Recombinant Hordeum vulgare Low molecular mass early light-inducible protein HV60, chloroplastic

What is Recombinant Hordeum vulgare Low molecular mass early light-inducible protein HV60?

Recombinant Hordeum vulgare Low molecular mass early light-inducible protein HV60, chloroplastic is a stress-responsive protein originally isolated from barley (Hordeum vulgare). The recombinant version is typically produced in expression systems such as E. coli for research purposes. It consists of amino acids 34-167 of the mature protein and is commonly fused to an N-terminal His-tag for purification purposes . The protein belongs to the light-harvesting complex (LHC)-like protein family that contains chlorophyll-binding motifs, similar to ELIPs studied in other plant species . The full protein is typically produced as a lyophilized powder with greater than 90% purity as determined by SDS-PAGE analysis .

What is the function of ELIP proteins in plants?

ELIP proteins play critical roles in protecting photosynthetic machinery from various environmental stresses. Based on comprehensive research with both land plants and algae, ELIPs demonstrate several key functions:

• Protection of photosystem II (PSII) from photooxidative damage during high light exposure

• Enhancement of cellular survival under cold stress conditions

• Modulation of the xanthophyll cycle under severe stress conditions

• Regulation of cellular redox state during combined stresses

Research with Chlamydomonas reinhardtii ELIP3 showed that these proteins are particularly important under combined stresses of high light and low temperature . The expression of ELIP genes is typically induced by light stress (including high light, blue light, and UV-A) in a light intensity-dependent manner, often regulated by photoreceptors such as cryptochrome1 (CRY1) . Notably, knockdown mutants of ELIP3 demonstrated much lower photosynthetic efficiency than wild type strains when exposed to low temperatures, while overexpression mutants survived significantly longer under combined high light and cold stress conditions .

How are ELIP proteins regulated in response to environmental stresses?

ELIP proteins demonstrate specific and complex regulation patterns in response to environmental stresses:

What experimental approaches can be used to study the role of HV60 in protecting photosynthetic machinery?

To comprehensively investigate HV60's role in photosynthetic protection, researchers should implement multiple complementary experimental approaches:

• Genetic manipulation strategies:

  • Generate knockdown mutants using RNAi or CRISPR-Cas9 to reduce HV60 expression

  • Create overexpression lines to enhance HV60 levels

  • Compare these genetically modified lines with wild-type plants under controlled stress conditions

  • Develop complementation lines to confirm phenotype specificity

• Chlorophyll fluorescence analysis protocols:

  • Measure quantum yield parameters (ΦPSII, Fv/Fm) to assess photosynthetic efficiency under stress

  • Construct rapid light curves (RLCs) to evaluate electron transport rates under increasing light intensities

  • Calculate non-photochemical quenching (NPQ) to quantify photoprotective mechanisms

  • Standard calculation methods include:

    • Effective quantum yield: ΦPSII = ΔF/Fm′ = (Fm′- F)/Fm′

    • Maximum quantum yield: Fv/Fm = (Fm - F0)/Fm

    • Relative electron transport rate: rETR = 0.84 × 0.5 × ΦPSII × light intensity

• Stress application methodologies:

  • Cold stress: Incubate cultures at 4°C compared to optimal temperature controls (e.g., 23°C)

  • High light stress: Expose to light intensities exceeding 500 μmol photons m⁻² s⁻¹

  • Combined stress protocols: Apply both high light and cold stress simultaneously

  • Monitor survival rates, photosynthetic parameters, and recovery kinetics over defined time periods

  • Standard concentration for cell experiments: 5 × 10⁶ cells/ml

• Biochemical and molecular analyses:

  • Perform Western blotting to quantify HV60 protein accumulation under different conditions

  • Measure reactive oxygen species (ROS) levels using fluorescent probes

  • Analyze lipid peroxidation as an indicator of membrane damage

  • Assess xanthophyll cycle pigments using HPLC to correlate with photoprotective capacity

  • Monitor expression of stress-responsive genes to identify regulatory networks

• Cellular response assessments:

  • Use confocal microscopy with fluorescent protein-tagged HV60 to track subcellular localization

  • Employ transmission electron microscopy to observe ultrastructural changes in thylakoids

  • Study movement responses (phototaxis) as indicators of stress perception and cell viability

  • Compare phototactic responses between wild-type, knockdown, and overexpression lines

How can researchers optimize the expression and purification of recombinant HV60?

Optimization of recombinant HV60 expression and purification requires systematic approach to multiple parameters:

• Expression system selection and optimization:

  • E. coli systems: Most commonly used for HV60 production with well-established protocols

  • Alternative systems: Yeast, baculovirus, or mammalian cell expression systems for specific applications

  • Cell-free expression: May offer advantages for difficult-to-express proteins or when rapid production is needed

  • Vector optimization: Select appropriate promoters, codon optimization, and fusion tags (His-tag is standard for HV60)

• Culture condition optimization matrix:

  Parameter	Variables to Test	Considerations
  Temperature	16°C, 25°C, 30°C, 37°C	Lower temperatures often reduce inclusion body formation
  Induction timing	OD600: 0.4, 0.6, 0.8, 1.0	Optimal cell density varies by system
  Inducer concentration	IPTG: 0.1mM, 0.5mM, 1.0mM	Lower concentrations may improve solubility
  Media composition	LB, TB, 2xYT, M9	Rich vs. minimal media affects expression
  Expression duration	4h, 8h, 16h, 24h	Longer times may increase yield but risk degradation

• Purification strategy development:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged HV60

  • Additional purification steps (ion exchange, size exclusion) to achieve >90% purity

  • Buffer optimization to maintain protein stability throughout purification

  • Endotoxin removal steps for sensitive applications (especially important as contamination can affect experimental results)

• Quality control and validation protocols:

  • SDS-PAGE analysis to confirm purity (target >90%)

  • Western blotting for identity confirmation

  • Mass spectrometry for accurate mass determination and sequence verification

  • Functional assays to verify biological activity of the purified protein

• Storage and handling recommendations:

  • Lyophilize the purified protein for long-term storage

  • Avoid repeated freeze-thaw cycles which can decrease activity

  • Store working aliquots at 4°C for up to one week

  • For long-term storage, keep at -20°C/-80°C with 5-50% glycerol (50% recommended)

  • Reconstitution protocol: add deionized sterile water to achieve 0.1-1.0 mg/mL concentration

What techniques are available to analyze HV60 protein-protein interactions in thylakoid membranes?

Analyzing protein-protein interactions of membrane-localized HV60 requires specialized techniques that account for the challenges of working with membrane proteins:

• In vivo proximity-based approaches:

  • Förster Resonance Energy Transfer (FRET):

    • Create fluorescent protein fusions (e.g., HV60-CFP paired with potential partner-YFP)

    • Measure energy transfer as indicator of molecular proximity (typically <10nm)

    • Quantify interaction strength through FRET efficiency calculations

  • Bimolecular Fluorescence Complementation (BiFC):

    • Split fluorescent protein fragments fused to potential interaction partners

    • Fluorescence occurs only when proteins interact, bringing fragments together

    • Provides direct visualization of interaction sites within the cell

  • Proximity Ligation Assay (PLA):

    • Detect endogenous protein interactions without overexpression artifacts

    • Provides spatial information about interaction location within chloroplasts

    • Higher sensitivity than conventional co-localization studies

• Co-immunoprecipitation strategies for membrane proteins:

  • Detergent optimization matrix:

    Detergent Type	Concentration Range	Membrane Protein Applications
    Digitonin	0.5-1.0%	Preserves supercomplexes and weak interactions
    n-Dodecyl β-D-maltoside	0.5-2.0%	Good for thylakoid membrane proteins
    Triton X-100	0.1-1.0%	Effective but may disrupt some interactions
    CHAPS	0.3-1.0%	Milder detergent for sensitive complexes

  • Cross-linking assisted co-IP:

    • Apply membrane-permeable cross-linkers before extraction

    • Stabilize transient interactions that might be lost during purification

    • Reverse cross-linking for SDS-PAGE analysis

• Mass spectrometry-based interaction profiling:

  • Affinity purification-mass spectrometry (AP-MS):

    • Purify HV60 complexes under native conditions with optimized detergents

    • Identify interacting proteins by tandem mass spectrometry

    • Implement appropriate controls to distinguish specific from non-specific interactions

  • Chemical cross-linking coupled with MS (XL-MS):

    • Cross-link proteins in their native thylakoid environment

    • Identify cross-linked peptides to map interaction interfaces at residue resolution

    • Provide distance constraints for structural modeling

• Membrane-specific yeast two-hybrid systems:

  • Split-ubiquitin membrane yeast two-hybrid:

    • Specifically designed for membrane protein interactions

    • Reconstitution of ubiquitin triggers reporter gene expression

    • Screen libraries of potential interaction partners

  • Membrane protein complementation assays:

    • Use reporter fragments that function only when brought together by interaction

    • Can be performed in yeast or bacterial systems

    • Allows high-throughput screening approaches

• Advanced microscopy techniques:

  • Super-resolution microscopy:

    • Overcome diffraction limit to visualize nanoscale protein clusters

    • Track dynamic interactions in living chloroplasts

    • Correlate with functional photosynthetic measurements

  • Single-molecule tracking:

    • Follow individual HV60 molecules within thylakoid membranes

    • Analyze diffusion patterns and interaction kinetics

    • Identify stable vs. transient interaction events

How can researchers measure the photoprotective efficiency of HV60 in vivo?

To comprehensively assess HV60's photoprotective efficiency in living systems, researchers should implement a multi-parameter experimental approach:

• Chlorophyll fluorescence parameter analysis:

  • Measure PSII maximum quantum yield (Fv/Fm) as the primary indicator of PSII health and functionality

  • Monitor effective quantum yield (ΦPSII) under illumination to assess operational efficiency

  • Track non-photochemical quenching (NPQ) capacity to quantify energy dissipation mechanisms

  • Calculate relative electron transport rates (rETR) to evaluate photosynthetic performance

• Rapid Light Curve (RLC) protocol:

  • Compare RLC parameters between wild-type and HV60-modified lines:

    • Initial slope (α) - light utilization efficiency

    • Maximum rETR (rETRmax) - photosynthetic capacity

    • Saturation irradiance (Ek) - photoacclimation status

    • Photoinhibition threshold - stress tolerance

• Stress recovery experimental design:

  • Subject plant material to standardized stress protocols:

    • Moderate stress: 4°C + 200 μmol photons m⁻² s⁻¹ for 2 hours

    • Severe stress: 4°C + 800 μmol photons m⁻² s⁻¹ for 2 hours

  • After stress removal, monitor recovery kinetics:

    • Fv/Fm recovery rate (% recovery per hour)

    • Time to 50% recovery (RT50)

    • Complete recovery time

  • Compare recovery parameters between wild-type, HV60 knockdown, and overexpression lines

• ROS quantification methodology:

  • Use fluorescent probes (e.g., DCFH-DA, DHE) to quantify specific ROS species

  • Measure lipid peroxidation products (e.g., MDA) as indicators of oxidative damage

  • Compare ROS accumulation with and without ROS quenchers like TEMPOL

  • Correlate ROS levels with HV60 expression levels and stress sensitivity

• Comparative survival analysis protocol:

  • Expose cells/plants to defined lethal stress combinations

  • Quantify survival using:

    • Cell viability staining techniques

    • Regrowth capacity after stress

    • Chlorophyll content retention

  • Construct survival curves for statistical comparison between genotypes

• Phototaxis assessment methodology:

  • Design light gradient apparatus for quantitative phototaxis measurements

  • Compare phototactic responses between genetic lines:

    • Wild-type controls typically show variable responses

    • HV60 knockdown mutants show negative phototaxis (light avoidance)

    • HV60 overexpression mutants show enhanced positive phototaxis

  • Correlate phototactic behavior with photosynthetic efficiency and stress tolerance

What are the molecular mechanisms by which HV60 protects thylakoid membranes during cold stress?

The molecular mechanisms underlying HV60-mediated protection of thylakoid membranes during cold stress involve multiple pathways that can be investigated through targeted experimental approaches:

• Membrane fluidity regulation hypothesis:
  HV60 may help maintain appropriate membrane fluidity at low temperatures, preventing phase transitions that disrupt photosynthetic protein complexes. This mechanism can be investigated through:

  • Fluorescence anisotropy measurements with membrane probes under cold stress

  • Differential scanning calorimetry (DSC) to measure membrane phase transition temperatures

  • Lipidomic analysis to detect changes in membrane lipid composition associated with HV60 expression

  • Comparison of membrane physical properties between wild-type and HV60-modified plants

• Protein-pigment interaction model:
  HV60 likely binds chlorophyll and carotenoid molecules to prevent formation of free radicals during photosynthetic imbalance caused by cold stress. This function can be studied through:

  • Spectroscopic analysis of pigment binding properties with purified HV60

  • Reconstitution experiments with isolated pigments and recombinant HV60

  • Site-directed mutagenesis of conserved pigment-binding residues followed by functional assays

  • Correlation with studies of Arabidopsis ELIPs showing association with PSII antenna complexes

• ROS management mechanisms:
  HV60 may directly or indirectly reduce ROS accumulation during cold stress, as suggested by experiments with the ROS quencher TEMPOL . This can be explored through:

  • Comparative ROS measurements in wild-type vs. HV60-modified plants during cold stress

  • Analysis of antioxidant enzyme activities in relation to HV60 expression

  • In vitro assessment of direct ROS scavenging capacity of purified HV60

  • Time-course studies correlating HV60 expression with ROS accumulation patterns

• Xanthophyll cycle enhancement:
  HV60 may promote efficient operation of the xanthophyll cycle, which dissipates excess excitation energy as heat. This hypothesis aligns with studies suggesting Arabidopsis ELIPs modulate the xanthophyll cycle , and can be tested through:

  • HPLC analysis of xanthophyll cycle pigment interconversion during cold stress

  • Measurement of de-epoxidation state (DEPS) in relation to HV60 levels

  • Correlation of NPQ capacity with HV60 expression during stress recovery

  • Combined analysis of HV60 and xanthophyll cycle enzyme expression patterns

• Photosystem stability mechanism:
  HV60 may physically associate with photosystem components to stabilize their structure during cold-induced membrane alterations. This can be investigated through:

  • Co-immunoprecipitation studies with photosystem components under cold stress

  • FRET analysis between fluorescently labeled HV60 and photosystem proteins

  • Electron microscopy of thylakoid membrane ultrastructure in HV60-modified plants

  • Assessment of photosystem integrity through blue-native PAGE analysis

• Integration with phototaxis response pathways:
  The observed differences in phototaxis between HV60 knockdown (negative phototaxis) and overexpression (positive phototaxis) mutants suggest integration with cellular signaling networks that can be explored through:

  • Analysis of photoreceptor activity in HV60-modified lines

  • Investigation of calcium signaling pathways during cold and light stress

  • Transcriptomic analysis of signaling components affected by HV60 levels

  • Correlation between phototactic responses and photosynthetic efficiency

How should researchers approach experimental design to resolve contradictory data about HV60 function?

Resolving contradictory findings regarding HV60 function requires rigorous experimental design and careful consideration of multiple factors:

• Standardization of experimental conditions:

  • Implement a comprehensive standard operating procedure specifying:

    • Plant material genetic background and growth stage

    • Growth conditions (light intensity, temperature, photoperiod, media composition)

    • Stress application protocols (intensity, duration, rate of change)

    • Sampling procedures and time points

    • Measurement techniques and instrumentation settings

  • Document all environmental parameters meticulously to enable true replication

  • Consider circadian and developmental effects on HV60 expression and function

• Multiple complementary methodological approaches:

  • Apply diverse techniques to investigate the same biological question:

    • Genetic approaches (knockout, knockdown, overexpression)

    • Biochemical assays (protein interaction, activity)

    • Physiological measurements (photosynthesis parameters, stress tolerance)

    • Imaging and localization studies

  • Look for convergence of evidence across multiple methodologies

  • Identify specific conditions where contradictions emerge for targeted investigation

• Genetic background considerations:

  • Test HV60 function across diverse genetic contexts:

    • Different barley cultivars/ecotypes

    • Near-isogenic lines differing only in HV60 alleles

    • Heterologous expression in model systems (Arabidopsis, Chlamydomonas)

  • Create isogenic lines differing only in HV60 expression level

  • Analyze potential genetic interactions with other stress-responsive systems

• Rigorous protein quality control:

  • Implement stringent purification protocols for recombinant HV60:

    • Multiple purification steps to ensure >90% purity

    • Endotoxin testing and removal (particularly important as contamination can affect experimental results)

    • Activity validation before experimental use

  • Include critical controls in all experiments:

    • Heat-denatured protein controls

    • Mock purifications from non-expressing cells

    • Dose-response relationships to identify potential artifacts

  • Consider the cautionary example from heat shock protein research:

    • Commercial recombinant HSP60 induced TNF-alpha release from macrophages

    • Highly purified preparations with low endotoxin activity showed no such effect

    • This demonstrates how contamination can lead to contradictory findings

• Comprehensive stress response characterization:

  • Test HV60 function across a matrix of environmental conditions:

    Temperature	Light Intensity	Duration	Additional Factors
    23°C (control)	50 μmol m⁻² s⁻¹	1-24h	None
    4°C (cold)	50 μmol m⁻² s⁻¹	1-24h	None
    23°C	800 μmol m⁻² s⁻¹ (high)	1-24h	None
    4°C	800 μmol m⁻² s⁻¹	1-24h	None
    4°C	800 μmol m⁻² s⁻¹	1-24h	+ ROS scavenger
    4°C	800 μmol m⁻² s⁻¹	1-24h	+ CO₂ enrichment

  • Identify specific conditions where HV60 function is most pronounced

  • Distinguish between protective effects under different stress combinations

• Data sharing and collaborative validation:

  • Establish multi-laboratory validation protocols

  • Share detailed methodologies, not just summarized results

  • Create repositories for raw data and experimental protocols

  • Implement pre-registration of experimental designs to reduce publication bias

  • Form collaborative networks to systematically address contradictory findings