Location of M13 Coat Protein in Sodium Dodecyl Sulfate Micelles As Determined by NMR

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)

NMR Studies of the Major Coat Protein of Bacteriophage M13

“…The 5-doxylstearate spinlabel also has a significant effect on residues 36 and 37, while residue 38 is also influenced by 16-doxylstearate. These same effects have also been observed in a similar study on the location of gVIIIp in SDS micelles and several explanations have been suggested to account for these effects (Papavoine et al, 1994). In that study, a model was proposed in which the micellar environment around the C-terminal part of protein is distorted.…”

“…The experiments performed to determine the location of gVIIIp in the DodPCho micelles have been described in detail in an earlier paper (Papavoine et al, 1994). Two detergent-like spin-labelled compounds were used, 5-doxylstearate and 16-doxylstearate (Aldrich), which were solubilized in [2H,]methano1 (Merck) to a final concentration of 0.1 M. The spin-labels differ in the location of the nitroxide radical relative to the alkyl chain of the stearate.…”

“…As the experiments and interpretation of the data are described in detail in an earlier study on the location of the major coat protein solubilized in SDS micelles (Papavoine et al, 1994), only the main results will be mentioned in the present paper.…”

“…From the latter study, a secondary structure was obtained which consisted of two a-helical strands, connected by a hinge region around residue 22. With the help of spin-label experiments, we proceeded to locate these two ahelices relative to the micelle structure (Papavoine et al, 1994). One hydrophobic helix (residues 24-46) spans the micelle, while the other is an amphipathic helix (residues 6-20) near the surface of the micelle.…”

The orientation and dynamics of substance P in lipid environments

“…Previous work has established that the 5-doxy1 moiety of the lipid 5-doxy1 stearic acid is located close to the surface of both SDS and DPC micelles (Brown, 1981;Mendz et al, 1988Mendz et al, , 1991Papavoine et al, 1994;Van den Hooven et al, 1996). The unpaired electron of the spin label broadens protons within -15 A sphere via electron-dipole nuclear-dipole interactions.…”