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Updated: Aug 13, 2026

Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins
12:47

Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins

Published on: December 27, 2016

Chemical shift assignments and folding topology of the Ras-binding domain of human Raf-1 as determined by

S D Emerson1, D S Waugh, J E Scheffler

  • 1Roche Research Center, Hoffmann-La Roche, Inc., Nutley, New Jersey 07110.

Biochemistry
|June 28, 1994
PubMed

Insights

The Raf55-132 protein fragment binds to Ras, a key component in cell growth signaling. Structural analysis reveals Raf55-132 folds into a distinct domain, surprisingly similar to ubiquitin.

Area of Science:

  • Molecular biology
  • Biochemistry
  • Structural biology

Background:

  • Raf-1 is a serine-threonine kinase and a downstream target of Ras in cell growth pathways.
  • Residues 51-131 of Raf mediate direct interaction with Ras.

Purpose of the Study:

  • To express and purify the Raf55-132 fragment.
  • To characterize the binding of Raf55-132 to Ras.
  • To determine the 3D structure of the Raf55-132 domain.

Main Methods:

  • Bacterial expression of Raf55-132 as a fusion protein.
  • Purification via affinity chromatography and protease cleavage.
  • Nuclear Magnetic Resonance (NMR) spectroscopy for structural determination.

Main Results:

  • Raf55-132 was successfully expressed, purified, and cleaved.
  • The Raf55-132 fragment demonstrated high-affinity binding to Ras.
  • NMR analysis revealed Raf55-132 is an independently folded domain with a five-stranded beta-sheet and alpha-helix.

Conclusions:

  • The Raf55-132 fragment represents a distinct, independently folded domain.
  • The structure of Raf55-132 shares similarities with ubiquitin despite low sequence homology.
  • This study provides structural insights into Ras-Raf interactions.

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