Determination of the Oligomeric Number and Intermolecular Distances of Membrane Protein Assemblies by Anisotropic ¹H-Driven Spin Diffusion NMR Spectroscopy

Abstract

Determination of the high-resolution quaternary structure of oligomeric membrane proteins requires knowledge of both the oligomeric number and intermolecular distances. The centerband-only detection of exchange (CODEX) technique has been shown to enable the extraction of the oligomeric number through the equilibrium exchange intensity at long mixing times. To obtain quantitative distances, we now provide an analysis of the mixing-time-dependent CODEX intensities using the 1H-driven spin diffusion theory. The exchange curve is fit to a rate equation, where the rate constants are proportional to the square of the dipolar coupling and the spectral overlap integral between the exchanging spins. Using a number of 13C- and 19F-labeled crystalline model compounds with known intermolecular distances, we empirically determined the overlap integrals of 13C and 19F CODEX for specific spinning speeds and chemical shift anisotropies. These consensus overlap integral values can be applied to structurally unknown systems to determine distances. Applying the 19F CODEX experiment and analysis, we studied the transmembrane peptide of the M2 protein (M2TMP) of influenza A virus bound to 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine bilayers. The experiment proved for the first time that M2TMP associates as tetramers in lipid bilayers, similar to its oligomeric state in detergent micelles. Moreover, the nearest-neighbor interhelical F-F distance between (4-19F)Phe30 is 7.9-9.5 angstroms. This distance constrains the orientation and the packing of the helices in the tetrameric bundle and supports the structural model derived from previous solid-state NMR 15N orientational data. Thus, the CODEX technique presents a general method for determining the oligomeric number and intermolecular distances in the approximately 10 angstroms range in membrane proteins and other complex biological assemblies.

Alamethicin Supramolecular Organization in Lipid Membranes from 19F Solid-State NMR

“…The typical radiofrequency field strengths were 70 kHz for 19 F and 50 kHz for 1 H. The recycle delay was 1 s. The 19 F chemical shifts were referenced to the Teflon 19 F signal at À122 ppm. 19 F-CODEX experiments were performed at a 15 kHz MAS speed as described by Luo and Hong (38). The normalized intensity, S/S 0 , was measured as a function of the mixing time until it reached a plateau.…”

“…The CODEX technique was originally developed to determine the number of aggregates and the intramolecular distances within the aggregated molecules, and it has been successfully applied to study the antimicrobial peptide protegrin-1 (54) and the transmembrane region of the influenza M2 protein in lipid membranes (38). The CODEX method is based on the proton-driven diffusion of 19 F-spin polarization under MAS conditions.…”

“…3). By homogeneously dispersing the 3,5-bis(trifluoromethyl)benzenesulfonyl compound in p-toluenesulfonyl chloride, one can ensure that intermolecular CF 3 -CF 3 couplings are negligible, whereas such second-moment contributions exceed the nearest-neighbor interactions 43-fold in 4 0 -19 F-2 0 -nitroacetanilide powder (38). Notably, the much larger overlap integral of the CF 3 groups indicates that due to the cumulated interactions between two pairs of chemically equivalent 19 F, the range of distances can be extended in comparison with a single 19 F on an aromatic ring system, where F(0) is 30-40 ms and distances are typically limited to <15 Å (38,39).…”

“…When it is possible to freeze out such motion, the only mechanism of spin exchange is spin diffusion. Under such conditions, the CODEX technique allows one to elucidate the aggregate structure through distances of up to 15 Å between specifically 19 F-labeled residues (38,39). In the same experiment, the number of spatially clustered 19 F spins can also be determined on the basis of their orientational inequivalence, thereby indicating the number of subunits in the aggregate structure (N).…”

“…Here, we conducted a 19 F MAS solid-state NMR investigation of ALM9 to study its aggregation in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) phospholipid membranes using the CODEX approach (38). The primary structures of ALM F50/5, ALM F30/3, and ALM9 are shown in Fig.…”