Related Experiment Video

Updated: Jul 4, 2026

Deciphering the Structural Effects of Activating EGFR Somatic Mutations with Molecular Dynamics Simulation
15:05

Deciphering the Structural Effects of Activating EGFR Somatic Mutations with Molecular Dynamics Simulation

Published on: May 20, 2020

Effect of mutations mimicking phosphorylation on the structure and properties of human 14-3-3zeta

Nikolai N Sluchanko1, Ivan S Chernik, Alim S Seit-Nebi

  • 1Department of Biochemistry, School of Biology, Moscow State University, Lenin Hills 1, Building 12, Moscow 119991, Russian Federation.

Archives of Biochemistry and Biophysics
|June 19, 2008
PubMed

Insights

Mimicking phosphorylation via mutations in human 14-3-3zeta protein alters its structure and stability. Ser58 mutations destabilized the protein, while Ser184 mutations enhanced its stability.

Area of Science:

  • Biochemistry
  • Structural Biology
  • Molecular Biology

Background:

  • 14-3-3 proteins are crucial regulators of cellular processes.
  • Phosphorylation is a key post-translational modification affecting protein function.
  • Understanding how phosphorylation affects 14-3-3zeta structure is vital for comprehending its regulatory roles.

Purpose of the Study:

  • To investigate the structural and stability effects of mutations mimicking phosphorylation on human 14-3-3zeta protein.
  • To analyze the impact of specific serine residue mutations (S58E, S184E, T232E) on 14-3-3zeta properties.

Main Methods:

  • Site-directed mutagenesis to introduce phosphomimetic mutations (S58E, S184E, T232E).
  • Intrinsic tryptophan fluorescence spectroscopy to assess protein conformation.
  • Bis-ANS binding assays to evaluate protein surface hydrophobicity.
  • Size-exclusion chromatography to determine Stokes radius.
  • Differential scanning fluorimetry for thermal stability analysis.
  • Limited proteolysis to assess protein susceptibility.

Main Results:

  • Mutation S58E increased intrinsic fluorescence and bis-ANS binding, indicating conformational changes and increased hydrophobicity.
  • At low concentrations, S58E mutation promoted 14-3-3zeta dissociation and proteolysis, suggesting destabilization.
  • Mutation S184E slightly increased Stokes radius and thermal stability, indicating stabilization.
  • Mutation T232E caused minor changes in Stokes radius and sedimentation coefficient.
  • The triple mutant (S58E/S184E/T232E) showed concentration-dependent dissociation and high susceptibility to proteolysis.

Conclusions:

  • Mimicking phosphorylation at Ser58 destabilizes the human 14-3-3zeta structure, increasing its susceptibility to proteolysis.
  • Mimicking phosphorylation at Ser184 stabilizes the human 14-3-3zeta structure.
  • The effects of phosphomimetic mutations are complex and can be concentration-dependent.

More Related Videos

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay12:26

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Published on: May 3, 2018

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...
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...
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.
Mutations01:39

Mutations

Overview
Mutations01:35

Mutations

Mutations are changes in the sequence of DNA. These changes can occur spontaneously or they can be induced by exposure to environmental factors. Mutations can be characterized in a number of different ways: whether and how they alter the amino acid sequence of the protein, whether they occur over a small or large area of DNA, and whether they occur in somatic cells or germline cells.
Chromosomal Alterations Are Large-Scale Mutations
While point mutations are changes in a single nucleotide in...
PI3K/mTOR/AKT Signaling Pathway01:22

PI3K/mTOR/AKT Signaling Pathway

The mammalian target of rapamycin  (mTOR) is a serine/threonine kinase that regulates growth, proliferation, and cell survival in response to hormones, growth factors, or nutrient availability. This kinase exists in two structurally and functionally distinct forms: mTOR complex 1  (mTORC1) and mTOR complex 2  (mTORC2). The first form (mTORC1) is composed of a rapamycin-sensitive Raptor and proline-rich Akt substrate, PRAS40. In contrast,  mTORC2 consists of a rapamycin-insensitive companion...