Related Experiment Video
Updated: Jul 19, 2026
Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels
Published on: July 4, 2016
Characterization of the linker 2 region in human vimentin using site-directed spin labeling and electron paramagnetic
John F Hess1, Madhu S Budamagunta, Rebecca L Shipman
1Department of Cell Biology and Human Anatomy, School of Medicine, University of California, Davis, California 95616, USA.
Abstract:
Site-directed spin labeling and electron paramagnetic resonance were used to probe residues 281-304 of human vimentin, a region that has been predicted to be a non-alpha-helical linker and the beginning of coiled-coil domain 2B. Though no direct test of linker structure has ever been made, this region has been hypothesized to be flexible with the polypeptide chains looping away from one another. EPR analysis of spin-labeled mutants indicates that (a) several residues reside in close proximity, suggesting that adjacent linker regions in a dimer run in parallel, and that (b) the polypeptide backbone is relatively rigid and inflexible in this region. However, this region does not show the characteristics of a coiled-coil as has been identified elsewhere in the molecule. Within this region, spectra from positions 283 and 291 are unique from all others thus far examined. These positions, predicted to be in a noncoiled-coil structure, display a significantly stronger interaction than the a-d contact positions of coiled-coil regions. Analysis of the early stages of assembly by dialysis from 8 M urea and progressive thermal denaturation shows the close apposition and structural rigidity at residues 283 and 291 occurs very early in assembly and with a relatively sudden onset, well before coiled-coil formation in other parts of the molecule. These features are inconsistent with hypotheses that envision the linkers as flexible regions, or as looping away from one another, and raise the possibility that the linker may be the site at which dimer alignment and/or formation is initiated. Spin labels placed further downstream yield spectra suggesting that the first regular heptad of rod domain 2 begins at position 302. In conjunction with our previous characterization of region 305-336 and the solved structure of rod 2B from 328-405, the full extent of coiled-coil domain in rod 2B is now known, spanning from vimentin positions 302-405.
Insights
Human vimentin linkers are rigid and parallel, not flexible. This suggests they initiate dimer formation, challenging previous hypotheses about vimentin assembly.
Area of Science:
- Biochemistry
- Structural Biology
- Biophysics
Background:
- Vimentin is a type III intermediate filament protein crucial for cellular structure.
- Residues 281-304 of vimentin were predicted to be a flexible linker preceding coiled-coil domain 2B.
- Previous hypotheses suggested this linker region allows polypeptide chains to loop away from each other.
Purpose of the Study:
- To investigate the structure and flexibility of the vimentin linker region (residues 281-304).
- To determine the role of this region in vimentin assembly and dimer formation.
- To precisely define the boundaries of coiled-coil domain 2B in vimentin.
Main Methods:
- Site-directed spin labeling to introduce paramagnetic probes at specific vimentin residues.
- Electron paramagnetic resonance (EPR) spectroscopy to analyze the local environment and dynamics of the spin labels.
- Analysis of vimentin assembly and denaturation under varying conditions (urea, thermal denaturation).
Main Results:
- EPR analysis revealed that adjacent linker regions in a vimentin dimer run in parallel and the backbone is relatively rigid.
- Residues 283 and 291 showed unique spectral characteristics, indicating strong interactions inconsistent with a flexible, looping structure.
- These interactions and rigidity were observed early in assembly, preceding coiled-coil formation.
- The coiled-coil domain 2B was confirmed to span vimentin positions 302-405.
Conclusions:
- The vimentin linker region (281-304) is structurally rigid and parallel, contradicting flexible linker hypotheses.
- The linker region's early structural rigidity suggests it may initiate vimentin dimer alignment and formation.
- The complete boundaries of vimentin coiled-coil domain 2B have been established from position 302 to 405.
More Related Videos
10:28Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy (NMR) and Microscale Thermophoresis (MST)
Published on: November 2, 2018
07:24Paramagnetic Relaxation Enhancement for Detecting and Characterizing Self-Associations of Intrinsically Disordered Proteins
Published on: September 23, 2021