Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Videos

Truncations at the NH2 terminus of rhodanese destabilize the enzyme and decrease its heterologous expression

R J Trevino1, T Tsalkova, G Kramer

  • 1Department of Biochemistry, the University of Texas Health Science Center, San Antonio, Texas 78284, USA.

The Journal of Biological Chemistry
|October 17, 1998
PubMed
Summary
This summary is machine-generated.

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Recombinant porcine sequence factor VIII (rpFVIII) for acquired haemophilia A: practical clinical experience of its use in seven patients.

Haemophilia : the official journal of the World Federation of Hemophilia·2016
Same author

Use and Effectiveness of Continuous Subcutaneous Insulin Infusion (CSII) and Multiple Daily Insulin Injection Therapy (MIT) in Children, Adolescents and Young Adults with Type 1 Diabetes Mellitus.

Experimental and clinical endocrinology & diabetes : official journal, German Society of Endocrinology [and] German Diabetes Association·2016
Same author

Metabolic control and hypoglycaemia in people with type 2 diabetes on conventional or intensified insulin therapy: a 22 year retrospective single centre survey.

Diabetes/metabolism research and reviews·2016
Same author

Quantitative proteomics of rat and human pancreatic beta cells.

Data in brief·2015
Same author

A literature review of the association between diffusion-weighted MRI derived apparent diffusion coefficient and tumour aggressiveness in pelvic cancer.

Cancer treatment reviews·2015
Same author

Potential of UCHL1 as biomarker for destruction of pancreatic beta cells.

Journal of proteomics·2015
Same journal

Isotope-Edited ESEEM: A New Method for Probing Copper Binding Sites in Neurodegenerative Proteins.

The Journal of biological chemistry·2026
Same journal

Introduction to the Thematic Review Series on Intracellular Protein Degradation. The ubiquitous biology of intracellular protein degradation: a tribute to Alfred L. ("Fred") Goldberg.

The Journal of biological chemistry·2026
Same journal

Correction: Aromatic residue-rich amino-terminal segments of temporin L self-assemble into collagen-mimetic peptides with cell-adhesion properties.

The Journal of biological chemistry·2026
Same journal

YhbO is a DJ-1 family glyoxalase and α-oxoaldehyde hydratase that confers resistance to reactive carbonyl stress (112).

The Journal of biological chemistry·2026
Same journal

ARMH3 acts as a central scaffold at the Golgi/TGN through interactions with Arl5, GBF1, and PI4KB.

The Journal of biological chemistry·2026
Same journal

PAX8 controls proximal tubule epithelial identity and stress response through epigenetic modification of distal regulatory elements.

The Journal of biological chemistry·2026
See all related articles

N-terminal deletions in rhodanese affect its stability and expression. While the first 11 residues are not essential for folding, they are crucial for an active and stable structure in E. coli.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protein Engineering

Background:

  • Rhodanese is an enzyme involved in cyanide detoxification.
  • The N-terminus of proteins often plays critical roles in folding, stability, and function.
  • Understanding protein stability is crucial for protein engineering and therapeutic applications.

Purpose of the Study:

  • To investigate the role of the N-terminus of rhodanese in protein expression, folding, and stability.
  • To determine the specific contributions of sequential N-terminal deletions to rhodanese structure and function.
  • To elucidate how N-terminal truncations impact the enzyme's active site stability and susceptibility to denaturation.

Main Methods:

  • Construction and expression of sequential N-terminal deletion mutants of rhodanese in Escherichia coli.

Related Experiment Videos

  • Purification and kinetic characterization of rhodanese mutants.
  • Assessment of protein stability using urea denaturation and dithiothreitol inactivation assays.
  • Analysis of protein degradation and reactivation capabilities.
  • In vitro transcription/translation assays at different temperatures to evaluate enzymatic activity.

    • Main Results:

    • The first 11 N-terminal residues are not essential for folding to an active conformation but are necessary for stability in E. coli.
    • Mutants with deletions up to 9 residues showed similar kinetic parameters to wild-type rhodanese.
    • Longer deletions (7-9 residues) resulted in increased inactivation by urea and dithiothreitol, reduced reactivation, and faster inactivation at 37°C.
    • Mutants lacking 10-23 N-terminal amino acids were expressed as inactive species and rapidly degraded.
    • In vitro studies indicated that N-terminal truncations affect the chemical stability of the active site, with activity being temperature-dependent.

    Conclusions:

    • The N-terminus of rhodanese, particularly residues Ser-11 through Gly-22 forming the N-proximal alpha-helix, is critical for achieving an active conformation, resisting degradation during heterologous expression, and maintaining in vitro chemical stability.
    • N-terminal truncations significantly impact the stability of the distantly located active site.
    • These findings provide insights into the structural requirements for rhodanese stability and function, relevant for protein engineering efforts.