Reverse micelle encapsulation of membrane-anchored proteins for solution NMR studies

Abstract

Perhaps 5%-10% of proteins bind to membranes via a covalently attached lipid. Posttranslational attachment of fatty acids such as myristate occurs on a variety of viral and cellular proteins. High-resolution information about the nature of lipidated proteins is remarkably sparse, often because of solubility problems caused by the exposed fatty acids. Reverse micelle encapsulation is used here to study two myristoylated proteins in their lipid-extruded states: myristoylated recoverin, which is a switch in the Ca(2+) signaling pathway in vision, and the myristoylated HIV-1 matrix protein, which is postulated to be targeted to the plasma membrane through its binding to phosphatidylinositol-4,5-bisphosphate. Both proteins have been successfully encapsulated in the lipid-extruded state and high-resolution NMR spectra obtained. Both proteins bind their activating ligands in the reverse micelle. This approach seems broadly applicable to membrane proteins with exposed fatty acid chains that have eluded structural characterization by conventional approaches.

Figure 1. Reverse micelle encapsulation of lipidated proteins for solution NMR

Schematic illustration of the structure of a reverse micelle surfactant assembly hosting a lipidated protein. A ribbon representation of the NMR structure of HIV-1 MA (-myr) (Doyle et al., 1998) is shown. The surfactant CTAB (orange) co-solubilizes with hexanol (green) to form the reverse micelle shell. The myristoylated protein HIV-1 MA inserts into the aqueous interior with the acyl chain extruded into the hydrophobic surfactants. The reverse micelle particle is solvated in a compatible low viscosity solvent such as the short chain alkanes (Wand et al., 1998). This sample is designed to avoid the poor solution behavior of lipidated proteins that often defeats detailed solution NMR studies.

Figure 2. Encapsulation of myristoylated recoverin

¹⁵N-HSQC spectra of recoverin (+myr) in (A) aqueous buffer in the Ca⁺² free apo state; (B) Ca⁺² free recoverin in CTAB/hexanol reverse micelles and (C) Ca⁺² bound state of recoverin in CTAB reverse micelles. The spectra in B and C are the identical sample without and with Ca⁺² collected with the same number of transients, the contour threshold is 5 times lower in spectrum B. The increased sensitivity and spectral dispersion of the Ca⁺² bound state in reverse micelles is a clear demonstration of the folding of encapsulated recoverin(+myr) with the addition of Ca⁺². The boxes include cross peaks that are followed during the titration of Ca⁺² into reverse micelles shown in Figure 3.

Figure 3. Titration of recoverin with Ca⁺² in reverse micelles

The ¹⁵N HSQC spectra of recoverin (+myr) in CTAB/hexanol reverse micelles in the top panels (A) through (E) are titration points following the amide N-H correlations of residues G79 and G115 in the native state with the additions of Ca⁺². The molar ratios of Ca⁺² : recoverin (+myr) are indicated above the panels. The bottom panels (F) through (J) show the disappearance of the indole N-H correlations of the tryptophan sidechain in the disordered apo-state and the appearance of their counterparts in the calcium-saturated state. The molar ratios are the same as in the top panels.

Figure 4. Encapsulation of myristoylated HIV-1 matrix protein

(A) ¹⁵N HSQC spectrum of HIV-1 MA(+myr) in CTAB/hexanol reverse micelles. Assigned amide correlations are labeled. (B) The chemical shift differences between amide NH correlations in aqueous HIV-1 MA (-myr) spectrum minus the corresponding resonances the HIV-1 MA (+myr) protein encapsulated in CTAB/hexanol reverse micelles. the ¹⁵N chemical shift differences have been scaled by a factor of ten to reflect the difference in gyromagnetic ratios of ¹H and ¹⁵N.

Figure 5. Titration of encapsulated HIV-1 MA (+myr) with PI(4,5)P₂

(A) Expansions of the ¹⁵N HSQC spectra of HIV-1 MA(+myr) in CTAB/hexanol reverse micelles illustrating amide N-H correlations that shift upon titration with PI(4,5)P₂. The molar ratios of PI(4,5)P₂: MA(+myr) are color coded as indicated. (B) A ribbon representation of the solution NMR structure of HIV-1 MA(-myr) (Massiah et al., 1994) (PDB code 2HMX) is shown on the right with the backbone colored in red at sites that demonstrate significant shifts in the HSQC spectra on titration with PI(4,5)P₂. Drawn with PyMol (DeLano, 2002).