Recombinant Neurospora crassa Mitochondrial import inner membrane translocase subunit tim-50 (tim-50)

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Cat. No.

BT2501426

Source

in vitro E.coli expression system

Synonyms

tim50; NCU02943; Mitochondrial import inner membrane translocase subunit tim50

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Description

Introduction to Recombinant Neurospora crassa Mitochondrial Import Inner Membrane Translocase Subunit Tim-50

Recombinant Neurospora crassa mitochondrial import inner membrane translocase subunit Tim-50 (Tim-50) is a purified protein critical for studying mitochondrial protein import mechanisms. It is a component of the TIM23 complex, a conserved translocase responsible for importing nuclear-encoded preproteins into the mitochondrial matrix and inner membrane . The recombinant form is engineered with a His-tag for enhanced solubility and purification efficiency, enabling structural and functional studies .

Domain Architecture

Transmembrane segment: Tim-50 spans the mitochondrial inner membrane once, anchoring its N-terminal domain in the matrix and exposing a large hydrophilic C-terminal domain to the intermembrane space (IMS) .
Amino acid sequence: The full-length mature protein (residues 39–540) includes an N-terminal His-tag and a 502-residue sequence rich in charged and polar amino acids (e.g., lysine, arginine) .
Conserved regions: A coiled-coil domain and a nuclear localization signal (NLS)-interacting factor (NIF) domain are critical for interactions with Tim23 and preproteins .

Functional Role in Mitochondrial Protein Import

Tim-50 is essential for the TIM23 complex’s ability to transfer preproteins from the translocase of the outer membrane (TOM) to the inner membrane. Key findings include:

Preprotein recognition: Tim-50 cross-links with preproteins arrested at the TOM complex, confirming its role as a receptor in the IMS .
Energy dependence: Depletion of Tim-50 in yeast (Saccharomyces cerevisiae) reduces preprotein import rates by >80%, demonstrating its necessity for maintaining import kinetics .
Membrane potential regulation: Tim-50 stabilizes the TIM23 complex’s interaction with preproteins and preserves the mitochondrial membrane potential during translocation .

Production and Biochemical Properties

The recombinant Tim-50 protein (UniProt ID: Q874C1) is produced under the following conditions :

Expression: Full-length mature protein (residues 39–540) fused to an N-terminal His-tag.
Purification: Affinity chromatography via Ni-NTA resin, followed by anion-exchange chromatography.
Reconstitution: Lyophilized protein is reconstituted in sterile water (0.1–1.0 mg/mL) with 50% glycerol for long-term storage at -80°C.

Critical Notes for Usage

Avoid repeated freeze-thaw cycles to prevent aggregation.
Functional assays require reconstitution in mitochondrial-like buffer systems (e.g., Tris/PBS pH 8.0).

Key Studies

Mechanistic insights into TIM23 function:
- Tim-50’s hydrophilic IMS domain facilitates preprotein transfer from TOM to TIM23, acting as a "gatekeeper" .
- Cross-linking experiments show Tim-50 interacts with preproteins both at the TOM complex and during translocation .
Evolutionary conservation:
- Homologs in yeast (S. cerevisiae) and humans (TIMM50) share structural and functional roles, though N. crassa Tim-50 has a longer N-terminal matrix domain .
Disease relevance:
- Mutations in human TIMM50 are linked to mitochondrial encephalopathy and impaired protein import .

Clinical and Biotechnological Implications

Therapeutic targets: Tim-50’s role in maintaining mitochondrial membrane potential makes it a candidate for addressing diseases linked to mitochondrial dysfunction .
Drug screening: Recombinant Tim-50 enables high-throughput assays to identify inhibitors/activators of the TIM23 pathway .

Product Specs

Form

Lyophilized powder.
Note: While we prioritize shipping the format currently in stock, please specify your preferred format in order notes for customized fulfillment.

Lead Time

Delivery times vary depending on the purchasing method and location. Please contact your local distributor for precise delivery estimates.
Note: All proteins are shipped with standard blue ice packs unless dry ice shipping is requested in advance. Additional fees apply for dry ice shipping.

Notes

Avoid repeated freeze-thaw cycles. Store working aliquots at 4°C for up to one week.

Reconstitution

Centrifuge the vial briefly before opening to collect the contents. Reconstitute the protein in sterile deionized water to a concentration of 0.1-1.0 mg/mL. We recommend adding 5-50% glycerol (final concentration) and aliquoting for long-term storage at -20°C/-80°C. Our standard glycerol concentration is 50%, but this can be adjusted per customer requirements.

Shelf Life

Shelf life depends on storage conditions, buffer composition, temperature, and protein stability. Generally, liquid formulations have a 6-month shelf life at -20°C/-80°C, while lyophilized forms have a 12-month shelf life at -20°C/-80°C.

Storage Condition

Upon receipt, store at -20°C/-80°C. Aliquoting is recommended for multiple uses. Avoid repeated freeze-thaw cycles.

Tag Info

The tag type is determined during the manufacturing process.
Should you require a specific tag, please inform us, and we will prioritize its development.

Synonyms

tim50; NCU02943; Mitochondrial import inner membrane translocase subunit tim50

Buffer Before Lyophilization

Tris/PBS-based buffer, 6% Trehalose.

Datasheet

Please contact us to get it.

Expression Region

39-540

Protein Length

Full Length of Mature Protein

Species

Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987)

Target Names

tim50

Target Protein Sequence

AKDNKPPKFSKPESTPAQKATPKAPEPAESEKAEQKQQQQQQQQTPAESEPEPEIDLSKL PDLRGGIPTTLEYEMAQKEAGKKPVAGEEAETQAEGAEGPEAATSGSGGGGRKKGQLPDS AYVSSTEKRRQKMANWAFIAAGLALVGGTIYLGREWDEEELEKHHDIPNGWGLGLWWKRA KARMTGTVSYYQEPAFEKLLPDPDPSFERPYTLCISLEDMLVHSEWTRDHGWRLAKRPGV DYFLRYLSQYYEIVLFTSVPFANAEPIVRKMDPYRFIMWPLFREATKYKDGEIVKDLSYL NRDLSKVIIIDTDPKHVRAQPENAIVLPKWKGDPKDTELVSLVPFLEFIHTMNFPDVRKV LKSFEGQHIPTEFARREAIARAEHNKLVAAKAKKAGLGSLGARFGIKPSKLNPMAMEGEE DPSEAFAKGKMIQDIARERGMRNYLAMEEEIKKNGEMWLKMEQEAQEKAQKEMMKNMQSS VFGWFGGAPSGEQQSGESEKKA

Uniprot No.

Q874C1

Target Background

Function

Recombinant Neurospora crassa Mitochondrial import inner membrane translocase subunit tim-50 (tim-50) is an essential component of the TIM23 complex. This complex mediates the translocation of transit peptide-containing proteins across the mitochondrial inner membrane. Tim-50 is required to guide preproteins during transit, directing them to the channel protein Tim23 and potentially facilitating the transfer of translocating proteins from the TOM complex to the TIM23 complex.

Database Links

KEGG: ncr:NCU02943

Protein Families

TIM50 family

Subcellular Location

Mitochondrion inner membrane; Single-pass membrane protein.

Q&A

**FAQs for Researchers on Recombinant Neurospora crassa Mitochondrial Import Inner Membrane Translocase Subunit Tim-50 (Tim-50)**

Advanced Research Questions

How do conflicting data on Tim-50’s enzymatic activity across species impact functional interpretations?

While fungal and mammalian Tim-50 lack enzymatic roles, Trypanosoma brucei Tim-50 (TbTim50) exhibits dual specificity phosphatase activity (preferring phosphotyrosine over phosphoserine/threonine) .

Resolution strategy:
- Perform phylogenetic analysis to identify evolutionary divergence.
- Use in vitro phosphatase assays (e.g., pNPP hydrolysis) with recombinant TbTim50 vs. N. crassa Tim-50 .

What experimental approaches resolve Tim-50’s role in presequence recognition vs. channel regulation?

Tim-50’s presequence-binding domain (PBD) is critical for signal-driven translocation. Photo-affinity labeling with engineered presequence probes mapped binding sites to Tim-50’s C-terminal domain .

Key findings:
- Antibodies against Tim-50’s PBD block presequence-driven import in mitoplasts .
- Tim-50 deletion mutants (ΔPBD) fail to bind presequences, causing lethal import defects .

How do contradictory results about Tim-50’s stoichiometry in the TIM23 complex affect mechanistic models?

Tim-50 is substoichiometric in N. crassa and yeast TIM23 complexes, suggesting a dynamic regulatory role rather than a core structural component .

Experimental design:
- Use quantitative immunoblotting with Tim23/Tim17 as internal standards.
- Apply single-molecule imaging (e.g., fluorescence correlation spectroscopy) to assess complex assembly .

Data Tables

Table 1: Comparative Properties of Tim-50 Across Species

Property	N. crassa Tim-50	Yeast Tim-50	T. brucei Tim-50
Molecular Mass (kDa)	56	50	50
Essential for Viability	Yes	Yes	Yes
Enzymatic Activity	None	None	Dual phosphatase
Presequence Binding	Yes	Yes	Not reported

Table 2: Key Experimental Approaches for Studying Tim-50

Method	Application	Example Finding
Cross-linking assays	Mapping preprotein-Tim-50 interactions	Tim-50 binds preproteins at TOM/TIM23
Gene deletion/knockdown	Assessing essentiality	TIM50 deletion is lethal in yeast
In organello import	Measuring import kinetics	Tim-50 depletion reduces import by >70%

Methodological Recommendations

For structural studies: Combine cryo-EM of the TIM23 complex with site-directed mutagenesis of Tim-50’s transmembrane domain.
For functional analysis: Use conditional knockdown strains (e.g., tetracycline-regulated) to study Tim-50’s role in dynamic import processes .
For evolutionary studies: Perform comparative genomics to identify conserved vs. divergent regions in Tim-50 across eukaryotes .

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Recombinant Neurospora crassa Mitochondrial import inner membrane translocase subunit tim-50 (tim-50)

Description

Introduction to Recombinant Neurospora crassa Mitochondrial Import Inner Membrane Translocase Subunit Tim-50

Domain Architecture

Functional Role in Mitochondrial Protein Import

Production and Biochemical Properties

Critical Notes for Usage

Key Studies

Clinical and Biotechnological Implications

Product Specs

Target Background

Q&A

**FAQs for Researchers on Recombinant Neurospora crassa Mitochondrial Import Inner Membrane Translocase Subunit Tim-50 (Tim-50)**

Advanced Research Questions

How do conflicting data on Tim-50’s enzymatic activity across species impact functional interpretations?

What experimental approaches resolve Tim-50’s role in presequence recognition vs. channel regulation?

How do contradictory results about Tim-50’s stoichiometry in the TIM23 complex affect mechanistic models?

Data Tables

Table 1: Comparative Properties of Tim-50 Across Species

Table 2: Key Experimental Approaches for Studying Tim-50

Methodological Recommendations

Quick Inquiry

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