Calumenin Human, His
Description
Production and Purification
Recombinant Calumenin Human, His is typically expressed in E. coli or HEK293 cells and purified via nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography .
Biochemical Properties
Calcium-Dependent Chaperone Activity
Calumenin exhibits chaperone-like activity in both disordered (apo) and structured (Ca²⁺-bound) states, stabilizing client proteins during ER stress . Pb²⁺ binding disrupts this activity by inducing a less stable conformation .
Regulatory Roles
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Calcium Homeostasis: Modulates SERCA2a activity and ryanodine receptor function, critical for ER calcium storage .
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Disease Links: Overexpression correlates with aggressive cancers and cardiac pathologies .
Research Applications
Calumenin Human, His is widely used to study:
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Calcium Signaling: Structural transitions and partner interactions (e.g., SERCA2a) .
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Heavy Metal Toxicity: Pb²⁺ binding disrupts its chaperone function, linking it to cellular stress .
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Cancer Mechanisms: Its role in cell migration and metastasis .
Comparative Analysis
| Feature | Calumenin Human, His | Native Calumenin |
|---|---|---|
| Structural State | Disordered (apo) → α-helical | Disordered (apo) → α-helical |
| Tag | His tag for purification | Untagged |
| Calcium Affinity | K<sub>d</sub> = 21–201 μM | Similar low-affinity binding |
| Research Use | In vitro assays, binding studies | Physiological context studies |
Future Directions
Current studies focus on:
Product Specs
MGSSHHHHHH SSGLVPRGSH MKPTEKKDRV HHEPQLSDKV HNDAQSFDYD HDAFLGAEEA KTFDQLTPEE SKERLGKIVS KIDGDKDGFV TVDELKDWIK FAQKRWIYED VERQWKGHDL NEDGLVSWEE YKNATYGYVL DDPDPDDGFN YKQMMVRDER RFKMADKDGD LIATKEEFTA FLHPEEYDYM KDIVVQETME DIDKNADGFI DLEEYIGDMY SHDGNTDEPE WVKTEREQFV EFRDKNRDGK MDKEETKDWI LPSDYDHAEA EARHLVYESD QNKDGKLTKE EIVDKYDLFV GSQATDFGEA LVRHDEF.
Q&A
What is the structural basis for calcium-induced folding in human calumenin?
Human calumenin undergoes a disorder-to-order transition upon calcium binding. In its apo state (Ca²⁺-free), HsCalu-1 is intrinsically disordered, as shown by circular dichroism (CD) spectroscopy and small-angle X-ray scattering (SAXS) . Calcium binding triggers a structural rearrangement, forming a compact, α-helical trilobal fold stabilized by six EF-hand motifs. The fourth EF-hand acts as a nucleation site for this transition .
Key methodological approaches:
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CD spectroscopy: Monitors shifts in secondary structure (e.g., increased α-helical content from 15% to 45% upon Ca²⁺ binding) .
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SAXS: Confirms global compaction (radius of gyration decreases from 4.8 nm to 3.2 nm) .
How does calumenin regulate ER calcium homeostasis?
HsCalu-1 interacts with Sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA2a) and ryanodine receptors (RyR) to modulate calcium flux. In its Ca²⁺-bound state, it stabilizes SERCA2a activity, enhancing calcium uptake into the ER .
Experimental validation:
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Co-immunoprecipitation: Demonstrates direct binding between HsCalu-1 and SERCA2a .
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Calcium flux assays: Show reduced ER calcium storage in calumenin-knockdown cell lines .
What experimental strategies are used to study calumenin’s chaperone activity?
Calumenin exhibits chaperone activity in both apo and Ca²⁺-bound states, preventing aggregation of client proteins like citrate synthase .
Methodological workflow:
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Thermal shift assay: Measures melting temperature (T<sub>m</sub>) to assess stability (e.g., T<sub>m</sub> = 67°C for Ca²⁺-bound vs. 43°C for Pb²⁺-bound HsCalu-1) .
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Client protein protection assay: Monitors aggregation kinetics using light scattering at 360 nm .
How does structural plasticity enable calumenin’s moonlighting functions?
HsCalu-1 adopts quasi-stable conformations to interact with diverse partners. For example, Pb²⁺ binding to EF-hand 2–4 induces a compact but less stable structure, attenuating chaperone activity while preserving calcium-sensing roles .
Comparative structural data:
| Condition | Secondary Structure (%) | T<sub>m</sub> (°C) | Chaperone Efficiency |
|---|---|---|---|
| Apo (Ca²⁺-free) | 15% α-helix | 35 | 70% |
| Ca²⁺-bound | 45% α-helix | 67 | 85% |
| Pb²⁺-bound | 30% α-helix | 43 | 50% |
| Data sourced from |
What explains contradictory reports on calumenin’s calcium affinity?
Discrepancies arise from species-specific variations. Vertebrate calumenin (e.g., human) requires Ca²⁺ for folding, while invertebrate homologs (e.g., C. elegans CeCalu) are structured even in the apo state. A single residue difference (Leu150 in humans vs. Gly150 in invertebrates) governs this divergence .
Mutational analysis:
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L150G HsCalu-1 mutant: Adopts a folded structure without Ca²⁺, mimicking invertebrate behavior .
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Evolutionary significance: Leu150 emerged in vertebrates, correlating with the development of cardiac systems .
How does calumenin contribute to disease pathways?
Calumenin dysregulation is implicated in:
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Heavy metal toxicity: Pb²⁺ competes with Ca²⁺ at EF-hand 2–4, disrupting chaperone activity .
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Cancer: Overexpression in glioblastoma linked to ER stress survival .
Functional assays:
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RNAi knockdown: Reduces tumor spheroid formation in glioblastoma cell lines .
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Electrophysiology: Prolongs calcium transients in cardiomyocytes .
Addressing Data Contradictions
| Conflict | Resolution Strategy | Example Study |
|---|---|---|
| Apo-state disorder vs. structure | Species-specific mutagenesis | L150G mutant |
| Chaperone activity modulation | Ion substitution experiments | Pb²⁺ vs. Ca²⁺ |
Future Research Directions
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Structural dynamics: Time-resolved crystallography of HsCalu-1 during Ca²⁺ binding.
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In vivo models: Tissue-specific knockout mice to dissect roles in cardiac vs. neural tissues.
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Therapeutic targeting: Screen small molecules stabilizing HsCalu-1’s chaperone activity.
Product Science Overview
Recombinant human calumenin, particularly the variant with a His tag, is produced using mammalian expression systems, such as HEK 293 cells . This recombinant protein typically has a high purity level, often exceeding 90% as determined by SDS-PAGE . The His tag, usually located at the C-terminus, facilitates purification and detection of the protein .
The recombinant human calumenin protein consists of 303 amino acids and has a predicted molecular mass of approximately 35.9 kDa. However, it migrates as a band of around 46-52 kDa in SDS-PAGE under reducing conditions .
Calumenin plays a crucial role in the calcium homeostasis within the ER. It contributes to the regulation of calcium levels, which is essential for various cellular functions, including muscle contraction, enzyme activity, and signal transduction . Additionally, calumenin is involved in the quality control of protein folding and sorting within the ER, ensuring that only properly folded proteins are transported to their final destinations .
