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

Abstract

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 gels 4 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 Waves 5 and the direct determination of the alignment tensors with λ-map plots 6 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, 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 micell...

Dynafold: A Dynamic Programming Approach to Protein Backbone Structure Determination From Minimal Sets of Residual Dipolar Couplings

“…Recent work has shown that the statistical distribution of an n-D RDC data set can be used to estimate the values of the order tensor matrices for each alignment medium 54–57 . These estimates have been shown to be of sufficiently high quality as to not distort protein structures significantly 36 . Therefore, the order tensors that describe the alignment of a protein can be assumed to have been determined.…”

“…RDCs have been the subject of a number of reviews 14–20 , and have been used in the study of carbohydrates 21–24 , nucleic acids 14,25–28 and proteins 29–35 . More importantly, they have recently demonstrated promise as a viable approach to the structural characterization of challenging proteins such as membrane proteins 36 .…”

Backbone resonance assignment and order tensor estimation using residual dipolar couplings

“…We assigned experimental data obtained from BMRB for three previously reported structures 2KLV (Park et al 2009), 1RWD (Tian et al 2001) and 1D3Z (Cornilescu et al 1998). Data from NMR based structures 1RWD and 1D3Z were assigned to their homologous X-ray structures 1BRF and 1UBQ respectively.…”

Structure and Dynamics of Proteins from Nuclear Magnetic Resonance Spectroscopy

“…As a result, the impact of these developments has enabled direct investigation of protein backbone structures (Bernado & Blackledge, 2004b;Clore et al, 1999;Fowler et al, 2000;Tian et al, 2001b;Valafar et al, 2005). Applications of RDCs have also extended into the structural elucidation of traditionally complex proteins such as membrane proteins (Opella & Marassi, 2004;Park et al, 2009) and homo-multimeric proteins (Wang et al, 2008). In fact, the utility of RDCs has extended well beyond the community of NMR spectroscopists.…”

“…This is in part due to their rich information content. RDCs also possess the potential for integrating structure determination by NMR spectroscopy (Bryson et al, 2008;Park et al, 2009;Prestegard et al, 2005;Tian et al, 2001b;Valafar et al, 2005), X-ray crystallography Ulmer et al, 2003;Valafar & Prestegard, 2003), and computational modeling Raman et al, 2010b;Valafar & Prestegard, 2003) methods into one unified approach for structural elucidation of biological macromolecules. Because RDCs may be used to characterize the structure and dynamics of challenging proteins, it presents a viable, costeffective method with the benefit of producing rapid, comprehensive and automated results (Al-Hashimi et al, 2002b;Bailor et al, 2007;Liu et al, 2010;Park et al, 2009;Prestegard et al, 2000;Tian et al, 2001a;Wang et al, 2007).…”