Phage-Induced Alignment of Membrane Proteins Enables the Measurement and Structural Analysis of Residual Dipolar Couplings with Dipolar Waves and λ-Maps

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Alignment is an essential component of contemporary protein NMR studies, especially for membrane proteins whose highly asymmetric structures are dominated by α-helices or β-sheets and are difficult to characterize based on short-range distance or intramolecular angle measurements. Solid-state NMR can take advantage of the immobilization and complete alignment of the protein in mechanically or magnetically aligned phospholipid bilayers.1 In contrast, weak alignment of detergent solubilized membrane proteins preserves the rapid molecular reorientation required for structure determination by solution NMR. Structure determination by solution NMR is complementary to that by X-ray diffraction of membrane proteins crystallized from the same types of detergent solutions, and it has the advantage that it can be performed on proteins that are resistant to crystallization.

Residual dipolar couplings (RDCs)2 are the principal source of structural constraints in solution NMR studies of membrane proteins because of the near-total absence of observable long-range NOEs, and the requirement of paramagnetic relaxation enhancement measurements for multiple mutations and chemical modifications.3 Unfortunately, the most widely used media for the alignment of globular proteins cannot be applied to membrane proteins, since they are immobilized by interactions with the long chain lipids in bicelles and the solubilizing detergents destroy the integrity of filamentous bacteriophages whose major coat protein is itself a membrane protein before particle assembly. These limitations led to the rapid adaptation of strained polyacrylamide gels4 as alignment media for membrane proteins. Presently, the limiting factor is obtaining a second alignment suitable for RDC measurements, since RDC data sets from two different protein alignments enable the direct analysis of the secondary structure with Dipolar Waves5 and the direct determination of the alignment tensors with λ-map plots6 without which the calculation of the three-dimensional structures can not proceed.

Although some success has been reported with the use of charged gels,7 additional alignment methods compatible with the detergents used to solubilize membrane proteins are needed. Binding of lanthanide ions to the membrane proteins, including those modified to interact specifically with metals, has been shown to induce alignment,8 however, there is always a concern about the effects of the paramagnetic metals or the requisite modifications. Recently, DNA-based liquid crystals have been shown to align detergent-solubilized membrane proteins.9

We describe here a general approach to obtaining a second alignment of membrane proteins that utilizes experimental conditions where fd bacteriophage remains intact in the presence of high concentrations (ca. 100 mM) of many of the detergents most commonly used to solubilize membrane protein, including short chain phospholipids, e.g., DHPC, isotropic bicelles (DMPC/DHPC mixtures), lyso lipids, e.g., LMPC, and dodecylphosphocholine (DPC) at pH ≥ 6.5. Measurements of RDCs for three different phage-aligned membrane proteins in DHPC micelles are illustrated in Figure 1. Notably, there is no detectable line broadening of the resonances under conditions that yield 1H-15N RDCs as large as 22 Hz.