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...

Advances in NMR instrumentation and pulse sequence design have resulted in easier acquisition of Residual Dipolar Coupling (RDC) data. However, computational and theoretical analysis of this type of data has continued to challenge the international community of investigators because of their complexity and rich information content. Contemporary use of RDC data has required a-priori assignment, which significantly increases the overall cost of structural analysis. This article introduces a novel algorithm that utilizes unassigned RDC data acquired from multiple alignment media (nD-RDC, n≥3) for simultaneous extraction of the relative order tensor matrices and reconstruction of the interacting vectors in space.Estimation of the relative order tensors and reconstruction of the interacting vectors can be invaluable in a number of endeavors. An example application has been presented where the reconstructed vectors have been used to quantify the fitness of a template protein structure to the unknown protein structure. This work has other important direct applications such as verification of the novelty of an unknown protein and validation of the accuracy of an available protein structure model in drug design. More importantly, the presented work has the potential to bridge the gap between experimental methods and computational methods of structure determination.

The rapid increase in the availability of RDC data from multiple alignment media in recent years has necessitated the development of more sophisticated analyses that extract the RDC data’s full information content. This article presents an analysis of the distribution of RDCs from two media (2D-RDC data), using the information obtained from a λ-map. This article also introduces an efficient algorithm, which leverages these findings to extract the order tensors for each alignment medium using unassigned RDC data in the absence of any structural information. The results of applying this 2D-RDC analysis method to synthetic and experimental data are reported in this article. The relative order tensor estimates obtained from the 2D-RDC analysis are compared to order tensors obtained from the program REDCAT after using assignment and structural information. The final comparisons indicate that the relative order tensors estimated from the unassigned 2D-RDC method very closely match the results from methods that require assignment and structural information. The presented method is successful even in cases with small datasets. The results of analyzing experimental RDC data for the protein 1P7E are presented to demonstrate the potential of the presented work in accurately estimating the principal order parameters from RDC data that incompletely sample the RDC space. In addition to the new algorithm, a discussion of the uniqueness of the solutions is presented; no more than two clusters of distinct solutions have been shown to satisfy each λ-map.

Residual dipolar couplings (RDCs) acquired by nuclear magnetic resonance (NMR) spectroscopy are an indispensable source of information in investigation of molecular structures and dynamics. Here, we present a comprehensive strategy for structure calculation and reconstruction of discrete-state dynamics from RDC data that is based on the singular value decomposition (SVD) method of order tensor estimation. In addition to structure determination, we provide a mechanism of producing an ensemble of conformations for the dynamical regions of a protein from RDC data. The developed methodology has been tested on simulated RDC data with ±1 Hz of error from an 83 residue α protein (PDB ID 1A1Z ) and a 213 residue α/β protein DGCR8 (PDB ID 2YT4 ). In nearly all instances, our method reproduced the structure of the protein including the conformational ensemble to within less than 2 Å. On the basis of our investigations, arc motions with more than 30° of rotation are identified as internal dynamics and are reconstructed with sufficient accuracy. Furthermore, states with relative occupancies above 20% are consistently recognized and reconstructed successfully. Arc motions with a magnitude of 15° or relative occupancy of less than 10% are consistently unrecognizable as dynamical regions within the context of ±1 Hz of error.

More than 90% of protein structures submitted to the PDB each year are homologous to some previously characterized protein structure. The extensive resources that are required for structural characterization of proteins can be justified for the 10% of the novel structures, but not for the remaining 90%. This report presents the 2D-PDPA method, which utilizes unassigned residual dipolar coupling in order to address the economics of structure determination of routine proteins by reducing the data acquisition and processing time. 2D-PDPA has been demonstrated to successfully identify the correct structure of an array of proteins that range from 46 to 445 residues in size from a library of 619 decoy structures by using unassigned simulated RDC data. When using experimental data, 2D-PDPA successfully identified the correct NMR structures from the same library of decoy structures. In addition, the most homologous X-ray structure was also identified as the second best structural candidate. Finally, success of 2D-PDPA in identifying and evaluating the most appropriate structure from a set of computationally predicted structures in the case of a previously uncharacterized protein Pf2048.1 has been demonstrated. This protein exhibits less than 20% sequence identity to any protein with known structure and therefore presents a compelling and practical application of our proposed work.