Suzanne Kiihne

Windowless dipolar recoupling: the detection of weak dipolar couplings between spin 12 nuclei with large chemical shift anisotropies

A new homonuclear dipolar recoupling technique is described which uses a sequence of phase-shifted, windowless irradiations applied synchronously with sample spinning. Experiments performed on a series of doubly labeled dicarboxylic acids, alanine-1,3-13C2, and 2′-deoxythymidine-4,6-13C2 demonstrate that this new windowless dipolar recoupling pulse sequence can accurately determine internuclear distances from polycrystalline solids in cases where the coupled spins have large chemical shift anisotropies and large differences in isotropic chemical shift.

(1)H and (13)C MAS NMR evidence for pronounced ligand-protein interactions involving the ionone ring of the retinylidene chromophore in rhodopsin.

Rhodopsin is a member of the superfamily of G-protein-coupled receptors. This seven α-helix transmembrane protein is the visual pigment of the vertebrate rod photoreceptor cells that mediate dim light vision. In the active binding site of this protein the ligand or chromophore, 11-cis-retinal, is covalently bound via a protonated Schiff base to lysine residue 296. Here we present the complete 1H and 13C assignments of the 11-cis-retinylidene chromophore in its ligand-binding site determined with ultra high field magic angle spinning NMR. Native bovine opsin was regenerated with 99% enriched uniformly 13C-labeled 11-cis-retinal. From the labeled pigment, 13C carbon chemical shifts could be obtained by using two-dimensional radio frequency-driven dipolar recoupling in a solid-state magic angle spinning homonuclear correlation experiment. The 1H chemical shifts were assigned by two-dimensional heteronuclear (1H-13C) dipolar correlation spectroscopy with phase-modulated Lee–Goldburg homonuclear 1H decoupling applied during the t1 period. The data indicate nonbonding interactions between the protons of the methyl groups of the retinylidene ionone ring and the protein. These nonbonding interactions are attributed to nearby aromatic acid residues Phe-208, Phe-212, and Trp-265 that are in close contact with, respectively, H-16/H-17 and H-18. Furthermore, binding of the chromophore involves a chiral selection of the ring conformation, resulting in equatorial and axial positions for CH3-16 and CH3-17.

Perspectives on solid state NMR in biology

Preface. List of Conference Organizers. List of Conference Speakers. Section I: Advances in MAS Techniques for Non-Aligned Systems. Using Symmetry to Design Pulse Sequences in Solid-State NMR A. Brinkmann, et al. Accurate 13C-15N Distance Measurements in Uniformly 13C,15N-Labeled Peptides C.P. Jaroniec, et al. Selectivity of Double-Quantum Filtered Rotational-Resonance Experiments on Larger-than-Two-Spin Systems M. Bechmann, et al. Multiple-Quantum Spectroscopy of Fully Labeled Polypeptides Under MAS: A Statistical and Experimental Analysis S. Luca, et al. Section II: Advances in Techniques for Aligned Systems. 2H, 15N and 31P Solid-State NMR Spectroscopy of Polypeptides Reconstituted Into Oriented Phospholipid Membranes B. Bechinger, et al. From Topology to High Resolution Membrane Protein Structures T.A. Cross, et al. Toward Dipolar Recoupling in Macroscopically Ordered Samples of Membrane Proteins Rotating at the Magic Angle C. Glaubitz, et al. Solid State 19F-NMR of Biomembranes S.L. Grage, et al. Section III: Important Technologies. Numerical Simulations for Experiment Design and Extraction of Structural Parameters in Biological Solid-State NMR Spectroscopy M. Bak, et al. An Ab-Initio Molecular Dynamics Modeling of the Primary Photochemical Event in Vision F. Buda, et al. Refolded G protein-coupled Receptors from E. coli Inclusion Bodies H. Kiefer, et al. Semliki Forest Virus Vectors: Versatile Tools for Efficient Large-Scale Expression of Membrane Receptors K. Lundstrom. G Protein-Coupled Receptor Expression in Halobacterium salinarum A.M. Winter-Vann, et al. Magnetic Resonance Microscopy for Studying the Development of Chicken and Mouse Embryos R.E. Poelmann, et al. Section IV: Applications in Membrane Proteins and Peptides. MAS NMR on a Uniformly 13C,15N Labeled LH2 Light-Harvesting Complex from Rhodopseudomonas Acidophila 10050 at Ultra-High Magnetic Fields T.A. Egorova-Zachernyuk, et al. Determination of Torsion Angles in Membrane Proteins J.C. Lansing, et al. Characterization and Assignment of Uniformly Labeled NT(8-13) at the Agonist Binding Site of the G-Protein Coupled Neurotensin Receptor P.T.F. Williamson, et al. Structural Insight into the Interaction of Amylod- Peptide with Biological Membranes by Solid State NMR G. Groebner, et al. Photochemically Induced Dynamic Nuclear Polarization in Bacterial Photosynthetic Reaction Centres Observed by 13C Solid-State NMR J. Matysik, et al. Index.

T1 relaxation in in vivo mouse brain at ultra‐high field

Accurate knowledge of relaxation times is imperative for adjustment of MRI parameters to obtain optimal signal‐to‐noise ratio (SNR) and contrast. As small animal MRI studies are extended to increasingly higher magnetic fields, these parameters must be assessed anew. The goal of this study was to obtain accurate spin‐lattice (T1) relaxation times for the normal mouse brain at field strengths of 9.4 and 17.6 T. T1 relaxation times were determined for cortex, corpus callosum, caudate putamen, hippocampus, periaqueductal gray, lateral ventricle, and cerebellum and varied from 1651 ± 28 to 2449 ± 150 ms at 9.4 T and 1824 ± 101 to 2772 ± 235 ms at 17.6 T. A field strength–dependent increase of T1 relaxation times is shown. The SNR increase at 17.6 T is in good agreement with the expected SNR increase for a sample‐dominated noise regime. Magn Reson Med 58:390–395, 2007.

Distance measurements in nucleic acids using windowless dipolar recoupling solid state NMR

A windowless, homonuclear dipolar recoupling pulse sequence (DRAWS) is described and a theoretical basis for describing its recoupling performance is developed using numerical techniques. It is demonstrated that DRAWS recouples weak dipolar interactions over a broad range of experimental and molecular conditions. We discuss two spectroscopic control experiments, which help to take into account effects due to insufficient proton decoupling, relaxation, and static dipolar couplings to nearby 13C spins at natural abundance. Finally DRAWS is used in combination with selective 13C labeling to measure 13C-13C distances in five doubly labeled DNA dodecamers, [d(CGCGAAT*T*CGCG)]2, which contain the binding site for the restriction enzyme EcoRI. The longest distance reported is 4.8 A. In most cases the distances agree well with those derived from X-ray crystallographic data, although small changes in hydration level can result in relatively large changes in internuclear distances.

Solid State NMR Investigation of the Interaction between Biomimetic Lipid Bilayers and de novo Designed Fusogenic Peptides

Regulated fusion of biological membranes can be induced by membrane-associated peptides with low-complexity sequences. Such peptides can drive fusion depending on their structural flexibility, but how the peptide changes the lipid phase is not known. Here we study the interaction of synthetic peptides with a biomimetic lipid mixture by using P solid-state NMR spectroscopy. We investigated two peptides: one previously shown to be rigid and virtually nonfusogenic, L16 (K3WL16K3), and one that is flexible and highly fusogenic, LV16G8P9 (K3WLVLVLVLGPVLVLVLVK3). [1] A mixture of brain phosphatidylethanolamine (PE), brain phosphatidylserine (PS) and egg phosphatidylcholine (PC) at a 3:1:1 ratio was used to mimic biological membrane composition. Here we show how the de novo designed peptides altered the phase behaviour of biomimetic lipid bilayers at a peptide to lipid ratio of 1:100. While L16 forms a rather stable a helix, LV16G8P9 can readily refold from a helix to b sheet and vice versa, by changing solvent polarity. Samples were prepared by dissolving 1-palmitoyl-2-oleoylsn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3[phospho-l-serine] (DOPS) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE; Avanti Polar Lipids, AL, USA) in chloroform. Peptide dissolved in TFE was added at a peptide/ lipid molar ratio of 0.01, as in previous fusion studies. In the case of POPC lipid-bilayer studies, the peptide was dissolved in TFE, and was then mixed with POPC in chloroform at the ACHTUNGTRENNUNGdesired peptide to lipid ratio. The mixtures were applied to 15 ACHTUNGTRENNUNGultrathin cover-glass slides, dried with a stream of N2 and kept in high vacuum, overnight. The plates were sprayed with fusion buffer (150 mm NaCl, 20 mm Tris-HCl, 0.2 mm EDTA, pH 7.4) and were equilibrated at 4 8C for 72 h at a relative humidity of 93% to yield samples with an estimated water content of ~0.2 mLmm . Stacks of plates were sealed with Teflon tape and plastic wrap, inserted into a multichannel flat-coil probe and placed in a Bruker AV750 spectrometer. Proton-decoupled P NMR spectra were recorded by using a Hahn-echo pulse sequence, with an echo delay of 40 ms and a recycle delay of 3 s. The orientation of lipids can be probed by using P NMR spectroscopy, since the observed line shape reflects the symmetry of the dynamic order of lipid molecules in a very characteristic manner. The chemical shift anisotropy (CSA) tensor was partially averaged to an effective tensor that was axially symmetric and had principal axis values sk ~28 ppm and s? ~ 19 ppm, for 08 orientation parallel to the rotation axis, and for 908 orientation perpendicular to the rotation axis, respectively. These values varied somewhat with lipid types and also depended on the exact orientation of the head-group with respect to the axis of rotation. The isotropic P chemical shifts of lipids in a buffer with pH 7.4 at 310 K were 2.7 ppm for POPC, 1.7 ppm for DOPE and 2.5 ppm for DOPS. The P NMR line shape of the pure lipid mixture was dominated by signals that are characteristic of oriented and cylindrical phospholipid phases (Figure 1A). Three narrow signals at 21.3, 22.5 and 32 ppm were assigned to 08 oriented PC, PE and PS in the bilayer phase, respectively, by comparison with values known for the pure lipids. 5,6] The broad signal at 20 ppm and the shoulder at 24 ppm revealed a cylindrical distribution of orientations in the unoriented phase, and the long axis of the cylinder was perpendicular to the field. The other extremes of the cylindrical signals were hidden under the bilayer signals. When the unoriented response was simulated and combined with a set of narrow lines that represented the bilayer signal, a remarkably good reproduction of the experimental data was obtained (Figure 1D). Given the overall sample composition of 60% PC, 20% PE and 20% PS, the ACHTUNGTRENNUNGsimulated curves in Figure 1 indicated that ~64% of the P signal accounted for cylindrical response at a ratio of ~36:13:15 (PC/PE/PS). The intensities of the narrow signals translated into relative fractions of ~24:7:5 (PC/PE/PS) for the aligned bilayer component (Figure 2). Provided that the underlying model of aligned bilayers and cylindrical fractions was correct, the composition analysis was accurate to about 5%. The bilayer defects that formed increased-curvature domains were likely due to multilamellar bilayers and might have contained water in the centre. These cylindrical fractions are expected to have relatively large diameters compared to the length scale of ~10 nm of lipid lateral diffusion. Small cylinder shapes are unlikely since these would give rise to the narrowing of the P CSA at a characteristic diffusion rate of ~10 11 ms . Furthermore, an inverted hexagonal phase does not fit our data since its cylindrical rods have a diameter of only two lipid molecules, and lateral diffusion of lipids over the curved surface gives rise to partial averaging of the NMR anisotropy. Also, unordered lipids would give rise to a narrow isotropic response due to rapid motional averaging. Small vesicles composed of two differently shaped lipids, such as PC and PE, are known to exhibit strong compositional asymmetry between the two monolayers of a highly curved bilayer. This asymmetry reduces the frustration between the [a] P. Agrawal, Dr. S. Kiihne, J. Hollander, Dr. F. Hulsbergen, Prof. H. de Groot Biophysical Organic Chemistry/Solid State NMR Leiden Institute of Chemistry, Leiden University Einsteinweg 55, 2333 CC Leiden (The Netherlands) Fax: (+31)71-5274603 E-mail : ssnmr@chem.leidenuniv.nl [b] Dr. M. Hofmann, Prof. D. Langosch Lehrstuhl Chemie der Biopolymere, Technische Universit;t M<nchen Weihenstephaner Berg 3, 85354 Freising (Germany)