Myristoylation as a general method for immobilization and alignment of soluble proteins for solid-state NMR structural studies.
N-terminal myristoylation of the immunoglobulin-binding domain of protein G (GB1) from group G Streptococcus provides the means to bind the protein to aligned phospholipid bilayers for solid-state NMR structural studies. The myristoylated protein is immobilized by its interactions with bilayers, and the sample alignment enables orientationally dependent 15N chemical shifts and 1H-15N-dipolar couplings to be measured. Spectra calculated for the average solution NMR structure of the protein at various orientations with respect to the magnetic field direction were compared to the experimental spectrum. The best fit identified the orientation of the myristoylated protein on the lipid bilayers, and demonstrated that the protein adopts a similar structure in both its myristoylated and non-myristoylated forms, and that the structure is not grossly distorted by its interaction with the phosholipid bilayer surface or by its location in the restricted aqueous space between bilayer leaflets. The protein is oriented such that its charged sides face the phosphatidylcholine headgroups of the lipids with the single amphiphilic helix running parallel to the bilayer surface.
Mesleh
M F
MF
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093, USA.
Valentine
K G
KG
Opella
S J
SJ
Louis
J M
JM
Gronenborn
A M
AM
D001426:Bacterial Proteins / Q000737:chemistry; D004134:Dimyristoylphosphatidylcholine; D008051:Lipid Bilayers; D008565:Membrane Proteins / Q000737:chemistry*; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009226:Myristates / Q000737:chemistry*; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D017433:Protein Structure, Secondary; D012995:Solubility
D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D008051:Lipid Bilayers; D008565:Membrane Proteins; D009226:Myristates; D009587:Nitrogen Isotopes; D004134:Dimyristoylphosphatidylcholine