Recombinant Photobacterium profundum Chaperone surA (surA)

How does the structure of SurA relate to its function?

SurA has a complex multi-domain architecture that is directly related to its chaperone function:

• The protein contains a core domain flanked by peptidyl-prolyl isomerase (PPIase) domains known as P1 and P2 .

• The first PPIase domain (P1) is primarily responsible for peptide recognition and binds preferentially to peptides with a high fraction of aromatic amino acids .

• Experimental data supports a model in which SurA binds unfolded OMPs (uOMPs) in a groove formed between the core and P1 domains .

• This binding event results in a dramatic expansion of the rest of the uOMP, which has significant biological implications for protein folding and assembly .

Crystal structure studies have shown that SurA binds peptides in an extended conformation, with the peptide assuming a specific orientation relative to the chaperone . This binding mode is critical for the proper recognition and processing of client proteins.

What methodologies are most effective for studying SurA-client interactions?

Researchers have employed several complementary approaches to study SurA-client interactions:

For example, a study used photo-crosslinking with p-azidophenylalanine (pAF) incorporated at various positions in SurA to identify binding sites on client uOMPs. This was followed by mass spectrometry analysis to identify the crosslinked peptides, revealing specific segments on uOMPs that preferentially interact with the SurA groove .

How can researchers measure and characterize the expansion of uOMPs when bound to SurA?

The unprecedented expansion of uOMPs when bound to SurA can be characterized using several biophysical techniques:

• Small-angle neutron scattering (SANS): This technique directly reports on the radius of gyration (RG) and maximum end-to-end distance (DMax) of macromolecules. In one study, SANS was used to measure the hydrodynamic properties of a SurA-uOMP complex, revealing that the bound uOMP adopts an expanded conformation compared to its unbound state .

• Selective contrast in SANS: By manipulating sample and buffer conditions, researchers can selectively visualize specific components within a complex. This approach was used to collect scattering profiles of photo-crosslinked complexes of protonated SurA with deuterated uOMP clients .

• Molecular modeling: Experimental data from crosslinking and scattering experiments can be used as restraints to generate integrative models of SurA-uOMP complexes. This approach has revealed that SurA can utilize multiple binding modes to interact with uOMPs .

The expansion of uOMPs when bound to SurA suggests a mechanism by which the chaperone prepares clients for delivery to the β-barrel assembly machinery (BAM) complex for insertion into the outer membrane .

How does pressure affect SurA expression and function in P. profundum?

P. profundum strain SS9 is a piezophilic bacterium that grows optimally at 28 MPa and 15°C, making it an excellent model for studying pressure adaptation mechanisms . While specific data on SurA expression under different pressure conditions is limited in the search results, we can draw insights from proteome-wide studies of P. profundum:

• Shotgun proteomic analysis of P. profundum grown at atmospheric pressure compared to high pressure (28 MPa) revealed differential expression of proteins involved in various cellular processes .

• Proteins involved in the glycolysis/gluconeogenesis pathway were up-regulated at high pressure, while several proteins involved in oxidative phosphorylation were up-regulated at atmospheric pressure .

• Several stress response genes, including molecular chaperones like htpG, dnaK, dnaJ, and groEL, are up-regulated in response to atmospheric pressure in P. profundum strain SS9 .

Given SurA's role in outer membrane protein biogenesis, its expression and function may be regulated in response to pressure changes to maintain membrane integrity and function. This represents an important area for future research, investigating how SurA activity is modulated under different pressure conditions to support P. profundum's piezophilic lifestyle.

What role might SurA play in P. profundum's adaptation to high-pressure environments?

The adaptation of P. profundum to high-pressure environments likely involves multiple mechanisms, including changes in membrane composition and protein function. SurA's role in this adaptation may include:

• Membrane protein homeostasis: SurA ensures proper folding and assembly of OMPs, which are crucial for membrane function under high pressure .

• Stress response: As a chaperone, SurA may help prevent protein misfolding under pressure stress, similar to how other chaperones (htpG, dnaK, dnaJ, groEL) are involved in the stress response .

• Transporter biogenesis: Proteomics studies have identified numerous differentially expressed ABC transporters involved in ion, sugar, and amino acid transport across the cell membrane under different pressure conditions . SurA may be involved in the biogenesis of these transporter proteins.

• Adaptation to nutrient availability: Different hydrostatic pressures represent distinct ecosystems with their own particular nutrient limitations and abundances . SurA's role in OMP biogenesis may contribute to the organism's ability to adapt to these varying nutrient conditions.

Large-scale transposon mutagenesis of P. profundum SS9 has identified genes involved in low-temperature and high-pressure growth, with many loci associated with pressure sensitivity involved in chromosomal structure and function, ribosome assembly, and signal transduction mechanisms . Future research could investigate whether SurA or its client proteins are among these pressure-sensitive loci.

How should one design experiments to study SurA function under different pressure conditions?

Designing experiments to study SurA function under different pressure conditions requires careful consideration of multiple factors:

• Pressure equipment: High-pressure microscopic chambers or high-pressure vessels are necessary for experiments at elevated pressures. For example, direct swimming velocity measurements of P. profundum were obtained using a high-pressure microscopic chamber capable of reaching pressures up to 150 MPa .

• Controls: Include appropriate controls such as pressure-sensitive organisms (e.g., E. coli) and pressure-adapted relatives (e.g., P. profundum strain 3TCK, which is less pressure-adapted than SS9) .

• Comparative approach: Compare SurA function in organisms with different pressure adaptations. For example, comparing SurA from P. profundum SS9 (optimal growth at 28 MPa) with SurA from P. profundum 3TCK (optimal growth at atmospheric pressure) .

• Molecular techniques: Methods such as in-frame deletions of genes, complementation analysis, and expression studies can be used to investigate SurA function. For example, deletion mutants of flagellar genes in P. profundum SS9 were constructed to study their role in high-pressure adaptation .

• Protein activity assays: Develop assays to measure SurA chaperone activity under different pressure conditions, potentially using fluorescence-based approaches to monitor client protein folding.

• Expression analysis: Use techniques such as qPCR or proteomics to measure changes in SurA expression under different pressure conditions .

What expression systems are recommended for producing recombinant P. profundum SurA?

Based on the principles of recombinant protein expression and the specific characteristics of SurA:

• E. coli expression system: This is the most common system for recombinant protein production and has been used successfully for expressing chaperones like SurA. The BL21(DE3) strain or its derivatives are commonly used for protein expression.

• Expression constructs: Design constructs with appropriate tags (e.g., His-tag, GST-tag) for purification. Consider the location of the tag to minimize interference with SurA function.

• Induction conditions: Optimize temperature, inducer concentration, and induction time. Lower temperatures (e.g., 18-25°C) may improve the solubility of the recombinant protein.

• Solubility considerations: SurA is normally located in the periplasm, so including the native signal peptide or using periplasmic expression systems may improve solubility and proper folding.

• Purification strategy: Design a purification scheme that typically includes affinity chromatography followed by size exclusion chromatography to obtain pure, homogeneous protein.

• Functional validation: Verify that the recombinant SurA is properly folded and functionally active using binding assays with known client peptides.

For specialized applications, such as structural studies using SANS, consider deuterated expression systems to facilitate contrast matching experiments .

How can integrative modeling approaches be used to study SurA-uOMP complexes?

Integrative modeling combines data from multiple experimental techniques to generate structural models of protein complexes. For studying SurA-uOMP complexes, the following approach has proven effective:

This integrative approach is particularly valuable for studying dynamic and heterogeneous systems like chaperone-client complexes, which may be difficult to characterize using a single technique .

What mass spectrometry approaches are most informative for studying SurA interactions?

Mass spectrometry (MS) offers powerful tools for studying protein-protein interactions, including those involving SurA:

• Crosslinking MS (XL-MS): This approach involves crosslinking proteins in their native state, followed by proteolytic digestion and MS analysis to identify crosslinked peptides. For SurA studies, both chemical crosslinking and photo-crosslinking have been used .

• Photo-crosslinking MS: By incorporating photo-activatable amino acids (e.g., p-azidophenylalanine) at specific positions in SurA, researchers can induce crosslinking upon UV exposure. MS analysis of these crosslinks provides precise information about binding interfaces .

• Hydrogen-deuterium exchange MS (HDX-MS): This technique measures the rate of hydrogen-deuterium exchange in protein backbones, providing information about protein dynamics and solvent accessibility. It could be used to study conformational changes in SurA upon client binding.

• Native MS: This approach analyzes intact protein complexes under native conditions, providing information about complex stoichiometry and stability. It could be used to study SurA-uOMP complexes with different client proteins.

• Quantitative proteomics: Label-free or labeled quantitative proteomics can be used to study changes in protein expression under different conditions. This approach has been used to study the proteome of P. profundum under different pressure conditions .

For studying SurA-client interactions, a combination of photo-crosslinking and LC-MS/MS analysis has proven particularly informative, revealing specific binding sites and preferred interaction segments .

How does P. profundum SurA compare to SurA from other bacterial species?

Comparing SurA from different bacterial species can provide insights into both conserved functions and specialized adaptations:

A detailed comparative analysis of P. profundum SurA with SurA from other bacterial species would contribute to our understanding of both universal chaperone mechanisms and specific adaptations to extreme environments.

What can we learn from studying SurA in a piezophilic organism like P. profundum?

Studying SurA in a piezophilic organism like P. profundum offers unique opportunities to understand:

• Pressure adaptation mechanisms: P. profundum grows optimally at 28 MPa, providing a model system to study how proteins adapt to function under high pressure .

• Membrane biogenesis under pressure: SurA's role in OMP biogenesis makes it an excellent target for investigating how membrane assembly is maintained under high-pressure conditions .

• Protein folding principles: The study of how SurA assists protein folding under pressure may reveal general principles about protein folding and stability in extreme environments.

• Evolutionary adaptations: Comparing SurA from piezophilic and non-piezophilic organisms can reveal evolutionary adaptations to high-pressure environments.

• Biotechnological applications: Insights from pressure-adapted proteins like P. profundum SurA may inform the development of pressure-stable enzymes or expression systems for biotechnological applications.

P. profundum SS9's ability to grow under a wide range of pressures (0.1 MPa to 90 MPa) makes it an excellent model organism for such studies, as it allows for both genetic manipulation at atmospheric pressure and phenotypic characterization under high pressure .

What are the most promising research avenues for understanding SurA function in piezophilic bacteria?

Several promising research directions could advance our understanding of SurA function in piezophilic bacteria:

• Structural studies under pressure: Develop methods to study SurA structure and dynamics under high pressure, potentially using high-pressure NMR, SANS, or other biophysical techniques.

• Client specificity under pressure: Investigate whether SurA's client specificity or binding mode changes under different pressure conditions, which could reveal adaptations for OMP biogenesis in deep-sea environments.

• Molecular evolution: Compare SurA sequences from bacteria adapted to different pressure environments to identify potential pressure-adaptive mutations.

• Functional genomics: Employ large-scale transposon mutagenesis or CRISPR-based approaches to identify genetic interactions with SurA in P. profundum, particularly those that affect pressure adaptation .

• Systems biology: Integrate proteomics, transcriptomics, and functional data to understand how SurA functions within the larger context of cellular adaptation to high pressure .

• Synthetic biology: Design and test SurA variants with enhanced or altered function under high pressure, potentially creating tools for biotechnological applications.

• In vivo visualization: Develop methods to visualize SurA-client interactions in live cells under different pressure conditions, possibly using fluorescence techniques adapted for high-pressure microscopy.

These approaches would contribute to a more comprehensive understanding of how essential cellular processes, such as membrane protein biogenesis, adapt to extreme environments.

What technological innovations might advance SurA research in the near future?

Several technological innovations could significantly advance SurA research:

• High-pressure structural biology techniques: Development of improved methods for studying protein structure and dynamics under high pressure, such as high-pressure cryo-EM, high-pressure NMR, or high-pressure X-ray crystallography.

• In situ high-pressure visualization: Advanced microscopy techniques that allow for the observation of cellular processes under high pressure in real-time.

• Computational methods: Enhanced molecular dynamics simulations that can accurately model protein behavior under high pressure, potentially predicting pressure-adaptive mutations or conformational changes.

• Deep-sea sampling and culturing technologies: Improved methods for sampling and culturing deep-sea microorganisms, expanding the diversity of piezophilic models available for comparative studies.

• Single-molecule techniques: Development of single-molecule methods adapted for high-pressure conditions to study individual SurA-client interactions.

• Synthetic biology tools: New genetic tools optimized for piezophilic organisms to facilitate genetic manipulation and functional studies.

• Protein engineering approaches: Methods to design pressure-stable proteins or to introduce pressure-sensing domains, potentially creating biosensors or pressure-regulated expression systems.

These technological innovations would not only advance SurA research but would also contribute more broadly to our understanding of life in extreme environments and the adaptation of biological processes to high-pressure conditions.