterize glycopeptides

• Compare fragmentation patterns with synthetic peptides of known sequence

• Employ multiple fragmentation techniques (CID, ETD, HCD) to increase sequence coverage

• Use specialized software designed for glycoprotein analysis

What are promising research directions for understanding MMP1685 function?

Based on current knowledge, several promising research directions for MMP1685 include:

• Comprehensive comparative analysis with homologous proteins in other archaea

• Investigation of environmental and metabolic factors that regulate MMP1685 expression

• Characterization of the complete pilus assembly pathway and the role of MMP1685

• Exploration of potential non-structural functions beyond pili formation

• Development of MMP1685 as a potential model for studying archaeal type IV pilin processing

How might advanced genetic tools enhance MMP1685 research?

M. maripaludis is a model methanogen with available genetic tools that can be leveraged for MMP1685 research:

• CRISPR-Cas9 adaptation for precise genome editing in M. maripaludis

• Development of inducible expression systems for controlled MMP1685 expression

• Reporter fusion constructs to monitor MMP1685 localization and expression patterns

• Creation of conditional knockdown strains to study MMP1685 essentiality

• Transposon mutagenesis screens to identify genetic interactors

These advanced genetic approaches can provide unprecedented insights into MMP1685 function and regulation.

What interdisciplinary approaches could yield new insights into MMP1685?

Interdisciplinary research approaches for MMP1685 could include:

• Synthetic biology: Engineering MMP1685 variants with novel properties or functions

• Evolutionary biology: Comparative analysis across archaeal species to understand evolutionary conservation

• Systems biology: Integration of MMP1685 into metabolic and protein interaction networks

• Biophysics: Advanced imaging and spectroscopy to characterize protein dynamics

• Computational biology: Machine learning approaches to predict interaction partners and functional domains

These interdisciplinary approaches can provide comprehensive understanding of MMP1685 beyond traditional biochemical characterization.