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Related Concept Videos

Protein Kinases and Phosphatases02:54

Protein Kinases and Phosphatases

Proteins undergo chemical modifications that trigger changes in the charge, structure, and conformation of the proteins. Phosphorylation, acetylation, glycosylation, nitrosylation, ubiquitination, lipidation, methylation, and proteolysis are various protein modifications that regulate protein activity. Such modifications are usually enzyme-driven.
Protein kinases
Many proteins in the cell are regulated by phosphorylation, the addition of a phosphate group. A family of enzymes called kinases...
Protein Kinases and Phosphatases02:54

Protein Kinases and Phosphatases

Proteins undergo chemical modifications that trigger changes in the charge, structure, and conformation of the proteins. Phosphorylation, acetylation, glycosylation, nitrosylation, ubiquitination, lipidation, methylation, and proteolysis are various protein modifications that regulate protein activity. Such modifications are usually enzyme-driven.
Protein kinases
Many proteins in the cell are regulated by phosphorylation, the addition of a phosphate group. A family of enzymes called kinases...
Phosphorylation01:02

Phosphorylation

The addition or removal of phosphate groups from proteins is the most common chemical modification that regulates cellular processes. These modifications can affect the structure, activity, stability, and localization of proteins within cells as well as their interactions with other proteins.
During phosphorylation, protein kinases transfer the terminal phosphate group of ATP to specific amino acid side chains of substrate proteins. Serine, threonine, and tyrosine are the most commonly...
Phosphorylation01:02

Phosphorylation

The addition or removal of phosphate groups from proteins is the most common chemical modification that regulates cellular processes. These modifications can affect the structure, activity, stability, and localization of proteins within cells as well as their interactions with other proteins.
During phosphorylation, protein kinases transfer the terminal phosphate group of ATP to specific amino acid side chains of substrate proteins. Serine, threonine, and tyrosine are the most commonly...
ATP and Macromolecule Synthesis01:28

ATP and Macromolecule Synthesis

Biological macromolecules are organic compounds, predominantly composed of carbon atoms. The carbon atoms are covalently bonded with hydrogen, oxygen, nitrogen, and other minor elements. There are four major biological macromolecule classes: carbohydrates, lipids, proteins, and nucleic acids.
Most macromolecules are composed of single subunits, or building blocks, called monomers. The monomers combine with each other using covalent bonds to form larger molecules known as polymers.
Conversion of...
Amplifying Signals via Enzymatic Cascade01:22

Amplifying Signals via Enzymatic Cascade

When a ligand binds to a cell-surface receptor, the receptor's intracellular domain changes shape, which may either activate its enzyme function or allow its binding to other molecules. The initial signal is amplified by most signal transduction pathways. This means that a single ligand molecule can activate multiple molecules of a downstream target. Proteins that relay a signal are most commonly phosphorylated at one or more sites, activating or inactivating the protein. Kinases catalyze the...

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Related Experiment Video

Updated: Jun 21, 2026

Oligopeptide Competition Assay for Phosphorylation Site Determination
09:16

Oligopeptide Competition Assay for Phosphorylation Site Determination

Published on: May 18, 2017

Protein phosphorylation by semisynthesis: from paper to practice.

Lawrence M Szewczuk1, Mary Katherine Tarrant, Philip A Cole

  • 1Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

Methods in Enzymology
|July 28, 2009
PubMed
Summary
This summary is machine-generated.

Investigating protein phosphorylation is challenging due to its reversibility. Protein semisynthesis using nonhydrolyzable phosphonate analogues provides a method to create stable phosphoproteins for studying signaling pathways.

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An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation
07:45

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation

Published on: June 6, 2022

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Last Updated: Jun 21, 2026

Oligopeptide Competition Assay for Phosphorylation Site Determination
09:16

Oligopeptide Competition Assay for Phosphorylation Site Determination

Published on: May 18, 2017

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation
07:45

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation

Published on: June 6, 2022

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Chemical Biology

Background:

  • Phosphorylation is a crucial post-translational modification regulating cellular signaling.
  • The dynamic and reversible nature of phosphorylation complicates the study of specific signaling events.
  • Existing methods struggle to isolate and analyze individual phosphorylation events due to reversibility.

Purpose of the Study:

  • To present protein semisynthesis with phosphonate analogues as a robust method for studying phosphorylation.
  • To enable the creation of site-specific, stoichiometric, and phosphatase-resistant phosphoproteins.
  • To facilitate the discrete examination of complex signaling pathways by controlling modification reversibility.

Main Methods:

  • Utilizing N- and C-terminal synthetic peptides containing nonhydrolyzable phosphonate analogues.
  • Ligating these modified peptides to recombinant proteins of interest.
  • Employing protein semisynthesis to generate stable phosphoprotein analogues.

Main Results:

  • Successfully generated semisynthetic proteins with site-specific, stoichiometric phosphonate modifications.
  • The resulting phosphoprotein analogues are completely resistant to endogenous phosphatase activity.
  • Demonstrated the potential to control stoichiometry, specificity, and reversibility of modifications.

Conclusions:

  • Protein semisynthesis with phosphonate analogues is a powerful technique for functional analysis of signaling pathways.
  • This approach overcomes the limitations imposed by the reversibility of natural phosphorylation.
  • Enables detailed investigation of complex signaling networks by dissecting individual phosphorylation events.