NGB Human, His

What is the structural relationship between histidine residues and heme in human neuroglobin?

Human neuroglobin exhibits unique structural properties involving histidine coordination with the heme group. Under normal conditions, neuroglobin exists as a hexacoordinated hemoprotein, where both the proximal and distal positions of the heme iron are coordinated by histidine residues (typically His64 and His96) . This bis-histidine coordination is critical for neuroglobin's function and distinguishes it from other globins like hemoglobin and myoglobin, which are pentacoordinated in their deoxy forms.

Methodologically, researchers can study this coordination using various spectroscopic techniques, including NMR spectroscopy, which has proven effective for probing ligand binding properties and structural characteristics. For instance, 1H NMR spectroscopy has been used to analyze cyanide binding properties of different Ngb species in solution, revealing important insights about heme orientation and histidine coordination .

How does oxidative stress influence the histidine coordination in human neuroglobin?

During oxidative stress, human neuroglobin undergoes significant conformational changes that affect its histidine coordination. Specifically, the ferrous oxygen-bound form that exists under normoxia is converted to the ferric bis-His conformation during oxidative stress, inducing large tertiary structural changes . This transformation is central to neuroglobin's stress-responsive function.

Researchers investigating this phenomenon typically employ redox manipulation protocols combined with spectroscopic analysis. The experimental approach often involves exposing neuroglobin to oxidizing agents while monitoring spectral changes that indicate shifts in histidine coordination. Additionally, site-directed mutagenesis targeting key histidine residues can provide valuable insights into the specific roles of different histidine positions in the oxidative stress response .

What experimental models are most effective for studying neuroglobin expression and function?

For studying human neuroglobin, several experimental models have proven effective. The SH-SY5Y neuroblastoma cell line is frequently used for in vitro studies, as demonstrated in research examining the effects of overexpressed NGB on neural cell functions .

The development of stable transfected cell lines expressing neuroglobin provides a reliable system for functional studies. According to research protocols, this can be achieved through transfection with Lipofectamine Plus reagent following manufacturer's instructions. After approximately 3 weeks of selection with 400 μg/mL G418, single clones can be isolated and screened for their capability to express human NGB .

For plasmid construction, the human NGB ORF (NCBI Reference Sequence: NM_021257.3) can be subcloned using appropriate primers and conventional PCR techniques. The resulting PCR fragment can then be inserted into suitable restriction sites of expression vectors like pcDNA3.1-FLAG .

How does heme orientation heterogeneity affect histidine dissociation and ligand binding in human neuroglobin?

Human neuroglobin exhibits heme heterogeneity with two alternative heme orientations within the heme pocket, which significantly impacts its functional properties. Research has demonstrated that in the disulfide-containing wild-type protein, cyanide ligation is fivefold faster for one of the two heme orientations (the A isomer) compared with the other isomer . This difference is attributed to the lower stability of the distal His64–iron bond and reduced steric hindrance at the bottom of the cavity for heme sliding in the A isomer configuration.

Methodologically, researchers can investigate this phenomenon using 1H NMR spectroscopy to probe ligand binding properties of different Ngb species, including wild-type Ngb and various mutants (e.g., single C120S and triple C46G/C55S/C120S mutants) . This approach allows for the detailed characterization of binding kinetics as influenced by heme orientation and cysteine oxidation state.

What molecular mechanisms underlie the interaction between ferric bis-His neuroglobin and G proteins during oxidative stress?

During oxidative stress, ferric bis-His neuroglobin engages in specific protein-protein interactions that contribute to its neuroprotective function. Research has shown that ferric bis-His Ngb, but not ferrous ligand-bound Ngb, specifically binds to flotillin-1 (a lipid raft microdomain-associated protein) and α-subunits of heterotrimeric G proteins (Gα(i/o)) . Moreover, human ferric bis-His Ngb acts as a guanine nucleotide dissociation inhibitor for Gα(i/o) that has been modified by oxidative stress.

To study these interactions, researchers can employ co-immunoprecipitation assays, fluorescence resonance energy transfer (FRET), or surface plasmon resonance (SPR) techniques. Additionally, site-directed mutagenesis targeting specific residues involved in G protein binding can help elucidate the structural basis of these interactions. The functional consequences can be assessed by measuring downstream signaling events, such as changes in cAMP concentration, which has been shown to be regulated by Ngb under oxidative stress conditions .

How do disulfide bridges modulate histidine coordination and ligand affinity in human neuroglobin?

A distinctive feature of most mammalian neuroglobins is their ability to form an internal disulfide bridge that increases ligand affinity . This redox-dependent structural element interplays with histidine coordination to modulate neuroglobin's functional properties.

Research approaches to investigate this phenomenon include:

• Site-directed mutagenesis to generate cysteine variants (e.g., C120S single mutant or C46G/C55S/C120S triple mutant)

• Comparative analysis of ligand binding kinetics under different redox conditions

• Structural characterization using X-ray crystallography or NMR to determine how disulfide formation affects the positioning of histidine residues relative to the heme group

These approaches can reveal how the redox state of cysteine residues influences the stability of histidine-iron bonds and consequently affects ligand binding properties.

What proteomics approaches can reveal the impact of neuroglobin overexpression on cellular functions?

Proteomic analysis offers powerful insights into how neuroglobin overexpression affects cellular pathways. A comprehensive approach involves shotgun label-free quantitative proteomics, as demonstrated in research examining NGB-FLAG cells .

The methodological workflow typically includes:

• Stable transfection of cells with NGB expression constructs

• Protein extraction and processing for mass spectrometry

• Label-free quantification (LFQ) of protein abundance

• Statistical analysis using appropriate software (e.g., MetaboAnalyst 5.0)

• Bioinformatic treatment of proteomics data using tools like FunRich to reveal functional enrichment of biological processes

This approach has revealed alterations in processes such as transport, cytoskeleton organization, and bioenergetic pathways in neural cells overexpressing neuroglobin .

What are the optimal PCR protocols for studying neuroglobin gene expression?

For quantitative analysis of neuroglobin gene expression and related mitochondrial genes, real-time PCR can be employed using specific primer sets. Based on research protocols, the following primer sequences have been validated for related studies:

These primers can be used to amplify specific regions of interest for quantitative analysis of gene expression related to neuroglobin and its effects on cellular function .

How can researchers effectively distinguish between different conformational states of neuroglobin?

Distinguishing between different conformational states of neuroglobin, particularly regarding histidine coordination, requires specialized spectroscopic techniques. The ferrous oxygen-bound form and the ferric bis-His conformation exhibit distinct spectral signatures that can be detected using various approaches.

Methodologically, researchers can employ:

• UV-visible spectroscopy to monitor changes in the heme absorption spectrum associated with different coordination states

• Resonance Raman spectroscopy to examine vibrational modes specific to different histidine-iron coordination configurations

• Electron paramagnetic resonance (EPR) to characterize the electronic state of the heme iron

• NMR spectroscopy to analyze structural features at atomic resolution, as demonstrated in studies of cyanide binding to different neuroglobin variants

These techniques, used individually or in combination, provide complementary information about the conformational dynamics of neuroglobin under different conditions.

How does the conversion between ferrous and ferric states of neuroglobin regulate its neuroprotective function?

The redox state of neuroglobin critically influences its neuroprotective capacity. Research has shown that the ferrous oxygen-bound form exists under normoxia, while oxidative stress triggers conversion to the ferric bis-His conformation . This conversion induces large tertiary structural changes that enable specific protein-protein interactions.

Functionally, ferric bis-His Ngb binds to flotillin-1 and Gα(i/o), acting as a guanine nucleotide dissociation inhibitor for Gα(i/o) that has been modified by oxidative stress . This interaction inhibits the decrease in cAMP concentration that occurs under oxidative stress, ultimately protecting against cell death.

Researchers can investigate this mechanism by:

• Using redox-sensitive fluorescent probes to monitor changes in the cellular redox environment

• Measuring changes in cAMP levels using ELISA or other quantitative assays

• Assessing cell viability under oxidative stress conditions with and without neuroglobin expression

• Employing mutated Ngb proteins that cannot form the bis-His conformation to demonstrate the essential nature of these oxidative stress-induced structural changes

What role does neuroglobin play in mitochondrial function and energy metabolism?

Proteomic and bioinformatic analyses have revealed that neuroglobin overexpression affects bioenergetic pathways in neural cells . Functional validation has demonstrated that neural cells with increased neuroglobin expression show positive regulation of energy metabolism, mitochondrial health, and lysosomal pathways.

The relationship between mitochondrial turnover, mitochondrial mass, and cell survival appears to be influenced by neuroglobin levels . This suggests a potential role for neuroglobin in maintaining mitochondrial homeostasis, particularly under stress conditions.

Research approaches to investigate this relationship include:

• Assessment of mitochondrial membrane potential using fluorescent dyes

• Measurement of ATP production and oxygen consumption rates

• Analysis of mitochondrial morphology and dynamics through live-cell imaging

• Quantification of mitochondrial mass and turnover rates

• Evaluation of reactive oxygen species production under different conditions

These methodologies provide a comprehensive view of how neuroglobin influences cellular energetics and mitochondrial function.

How might targeted manipulation of neuroglobin's histidine coordination be exploited for neuroprotective therapies?

Understanding the structural basis of neuroglobin's neuroprotective function, particularly the role of histidine coordination, provides potential avenues for therapeutic intervention. Since the oxidative stress-induced structural changes involving bis-His coordination are essential for neuroprotective activity , strategies that stabilize or promote this conformation might enhance neuroglobin's protective effects.

Potential research approaches include:

• Development of small molecules that selectively bind to neuroglobin and modulate histidine-iron coordination

• Design of peptide mimetics that target interaction sites between neuroglobin and its binding partners

• Gene therapy approaches to deliver modified neuroglobin variants with enhanced stress-responsive properties

• Screening for compounds that influence the redox state of neuroglobin and its disulfide bridge formation

These translational research directions could lead to novel therapeutic strategies for neurodegenerative conditions where oxidative stress plays a significant role.

What are the implications of neuroglobin research for understanding human neurological disorders?

Neuroglobin's role as an oxidative stress-responsive sensor with neuroprotective functions suggests its potential involvement in various neurological disorders characterized by oxidative stress and mitochondrial dysfunction.

Research in this area might explore:

• Changes in neuroglobin expression and function in neurodegenerative diseases such as Alzheimer's, Parkinson's, and stroke

• Correlation between neuroglobin levels and clinical outcomes in patients with neurological disorders

• Genetic variations in the neuroglobin gene and their association with disease susceptibility or progression

• The interplay between neuroglobin and other neuroprotective mechanisms in the context of specific pathological conditions

Understanding these relationships could provide valuable insights into disease mechanisms and potentially identify neuroglobin as a biomarker or therapeutic target for neurological disorders.