Related Experiment Videos
Location of M13 coat protein in sodium dodecyl sulfate micelles as determined by NMR
C H Papavoine1, R N Konings, C W Hilbers
1Laboratory of Biophysical Chemistry, NSR Center, University of Nijmegen, The Netherlands.
Abstract:
The major coat protein (gVIIIp) of bacteriophage M13 solubilized in sodium dodecyl sulfate (SDS) detergent micelles was used as a model system to study this protein in the lipid-bound form. In order to probe the position of gVIIIp relative to the SDS micelles, stearate was added, spin-labeled at the 5- or 16-position with a doxyl group containing a stable nitroxide radical. The average position of the spin-labels in the micelles was derived from the line broadening of the resonances in the 13C spectrum of SDS. Subsequently, we derived a model of the relative position of gVIIIp in the SDS micelle from the effect of the spin-labels on the gVIIIp resonances, monitored via 1H-15N HSQC and TOCSY experiments. The results are consistent with the structure of gVIIIp having two helical strands. One strand is a long hydrophobic helix that spans the micelle, and the other is a shorter amphipathic helix on the surface of the micelle. These results are in good agreement with the structure of gVIIIp in membranes proposed by McDonnell et al. on the basis of solid state NMR data [McDonnell, P. A., Shon, K., Kim, Y., & Opella, S. J. (1993) J. Mol. Biol. 233, 447-463]. This study indicates that high-resolution NMR on this membrane protein, solubilized in detergent micelles, is a very suitable technique for mimicking these proteins in their natural environment. Furthermore, the data indicate that the structure of the micelle near the C-terminus of the major coat protein is distorted.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Researchers studied the bacteriophage M13 major coat protein (gVIIIp) in detergent micelles. Using spin-labeled stearate and NMR, they determined gVIIIp has two helical strands, one spanning the micelle and another on the surface.
Area of Science:
- Structural Biology
- Biophysics
- Membrane Protein Chemistry
Background:
- The major coat protein (gVIIIp) of bacteriophage M13 is a model for studying membrane proteins.
- Understanding gVIIIp's structure in a lipid-bound form is crucial for membrane protein research.
Purpose of the Study:
- To determine the structural model of bacteriophage M13 major coat protein (gVIIIp) within sodium dodecyl sulfate (SDS) micelles.
- To investigate the positioning of gVIIIp relative to SDS micelles using spin-labeling techniques.
Main Methods:
- Utilized spin-labeled stearate at specific positions (5- or 16-) with a doxyl group.
- Analyzed 13C NMR spectra of SDS for spin-label position determination.
- Employed 1H-15N HSQC and TOCSY experiments to monitor gVIIIp resonances and derive structural models.
Main Results:
- Established a model for gVIIIp structure within SDS micelles, revealing two helical strands.
- Identified a long hydrophobic helix spanning the micelle and a shorter amphipathic helix on the micelle surface.
- Observed micelle distortion near the C-terminus of gVIIIp.
Conclusions:
- High-resolution NMR of detergent-solubilized membrane proteins is a suitable method for mimicking their natural membrane environment.
- The determined structure of gVIIIp in SDS micelles aligns with previous findings in biological membranes.
- The study provides insights into the structural organization of viral coat proteins in lipidic environments.