Recombinant Desulfovibrio vulgaris Lipoprotein-releasing system ATP-binding protein LolD (lolD)

Basic Research Questions

• What is LolD and what is its functional role in bacterial physiology?

  LolD is an essential component of the Lol system, which is responsible for the localization of lipoproteins from the inner membrane to the outer membrane in gram-negative bacteria. In Desulfovibrio vulgaris, LolD functions as the ATP-binding protein of the ABC transporter complex involved in lipoprotein release. It provides the energy through ATP hydrolysis that allows LolF (another component of the system) to extract lipoproteins from the membrane for localization to the outer membrane .

  Structurally, LolD belongs to the ABC family of ATP-binding proteins. Comparative analysis with E. coli LolD shows structural similarity with an RMSD value of 2.1 Å between aligned pairs of backbone C-alpha atoms, which is comparable to the similarity between E. coli LolD and other ABC family ATP-binding proteins like LptB (2.4 Å) and MalK (2.2 Å) .

• How can I properly store and handle recombinant D. vulgaris LolD protein to maintain activity?

  Proper storage and handling are critical for maintaining the structural integrity and functional activity of recombinant LolD. According to established protocols:

  • The shelf life of liquid form is generally 6 months at -20°C/-80°C

  • The shelf life of lyophilized form is 12 months at -20°C/-80°C

  • Repeated freezing and thawing is not recommended

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

  • For reconstitution, briefly centrifuge the vial prior to opening

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

  • Addition of 5-50% glycerol (final concentration) is recommended for long-term storage

  • Aliquoting the reconstituted protein is advisable to avoid repeated freeze-thaw cycles

• What experimental approaches are most effective for identifying LolD interaction partners?

  Affinity purification-mass spectrometry (AP-MS) has proven to be a highly effective approach for identifying interaction partners of LolD and other Lol system components. The methodology involves:

  1. Creating a tagged version of the protein (e.g., DDK-tagged LolF was used to identify LolD in H. pylori)

  2. Verifying expression of the tagged protein through immunoblot analysis

  3. Immunoprecipitating the tagged protein using an appropriate antibody

  4. Analyzing samples by mass spectrometry to identify co-purified proteins

  5. Using appropriate controls (e.g., unrelated inner membrane proteins tagged similarly)

  6. Including ATP in the buffer to enhance interactions between LolF and ABC family ATP-binding proteins

  7. Testing various detergents, with ionic or zwitterionic detergents typically disrupting these interactions

  8. Using statistical analysis tools like SAINTexpress to compute the probability that a prey protein is a true interaction partner

Intermediate Research Questions

• How can I optimize the expression conditions for recombinant D. vulgaris LolD?

  Optimizing expression conditions for recombinant LolD requires a systematic approach using design-of-experiments (DoE) methodology. This approach is more efficient than the traditional univariant method where variables are changed one at a time.

  Recommended DoE approach:

  1. Identify key variables: Temperature, IPTG concentration, medium composition, induction time, initial OD, and pH are typically important

  2. Design factorial experiments: Use a fractional factorial design (e.g., 2^8-4 for 8 variables) with replicas at central points to reduce the number of experiments while maintaining statistical validity

  3. Define response variables: Typically protein yield, solubility, and functional activity

  4. Execute experiments and analyze data: Use statistical software to identify significant factors and their interactions

  5. Optimize significant factors: Once identified, conduct further experiments to fine-tune these variables

  Recent studies using this approach have achieved high levels (250 mg/L) of soluble expression of recombinant proteins in E. coli with 75% homogeneity while maintaining functional activity .

• What are the key differences between LolD in D. vulgaris and other bacterial species?

  Comparing LolD across different bacterial species reveals important evolutionary adaptations. In H. pylori, for instance, a LolD-like protein (HP0179) was identified through affinity purification-mass spectrometry using LolF as bait.

  Comparative analysis:

  Species	LolD Protein	Sequence identity to E. coli LolD	Key features
  E. coli	LolD	Reference	Well-characterized prototype
  D. vulgaris	LolD (Q729H7)	<40%	Contains characteristic ABC ATP-binding domains
  H. pylori	HP0179	<40%	Annotated as "putative ABC transport system ATP-binding protein"

  While sequence identity between these proteins is relatively low (<40%), structural comparisons show significant similarities in their three-dimensional conformations. The RMSD between D. vulgaris LolD and E. coli LolD is comparable to that between E. coli LolD and other ABC family ATP-binding proteins, suggesting functional conservation despite sequence divergence .

• How can I determine if recombinant LolD is properly folded and functional?

  Assessing proper folding and functionality of recombinant LolD involves multiple complementary approaches:

  1. ATP binding and hydrolysis assays: As an ABC-type ATPase, functional LolD should bind and hydrolyze ATP. Measure ATPase activity using:

     • Malachite green phosphate assay

     • Coupled enzyme assays (e.g., pyruvate kinase/lactate dehydrogenase)

     • Radioactive [γ-32P]ATP hydrolysis assays

  2. Protein-protein interaction studies: Functional LolD should interact with other components of the Lol system. Use:

     • Pull-down assays with tagged LolF

     • Surface plasmon resonance (SPR)

     • Isothermal titration calorimetry (ITC)

  3. Circular dichroism (CD) spectroscopy: Compare the CD spectrum with properly folded reference proteins to assess secondary structure elements

  4. Thermal shift assays: Properly folded proteins typically show cooperative unfolding with higher melting temperatures

  5. Functional complementation: Test if the recombinant protein can rescue growth in conditional LolD mutants

Advanced Research Questions

• How can Design of Experiments (DoE) methodology be applied to optimize purification of recombinant LolD?

  Design of Experiments methodology provides a systematic framework for optimizing purification protocols with fewer experiments while gaining more information about parameter interactions:

  1. Define response variables: Typically purity (%), yield (mg/L), and specific activity (units/mg)

  2. Identify critical parameters: For immobilized metal affinity chromatography (IMAC):

     • Imidazole concentration in equilibration buffer

     • Imidazole concentration in wash buffer

     • pH

     • Flow rate

     • Sample loading volume

     • Column dimensions

  3. Design an experimental matrix: Use factorial design software to create an orthogonal experimental plan

  4. Statistical analysis: Analyze results to:

     • Identify statistically significant parameters

     • Quantify interactions between parameters

     • Create response surface models

     • Predict optimal conditions

  5. Validation: Confirm predicted optimal conditions experimentally

  This approach has demonstrated superior results compared to traditional one-factor-at-a-time optimization for bioprocess development, reducing development time while increasing process understanding .

• What molecular mechanisms explain ATP-dependent lipoprotein release by the LolD/LolF complex?

  The LolD/LolF complex functions as an ATP-dependent lipoprotein extraction machinery. Current mechanistic models suggest:

  1. ATP binding to LolD induces conformational changes that are transmitted to the transmembrane domains of LolF

  2. These conformational changes alter the arrangement of LolF's transmembrane helices, decreasing their affinity for lipoprotein acyl chains

  3. Lipoproteins are released from the inner membrane and transferred to a carrier protein (LolA in E. coli)

  4. ATP hydrolysis resets the system for another cycle

  Site-directed mutagenesis studies of LolD's Walker A and Walker B motifs, which are essential for ATP binding and hydrolysis, respectively, have shown that mutations in these regions abolish lipoprotein release activity. In H. pylori, conditional hp0179 mutants demonstrated that both HP0179 (the LolD homolog) and amino acids predicted to be required for ATP binding and ATP hydrolysis are essential for bacterial growth .

• What are the implications of LolD function for developing new antimicrobial strategies against Desulfovibrio species?

  LolD represents a promising target for novel antimicrobial strategies based on several key considerations:

  1. Essentiality: Studies with conditional hp0179 mutants in H. pylori demonstrated that LolD-like proteins are essential for bacterial growth. Transposon mutagenesis studies suggest similar essentiality in other species .

  2. Conservation vs. divergence: While LolD is conserved across gram-negative bacteria, there are significant sequence differences between bacterial and human ABC transporters, potentially allowing selective targeting.

  3. Systems approach: Targeting the Lol system could disrupt proper lipoprotein localization, affecting numerous cellular functions simultaneously.

  4. Clinical relevance: Desulfovibrio species have been implicated in several human diseases, including ulcerative colitis, type 2 diabetes, inflammatory bowel diseases, liver abscesses, and appendicitis. More recently, D. desulfuricans has been connected to atherosclerosis by increasing intestinal permeability and downregulating tight junction proteins .

  Potential therapeutic strategies include:

  • Small molecule inhibitors of LolD ATPase activity

  • Peptides disrupting LolD-LolF interactions

  • Compounds preventing lipoprotein recognition

• How can genome-scale approaches be integrated with biochemical studies of LolD to understand its role in Desulfovibrio metabolism?

  Integrating genome-scale approaches with targeted biochemical studies provides a comprehensive understanding of LolD's role in Desulfovibrio metabolism:

  1. Transcriptomic analysis: RNA-seq data can reveal co-expression patterns between lolD and other genes, particularly under different growth conditions. In D. vulgaris Hildenborough, microarray studies have provided insights into gene expression changes under different electron donors and acceptors .

  2. Transposon mutagenesis libraries: The absence of transposon insertions in particular genes can indicate essentiality. The Wall lab has created 8,869 D. vulgaris mutants with identified insertion sites, covering 62% of predicted genes. Analysis of these libraries can reveal connections between LolD and other cellular pathways .

  3. Interactome studies: Affinity purification-mass spectrometry approaches have successfully identified protein-protein interactions in the Lol system. Expanding these studies to capture broader interaction networks can place LolD in a systems context .

  4. Metabolic modeling: Genome-scale metabolic models can predict the systemic effects of LolD dysfunction on cellular metabolism.

  5. Conditional expression systems: For essential genes like lolD, regulated expression systems allow titrated depletion to study physiological responses before lethality.

  Integration of these approaches has revealed unexpected relationships between lipoprotein transport and energy metabolism in Desulfovibrio species, highlighting the interconnected nature of bacterial cellular processes .