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 Concept Videos

Covalently Linked Protein Regulators02:04

Covalently Linked Protein Regulators

Proteins can undergo many types of post-translational modifications, often in response to changes in their environment. These modifications play an important role in the function and stability of these proteins. Covalently linked molecules include functional groups, such as methyl, acetyl, and phosphate groups, and also small proteins, such as ubiquitin. There are around 200 different types of covalent regulators that have been identified.
These groups modify specific amino acids in a protein.
Covalently Linked Protein Regulators02:04

Covalently Linked Protein Regulators

Proteins can undergo many types of post-translational modifications, often in response to changes in their environment. These modifications play an important role in the function and stability of these proteins. Covalently linked molecules include functional groups, such as methyl, acetyl, and phosphate groups, and also small proteins, such as ubiquitin. There are around 200 different types of covalent regulators that have been identified.
These groups modify specific amino acids in a protein.
Protein Complexes with Interchangeable Parts01:57

Protein Complexes with Interchangeable Parts

Groups of proteins may form a complex where each protein in this complex has a different role in the overall execution of the complex’s function. Often some of the proteins in the complex can be replaced by a closely related variant to give a complex that contains many of the same components yet is functionally distinct.
The SCF ubiquitin ligase is a protein complex of five individual proteins. This complex attaches ubiquitin to other target proteins to mark them for degradation. In order to...
Ligand Binding and Linkage00:49

Ligand Binding and Linkage

Allosteric proteins have more than one ligand binding site; the binding of a ligand to any of these sites influences the binding of ligands to the other sites. When a protein is allosteric, its binding sites are called coupled or linked.  In the case of enzymes, the site that binds to the substrate is known as the active site and the other site is known as the regulatory site. When a ligand binds to the regulatory site, this leads to conformational changes in the protein that can influence the...
Ligand Binding and Linkage00:49

Ligand Binding and Linkage

Allosteric proteins have more than one ligand binding site; the binding of a ligand to any of these sites influences the binding of ligands to the other sites. When a protein is allosteric, its binding sites are called coupled or linked.  In the case of enzymes, the site that binds to the substrate is known as the active site and the other site is known as the regulatory site. When a ligand binds to the regulatory site, this leads to conformational changes in the protein that can influence the...
Histone Variants at the Centromere02:30

Histone Variants at the Centromere

Histone variants are the histone proteins with structural and sequence variations. These variants may be regarded as “mutant” forms that replace their canonical histone counterparts in the nucleosomes. Specific post-translational modifications on the histone variants enable further chromatin complexity and regulate tissue-specific gene expression. The most common histone variants are from histone H2A, H2B, and linker histone H1 families. However, several variants of histone H3 variants are also...

You might also read

Related Articles

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

Sort by
Same author

Mapping the dialogue: Decoding alveolar stem-niche interactions.

Proceedings of the National Academy of Sciences of the United States of America·2026
Same author

Architecture and function of holocentric CENP-A-independent inner kinetochores.

Science advances·2026
Same author

Direct RNA Sequencing reveals epitranscriptomic regulation of brain cells and Alzheimer's Disease pathology.

bioRxiv : the preprint server for biology·2026
Same author

Template-driven scaffolding of SCF<sup>FBXO42</sup> regulates PP2A degradation.

Nature·2026
Same author

Ubiquitin Ligase COP1 Suppresses Neuroinflammation by Degrading c/EBPβ in Microglia.

Cell·2026
Same author

cFLIP suppresses caspase-1- and MLKL-independent perinatal lethality driven by auto-processing impaired caspase-8 D387A.

Cell death and differentiation·2025

Related Experiment Video

Updated: Jun 3, 2026

In Vitro Analysis of E3 Ubiquitin Ligase Function
06:06

In Vitro Analysis of E3 Ubiquitin Ligase Function

Published on: May 14, 2021

Modulation of K11-linkage formation by variable loop residues within UbcH5A.

Ivan Bosanac1, Lilian Phu, Borlan Pan

  • 1Department of Structural Biology, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.

Journal of Molecular Biology
|March 15, 2011
PubMed
Summary

This study reveals how the UbcH5A enzyme forms specific ubiquitin chains. Mutating key residues near the active site altered chain formation, favoring K63-linked chains over K11-linked chains.

More Related Videos

A Scalable, Cell-Based Method for the Functional Assessment of Ube3a Variants
06:35

A Scalable, Cell-Based Method for the Functional Assessment of Ube3a Variants

Published on: October 10, 2022

Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates
09:47

Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates

Published on: May 10, 2022

Related Experiment Videos

Last Updated: Jun 3, 2026

In Vitro Analysis of E3 Ubiquitin Ligase Function
06:06

In Vitro Analysis of E3 Ubiquitin Ligase Function

Published on: May 14, 2021

A Scalable, Cell-Based Method for the Functional Assessment of Ube3a Variants
06:35

A Scalable, Cell-Based Method for the Functional Assessment of Ube3a Variants

Published on: October 10, 2022

Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates
09:47

Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates

Published on: May 10, 2022

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Proteomics

Background:

  • Ubiquitination is a crucial post-translational modification involving E1, E2, and E3 enzymes.
  • Mass spectrometry advances enable detailed analysis of ubiquitin linkages.
  • UbcH5A is an E2 enzyme known for forming K11, K48, and K63 ubiquitin chains.

Purpose of the Study:

  • To investigate the mechanisms governing E2 enzyme linkage specificity.
  • To probe the role of UbcH5A in forming specific polyubiquitin chains.
  • To understand how active site residues influence ubiquitin linkage formation.

Main Methods:

  • Crystal structure determination of ubiquitin (Ub) and UbcH5A complex.
  • Structure-guided mutagenesis of UbcH5A.
  • In vitro ubiquitination assays.
  • Quantitative mass spectrometry for linkage analysis.

Main Results:

  • The crystal structure revealed a novel interaction surface on Ub near K11 and UbcH5A active site residues.
  • Mutagenesis of interface residues altered UbcH5A's linkage specificity.
  • Mutated UbcH5A generated more K63-linked chains, reducing K11-linkage synthesis.

Conclusions:

  • Active site residues play a critical role in determining E2 enzyme linkage specificity.
  • E2 enzyme linkage specificity can be modulated through targeted mutagenesis.
  • This provides direct evidence for altering E2 enzyme specificity via active-site modification.