James H. Prestegard

NMR structures of biomolecules using field oriented media and residual dipolar couplings

2. Theoretical treatment of dipolar interactions 376 2.1 Anisotropic interactions as probes of macromolecular structure and dynamics 376 2.1.1 The dipolar interaction 376 2.1.2 Averaging in the solution state 377 2.2 Ordering of a rigid body 377 2.2.1 The Saupe order tensor 378 2.2.2 Orientational probability distribution function 380 2.2.3 The generalized degree of order 380 2.3 Molecular structure and internal dynamics 381

Magnetically-oriented phospholipid micelles as a tool for the study of membrane-associated molecules

Introduction: Structure of Membrane-Associated Molecules by NMR High Resolution Solid State NMR Spectroscopy of Membrane Samples 2.1. Sampling spinning methods 2.2. Mechanical orientation of bilayers 2.3. Magnetic orientation of bilayers 2.3.1. Oriented phospholipid bilayers 2.3.2. Incorporation of membrane-associated molecules 2.3.3. Future development of magnetically orientable lipid media Experimental Considerations for Magnetically-Orientable Membrane Systems 3.1. Spectrometer requirements 3.2. Isotopic labeling 3.3. Strong coupling Anisotropic Spin Interactions: The Source of Orientation-Based Structural Data 4.1. Dipolar coupling 4.2. Quadrupolar coupling 4.3. Chemical shift anisotropy 4.4. Spin relaxation Determining Structure and Dynamics of Membrane-Bound Molecules 5.1. Structure and dynamics directly from experimental measurements 5.1.1. Order matrix analysis 5.1.2. Torsion angle analysis 5.2. NMR data as structural constraints in molecular modeling 5.3. Molecular dynamics simulations followed by back calculation of data Future Prospects Acknowledgements References 421 422 422 423 423 424 429 429 430 430 430 432 432 433 433 434 436 437 437 437 438 439 440 442

NMR structure determination for larger proteins using backbone-only data.

Examining the Backbone Determination of tertiary protein structures by nuclear magnetic resonance (NMR) currently relies heavily on side-chain NMR data. The assignment of side-chain atoms is challenging. In addition, proteins larger than 15 kilodaltons (kD) must be deuterated to improve resolution and this eliminates the possibility of measuring long-range interproton distance constraints. Now Raman et al. (p. 1014, published online 4 February) use backbone-only NMR data—chemical shifts, residual dipolar coupling, and backbone amide proton distances—available from highly deuterated proteins to guide conformational searching in the Rosetta structure prediction protocol. Using this new protocol, they were able to generate accurate structures for proteins of up to 25 kD. Protein structures can be determined by using the limited nuclear magnetic resonance information obtainable for larger proteins. Conventional protein structure determination from nuclear magnetic resonance data relies heavily on side-chain proton-to-proton distances. The necessary side-chain resonance assignment, however, is labor intensive and prone to error. Here we show that structures can be accurately determined without nuclear magnetic resonance (NMR) information on the side chains for proteins up to 25 kilodaltons by incorporating backbone chemical shifts, residual dipolar couplings, and amide proton distances into the Rosetta protein structure modeling methodology. These data, which are too sparse for conventional methods, serve only to guide conformational search toward the lowest-energy conformations in the folding landscape; the details of the computed models are determined by the physical chemistry implicit in the Rosetta all-atom energy function. The new method is not hindered by the deuteration required to suppress nuclear relaxation processes for proteins greater than 15 kilodaltons and should enable routine NMR structure determination for larger proteins.

Magnetically orientable phospholipid bilayers containing small amounts of a bile salt analogue, CHAPSO

Buffered mixtures of the detergent 3-(cholamidopropyl)dimethylammonio-2-hydroxy-1-propanesulfonate (CHAPSO) and dimyristoylphosphatidylcholine (DMPC) orient in the presence of a strong magnetic field over a wide range of water contents (at least 65-85%) and CHAPSO:DMPC molar ratios (typically 1:10-1:3). 31P NMR studies show that the phospholipid in such mixtures is oriented with its director axis perpendicular to the magnetic field. 31P and 2H NMR results also suggest that the structure and dynamics of the DMPC molecules are similar to that of pure phospholipids existing in the liquid crystalline (L alpha) bilayer phase. The ability of 1:5 CHAPSO:DMPC samples to orient is highly tolerant of large changes in temperature, pH, and ionic strength, as well as to the addition of substantial amounts of charged amphiphiles or soluble protein. However, 2H NMR studies of deuterated beta-dodecyl melibiose (DD-MB) solubilized in the system indicate the head group conformation and/or dynamics of this glycolipid analogue is dependent upon the CHAPSO concentration. Despite the latter results, the orientational versatility of the system, together with the nondenaturing properties of CHAPSO, makes this system useful in spectroscopic studies of membrane-associated phenomena.

Measurement of vicinal couplings from cross peaks in COSY spectra

Abstract Vicinal proton-proton couplings can provide useful structural information on both small and large molecules. For large molecules, couplings are in principle accessible from cross-peak multiplets in two-dimensional phase-sensitive COSY spectra. Direct measurement, however, often provides unrealistically large values because of cancellation of antiphase components. A method which allows accurate calculation of scalar couplings from measurement of separations of extrema in dispersive and absorptive plots of rows through cross peaks is presented. The method is applied to both simulated and real data on a small protein and is shown to be effective in analyzing spectra with moderate to high signal-to-noise ratios.

New techniques in structural NMR — anisotropic interactions

Structure determination of biomolecules by NMR has traditionally been based on nuclear Overhauser effects (NOEs). Now there are additional sources of information that can complement NOEs in cases where positioning of remote parts of molecules is important, and where extension to larger and more complex systems is desired.

Improved dilute bicelle solutions for high-resolution NMR of biological macromolecules

Dissolving biological macromolecules in dilute bicelle solutions, which form oriented liquid crystals in the presence of a magnetic field, permits measurement of anisotropic spin interactions such as dipolar couplings [Tjandra, N. and Bax, A., Science, 278, 1111–1114]. However, the lifetimes and temperature ranges of orientation for these samples are critically dependent on sample composition and experimental conditions. This paper demonstrates that doping dilute bicelle solutions with small amounts of charged amphiphiles substantially improves the stability and degree of alignment, as well as extends the temperature range of orientation for these systems. An explanation of the dependence of bicelle aggregation on sample composition is proposed based on the DLVO theory of colloids.

Structural studies of the transmembrane C-terminal domain of the amyloid precursor protein (APP): does APP function as a cholesterol sensor?

The amyloid precursor protein (APP) is subject to alternative pathways of proteolytic processing, leading either to production of the amyloid-beta (Abeta) 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 beta-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 alpha-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 alpha-secretase cleavage that helps to organize the connecting loop to the transmembrane domain, which is known to be essential for Abeta 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 beta- and gamma-secretase and resulting amyloid-beta production. It is proposed that APP may serve as a cellular cholesterol sensor that is linked to mechanisms for suppressing cellular cholesterol uptake.

Magnetic field induced ordering of bile salt/phospholipid micelles: new media for NMR structural investigations

Micelles formed from sodium glycocholate and dimyristoylphosphatidylcholine are demonstrated to form a magnetic field orientable liquid crystal within narrow ranges of composition and temperature. The utility of this medium in structural investigations of biological membrane components using deuterium NMR is discussed.

NMR analysis demonstrates immunoglobulin G N-glycans are accessible and dynamic.

The N-glycan at Asn297 of the immunoglobulin G Fc fragment modulates cellular responses of the adaptive immune system. However, the underlying mechanism remains undefined, as existing structural data suggest the glycan does not directly engage cell surface receptors. Here we characterize the dynamics of the glycan termini using solution NMR spectroscopy. Contrary to previous conclusions based on X-ray crystallography and limited NMR data, our spin relaxation studies indicate that the termini of both glycan branches are highly dynamic and experience considerable motion in addition to tumbling of the Fc molecule. Relaxation dispersion and temperature-dependent chemical shift perturbations demonstrate exchange of the α1-6Man-linked branch between a protein-bound and a previously unobserved unbound state, suggesting the glycan samples conformational states that can be accessed by glycan-modifying enzymes and possibly glycan recognition domains. These findings suggest a role for Fc-glycan dynamics in Fc-receptor interactions and enzymatic glycan remodeling.