The three-dimensional structure of the dimeric transmembrane domain of glycophorin A (GpA) was determined by solution nuclear magnetic resonance spectroscopy of a 40-residue peptide solubilized in aqueous detergent micelles. The GpA membrane-spanning alpha helices cross at an angle of -40 degrees and form a small but well-packed interface that lacks intermonomer hydrogen bonds. The structure provides an explanation for the previously characterized sequence dependence of GpA dimerization and demonstrates that van der Waals interactions alone can mediate stable and specific associations between transmembrane helices.

The measurement ofdipolar contributions to the splitting of 15N resonances of 1H-15N amide pairs in multidimensional high-field NMR spectra of field-oriented cyanometmyoglobin is reported. The splittings appear as small field-dependent perturbations of normal scalar couplings. Assignment of more than 90 resonances to specific sequential sites in the protein allows correlation of the dipolar contributions with predictions based on the known susceptibility and known structure of the protein. Implications as an additional source of information for protein structure determination in solution are discussed.Within the past 15 years, NMR has emerged as a powerful approach to the study of protein structure in solution (1, 2). The approach relies on distance constraints extracted from nuclear Overhauser effects (NOEs) and typically proceeds in three stages: assignment of backbone resonances to specific sequential sites, identification of secondary structure elements, and determination of a tertiary fold. The latter stage is particularly demanding because it requires that a large number of long-range distance constraints be extracted from NOE data and assigned to specific proton pairs. Because NOEs drop off with the inverse 6th power of the internuclear distance, the pairs tend to arise from direct side-chain-side-chain contacts, contacts involving protons which are among the most difficult to assign. Opportunities to supplement long-range distance constraints with other types of structural data would, therefore, be welcome. We demonstrate here that NMR spectra of certain proteins, taken at very high field, may contain data that can usefully complement NOEs in determining a tertiary fold. The data come from residual dipolar contributions to the scalar couplings normally seen in high-resolution spectra. These appear when the protein has a slightly preferred orientation in a magnetic field. The contributions are angle dependent and can yield constraints for the orientation of one structural element relative to another structural element. In our case, we use a 17.5-kDa protein, cyanometmyoglobin, which has a very highly anisotropic paramagnetic susceptibility to achieve preferred orientation. Myoglobin crystals have been previously shown to orient in a magnetic field because of their anisotropy (3). Bothner-By and co-workers (4) also showed years ago that orientational effects on NMR spectra of single molecules in solution could be observed if fields and resolution were high enough. That these effects can be observed in an isolated protein molecule, and that the effects can provide useful structural constraints, has awaited higher fields (17.5 T) and multidimensional NMR experiments for the detection and assignment of 15N resonances in an isotopically labeled protein.Theory. The dipolar interaction between two spin 1/2 nuclei in the high-field limit is given by the formulawhere the -y values are the gyromagnetic ratios for the nuclei, h is Plank's constant, r is the distance between the nuclei, 0 is the angle between...

The measurement of anisotropic spin interactions, such as residual dipolar couplings, in partially ordered solutions can provide valuable information on biomolecular structure. While the information can be used to refine local structure, it can make a unique contribution in determining the relative orientation of remote parts of molecules, which are locally well structured, but poorly connected based on NOE data. Analysis of dipolar couplings in terms of Saupe order matrices provides a concise description of both orientation and motional properties of locally structured fragments in these cases. This paper demonstrates that by using singular value decomposition as a method for calculating the order matrices, principal frames and order parameters can be determined efficiently, even when a very limited set of experimental data is available. Analysis of 1H-15N dipolar couplings, measured in a two-domain fragment of the barley lectin protein, is used to illustrate the computational method.

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1. Introduction 3721.1 Residual dipolar couplings as a route to structure and dynamics 3721.2 A brief history of oriented phase high resolution NMR 3742. Theoretical treatment of dipolar interactions 3762.1 Anisotropic interactions as probes of macromolecular structure and dynamics 3762.1.1 The dipolar interaction 3762.1.2 Averaging in the solution state 3772.2 Ordering of a rigid body 3772.2.1 The Saupe order tensor 3782.2.2 Orientational probability distribution function 3802.2.3 The generalized degree of order 3802.3 Molecular structure and internal dynamics 3813. Inducing molecular order in high resolution NMR 3833.1 Tensorial interactions between the magnetic field and anisotropic magnetic susceptibilities 3833.2 Dilute liquid crystal media: a tunable source of order 3843.2.1 Bicelles : from membrane mimics to aligning media 3853.2.2 Filamentous phage 3873.2.3 Transfer of alignment from ordered media to macromolecules 3883.3 Magnetic field alignment 3893.3.1 Paramagnetic assisted alignment 3893.3.2 Advantages of using magnetic alignment 3894. The measurement of residual dipolar couplings 3914.1 Introduction 3914.2 Frequency based methods 3924.2.1 Coupling enhanced pulse schemes 3924.2.2 In phase anti-phase methods (IPAP): 1DNH couplings in proteins 3934.2.3 Exclusive correlated spectroscopy (E-COSY): 1DNH, 1DNC′ and 2DHNC′ 3954.2.4 Extraction of splitting values from the frequency domain 3964.3 Intensity based experiments 3974.3.1 J-Modulated experiments: the measurement of 1DCαHα in proteins 3974.3.2 Phase modulated methods 3994.3.3 Constant time COSY – the measurement of DHH couplings 3994.3.4 Systematic errors in intensity based experiments 4005. Interpretation of residual dipolar coupling data 4015.1 Structure determination protocols utilizing orientational constraints 4015.1.1 The simulated annealing approach 4015.1.2 Order matrix analysis of dipolar couplings 4025.1.3 A discussion of the two approaches 4025.2 Reducing orientational degeneracy 4035.2.1 Multiple alignment media in the simulated annealing approach 4045.2.2 Multiple alignment media in the order matrix approach 4055.3 Simplifying effects arising due to molecular symmetry 4065.4 Database approaches for determining protein structure 4076. Applications to the characterization of macromolecular systems 4086.1 Protein structure refinement 4086.2 Protein domain orientation 4096.3 Oligosaccharides 4136.4 Biomolecular complexes 4156.5 Exchanging systems 4167. Acknowledgements 4188. References 419Within its relatively short history, nuclear magnetic resonance (NMR) spectroscopy has managed to play an important role in the characterization of biomolecular structure. However, the methods on which most of this characterization has been based, Nuclear Overhauser Effect (NOE) measurements for short-range distance constraints and scalar couplings measurements for torsional constraints, have limitations (Wüthrich, 1986). For extended structures, such as DNA helices, for example, propagation of errors in the short distance constraints derived from NOEs leaves the relative orientation of remote parts of the structures poorly defined. Also, the low density of observable protons in contact regions of molecules held together by factors other than hydrophobic packing, leads to poorly defined structures. This is especially true in carbohydrate containing complexes where hydrogen bonds often mediate contacts, and in multi-domain proteins where the area involved in domain–domain contact can also be small. Moreover, most NMR based structural applications are concerned with the characterization of a single, rigid conformer for the final structure. This can leave out important mechanistic information that depends on dynamic aspects and, when motion is present, this can lead to incorrect structural representations. This review focuses on one approach to alleviating some of the existing limitations in NMR based structure determination: the use of constraints derived from the measurement of residual dipolar couplings (D).

The amyloid precursor protein (APP) is subject to alternative pathways of proteolytic processing, leading either to production of the amyloid-β (Aβ) peptides or to non-amyloidogenic fragments. Here, we report the first structural study of C99, the 99-residue transmembrane C-terminal domain of APP liberated by β-secretase cleavage. We also show that cholesterol, an agent that promotes the amyloidogenic pathway, specifically binds to this protein. C99 was purified into model membranes where it was observed to homodimerize. NMR data show that the transmembrane domain of C99 is an α-helix that is flanked on both sides by mostly disordered extramembrane domains, with two exceptions. First, there is a short extracellular surface-associated helix located just after the site of α-secretase cleavage that helps to organize the connecting loop to the transmembrane domain, which is known to be essential for Aβ production. Second, there is a surface-associated helix located at the cytosolic C-terminus, adjacent to the YENPTY motif that plays critical roles in APP trafficking and protein-protein interactions. Cholesterol was seen to participate in saturable interactions with C99 that are centered at the critical loop connecting the extracellular helix to the transmembrane domain. Binding of cholesterol to C99 and, most likely, to APP may be critical for the trafficking of these proteins to cholesterol-rich membrane domains, which leads to cleavage by β-and γ-secretase and resulting amyloid-β production. It is proposed that APP may serve as a cellular cholesterol sensor that is linked to mechanisms for suppressing cellular cholesterol uptake.The human amyloid precursor protein (APP) 1 is a single-span membrane protein that is alternatively processed by either α-or β-secretase to release its large ectodomain from the cell surface, a process referred to as "shedding". β-Secretase (β-site APP cleaving enzyme 1, BACE1) cleaves APP after Met671, leading to production of the C-terminal 99-residue domain of APP, C99, a single-span membrane protein. Subsequent cleavage of C99 at membranedisposed sites by γ-secretase leads to release of both the amyloid-β (Aβ) peptides and the water-

The measurement of residual dipolar couplings in weakly aligned proteins can potentially provide unique information on their structure and dynamics in the solution state. The challenge is to extract the information of interest from the measurements, which normally reflect a convolution of the structural and dynamic properties. We discuss here a formalism which allows a first order separation of their effects, and thus, a simultaneous extraction of structural and motional parameters from residual dipolar coupling data. We introduce some terminology, namely a generalized degree of order, which is necessary for a meaningful discussion of the effects of motion on residual dipolar coupling measurements. We also illustrate this new methodology using an extensive set of residual dipolar coupling measurements made on (15)N,(13)C-labeled human ubiquitin solvated in a dilute bicelle solution. Our results support a solution structure of ubiquitin which on average agrees well with the X-ray structure (Vijay-Kumar, et al., J. Mol. Biol. 1987, 194, 531--544) for the protein core. However, the data are also consistent with a dynamic model of ubiquitin, exhibiting variable amplitudes, and anisotropy, of internal motions. This work suggests the possibility of primary use of residual dipolar couplings in characterizing both structure and anisotropic internal motions of proteins in the solution state.

Residual dipolar couplings observed in NMR spectra at very high magnetic fields have been measured to a high degree of accuracy for the paramagnetic protein cyanometmyoglobin. Deviations of these measurements from predictions based on available crystallographic and solution structures are largely systematic and well correlated within a given helix of this highly alpha-helical protein. These observations can be explained by invoking collective motion and small displacements of representative helices from their reported average positions in the solid state. Thus, the measurements appear to be capable of providing important insights into slower, collective protein motions, which are likely to be important for function, and which have been difficult to study using established experimental techniques.