Monday and Tuesday Posters 3

062
Session Assigned: M&T P
Title: Structure and Dynamics Analysis of an RNA Binding Site for Phage GA Coat Protein

Institute: Rice University Houston, TX. USA
Keywords: deuterium label, phosphorus, T1, T2
Abstract:
The solution structure and dynamics of an RNA binding site for the coat protein of bacteriophage GA has been characterized using heteronuclear NMR spectroscopic methods. Translation of the replicase gene of Escherichia coli bacteriophage GA is regulated by the binding of the phage coat protein proximal to the translation start site. A 23 nucleotide RNA molecule containing the binding site for the bacteriophage coat protein has been isotopically enriched with ¹³C and ¹⁵N and its three dimensional structure determined using heteronuclear multidimensional NMR techniques. In solution, the RNA recognition element forms a hairpin structure containing a bulged adenine residue in the stem and four adenine nucleotides in the loop. Although we have obtained a structure for the RNA hairpin, a low number of structural constraints around the loop and bulge, attributed to the mobility of several residues, leads to a poorly defined conformation around these regions. Indeed, nucleotides in the bulge region exhibit two conformations with a ration of 4:1.

The motional properties of the RNA recognition element were examined using a fractionally ¹³C enriched molecule for heteronuclear relaxation analysis. Various motional models were sampled to obtain order parameters for positions on the base and ribose moieties. Although the calculations suggest that the ribose groups in the loop are relatively rigid (s² ~ 0.85)--similar to those in the stem--the base moieties are flexible (s² ~ 0.6). The base and ribose groups of the nucleotides in the bulge region exhibit dynamic properties similar to nucleotides in the stem have order parameters s² ~ 0.85 for both the base and ribose.

Several structural similarities exist between the GA and bacteriophage Ms2 RNA recognition elements. The stem of the Ms2 element is made up of five base pairs, a bulged adenine, and two more base pairs between the bulged base and the four nucleotide loop. The nucleotide sequence of the GA RNA element allows either two or three base pairs between the bulged base and the loop. In contrast to GA, the upper stem (two base pairs) of the Ms2 RNA hairpin is well defined. The relaxation analysis and the solution state structure of a hairpin that is a hybrid between the GA and Ms2 sequences supports the hypothesis that the mobility of nucleotides in the loop region is the principal factor contributing to the overall instability in the upper stem.

063
Session Assigned: M&T P
Title: NMR Investigation of the Dynamics of the Rev Binding Element Internal Loop

Institute: University of California, Los Angeles, CA, USA
Keywords: Rna Dynamics
Abstract:
The internal dynamics of RBE3-A, an RNA oligonucleotide which contains the high-affinity Rev-binding site from the Rev Responsive Element (RRE), were investigated using heteronuclear NMR. RBE3-A consists of two A-form stems and a structured internal loop. Our earlier work suggested that while the internal loop is structured, it also undergoes motion which is different than the motion in the stem regions..T1, T2, and sup13/supC-sup1/supH steady state NOE values were measured on the C8 carbons of a sample of RBE3-A which was sup13/supC, sup15/supN-labeled at the guanosine residues only. This relaxation data was analyzed using the Lipari-Szabo "model-free" approach. The relaxation data of the stem residues could be fit, using the formalism of Lipari and Szabo, with the order parameters Ssup2/sup and .TAU.e. However, the internal loop residues, G5, G6, and G24 could not be fit using Ssup2/sup and .TAU.e alone, but required an additional exchange parameter. The fact that the relaxation data of the internal loop residues could not be explained with just the order parameters indicates that some other process, such as chemical or conformation all exchange, is responsible for their relaxation behavior. This is consistent with the idea that the internal loop undergoes conformational exchange on a timescale that is slow compared to the tumbling rate of the entire molecule.

064
Session Assigned: M&T P
Title: Dynamics Investigation of a Deuterium Labeled Qin Protein in High Field

Institute: NHMFL, Tallahassee, Florida, USA
Keywords: DFS Cross-correlation Dynamics Relaxation Quadrupolar
Abstract:
The longitudinal quadrupolar relaxation time, T_1Q, depends on the product of the spectrometer frequency (w₀) and the rotational correlation time (t_c) and thus it varies with the changes in the field and (or) the dynamics of the spins. T_1Q can also influence the multiplet structure of a coupled spin ½ nucleus. When T_1Q is rapid, the line widths of any attached spin ½ nuclei have additional contributions via a time dependent modulation of the through bond J couplings and the spectrum of the spin ½ nucleus appear as a singlet. When T_1Q becomes longer, as in macro-molecular systems with longer correlation times, J couplings that are unobservable at lower fields (from line broadening) often become resolved at high field. In addition, the simultaneous presence of multiple relaxation mechanisms (dipolar, quadrupolar, and shift anisotropy) can produce, depending upon the product of w₀ and t_c, pronounced interference effects such as differential line broadening and dynamic frequency shifts that influence the lineshapes and positions, respectively, of the observed transitions. Thus, these two features (T_1Q and interference effects) will lead to spin ½ multiplet structures which will appear as a symmetric triplet in the small molecule motional limit, as collapsed singlet in the intermediate motional regime, and finally as an asymmetric triplet in the large molecule motional limit. In the case of a large protein, in which mobility varies along the backbone, the lineshapes of ²H coupled nuclei such as ¹⁵N or ¹³C will directly report the nature of the local dynamics. At sufficiently high field strengths, a wide range of dynamical regimes can be probed within a given molecular system.

In this presentation we take advantage of both high magnetic fields and deuterium substitution effects on relaxation for the investigation of protein dynamics. Dynamics of the "winged-helix" domain (in ²H-¹⁵N-¹³C labeled form) of the cellular proto-oncoprotein Qin will be elucidated by lineshape analysis in heteronuclear multi-dimensional NMR experiments. Development of NMR pulse sequences and the development of computer software for analysis of spin ½ multiplet structure with respect to dynamics (and exchange) will also be presented.

065
Session Assigned: M&T P
Title: Deuterium Spin-Lattice Relaxation and Quadrupolar Coupling Constants in Isotopically-Labeled Saccharides.

Institute: University of Notre Dame, Notre Dame, IN, USA
Keywords:
Abstract:
Deuterium spin-lattice relaxation times, T₁, have been shown to be valuable parameters in NMR investigations of molecular motions. Since the main determinant of the field gradient at ²H is not the electrons in the hydrogen 1s orbital but the nucleus to which it is directly bonded and by the electrons donated by this nucleus, ²H quadrupolar coupling constants (QCC) report on the electronic environment of deuterium nuclei in the ground state. Therefore, ²H QCCs might be useful probes of electronic differences at the anomeric carbon of saccharides where stereoelectronic factors play an important role. Little data on ²H T₁s and QCCs have been reported in carbohydrates.

We have conducted solution measurements of these parameters in a wide range of cyclic and acyclic saccharides which were site-specifically labeled with ²H at anomeric and non-anomeric methine carbons. In order to calculate ²H QCC values, complementary ¹³C T₁ measurements were made using site-specific ¹³C-labeled compounds. Solutions containing a 50/50 mixture of ¹³C- and ²H-labeled compounds (0.2 M in each component) in ²H-depleted water and 5 mM EDTA were prepared to permit the measurement of ¹³C and ²H T₁s in the same sample under identical experimental conditions. At 30 ^oC, on a Varian UNITYplus spectrometer (B₀=14.1 T), ¹³C T₁s ranged from 0.57 to 8.20 sec and ²H T₁s ranged from 0.068 to 0.45 sec, depending on molecular structure, weight and shape. ²H QCCs ranged from 139 to 179 kHz. ²H QCCs for the C1-D1 fragment of D-aldopyranoses (⁴C₁) appear to depend on anomeric configuration, with larger values observed for the alpha-anomers. In addition, the dependence of ¹³C T₁ on molecular weight is influenced by molecular structure/shape, with cyclic forms (pyranoses, furanoses) exhibiting a stronger dependence than acyclic forms (hydrates, aldehydes). Within the latter group, a stronger dependence is observed for the hydrates. This behavior may, in part, reflect differences in solute-water interactions within different saccharide structures.

066
Session Assigned: M&T P
Title: Amide Exchange Rates as a Probe of the Origin of Hyperthermostability of P. furiosus Rubredoxin

Institute: CCRC University of Georgia, Athens GA, USA
Keywords:
Abstract:
Rubredoxin (Rd) is a small, Fe-containing protein with no known function. The rubredoxin from Pyrococcus furiosus (Pf) is contains 53 amino acids and is hyperthermostable, with a half-life at 95 C of about 10 days. In contrast, the most stable Rd from a mesophilic bacterium has a half life at 95 C of about 2 hrs. Despite very high sequence identity (55%), the factors that contribute to the dramatic differences in stability of the two types of Rd remain unknown. Very high resolution X-ray crystal structures (0.9 Å) of wild-type (no N-terminal methionine), met, and formyl-met-Pf Rd have been solved and there is no appreciable difference between these structures and the NMR solution structure of the wild type form in which the Fe atom has been replaced by Zn. The Rd structure consists of a three-stranded anti-parallel beta sheet connected by a number of turns. The metal is ligated by four cysteines, two of which are on the first beta strand.

We have undertaken amide exchange studies on wild-type and met Pf Rd in the Zn(II), Fe(II), and Fe(III) forms in order to investigate their relative thermostabilities. For simple mechanisms in which base catalyzed exchanges are rate limiting, rates can be related to free energies of secondary structure unfolding. At 55 C, wild-type and met-Rd(Zn)have amide exchange rates that are slower than the Fe(III) form, by several orders of magnitude. The wild-type and met-Rd(Zn) show amide exchange rates which can be classified into three groups: fast (t_1/2 &lt; 10 min.), medium (10 min. < t_1/2 < 100 min.), and slow (100 min. < t_1/2). In both wt and met- PfRd(Zn), 17 of 48 backbone amides are in the slow regime; 13 of these slow exchanging amides show minimal exchange after 48 hours at 55 C, t_1/2 > 500 min. In stark contrast, the met-Rd(Fe(III)) shows complete exchange in one hour at 55 C. In fact, most of the amides have exchanged in the mixing time of the experiment (5 min.). This is the first demonstration of metal-specific stability in Rds. Experimental methods based on stopped-flow NMR experiments are being developed to more accurately describe differences associated with rapidly exchanging sites.

067
Session Assigned: M&T P
Title: Backbone Dynamics of Cdc42: Binding of P21-Activated Kinase

Institute: Cornell University, Ithaca, New York USA
Keywords: Protein Dynamics, Signal Transduction
Abstract:
The Ras superfamily of GTP-binding proteins are molecular switches that are involved in a variety of cellular processes including tumorigenesis, intracellular trafficking, and cytoskeletal organization. The Rho subfamily plays a role in cell morphogenesis through a GTPase cascade, which regulates cytoskeletal changes. In particular, Cdc42Hs initiates a protein kinase cascade that begins with the activation of p21-activated kinase (PAK). We have previously determined the structure of Cdc42Hs (Feltham et al. (1997) Biochemistry 36:8755) and defined the binding surface with a 46 amino acid peptide (PBD46) derived from PAK (Guo et al. (1998) Biochemistry 37:14030). The binding surface on Cdc42Hs for PBD46 includes the beta2 strand and a portion of Switch I. This structure indicated that the two regions thought to be involved in effector (Switch I) and regulator (Switch II) actions are at least partially disordered. Here we describe the backbone dynamics of three constructs of ¹⁵N-labeled Cdc42Hs (GDP-bound, GMPPCP-bound, and PBD46-bound) using two-dimensional ¹⁵N-¹H NMR spectroscopy. Values of T₁ (longitudinal relaxation time), T_1rho (transverse relaxation time measured in the rotating frame), and steady-state NOE have been measured for a majority of the ¹⁵N nuclei at three magnetic field strengths (400, 500 and 600 MHz). The values of the relaxation parameters for each construct were analyzed using an extension of the Lipari-Szabo formalism in order to determine values of the global correlation time (tau_m), as well as values of the generalized order parameter (S²), local correlation time (tau_e), and chemical exchange rate (R_ex) for each residue. Switch I exhibits dynamics which can be characterized by motions on two timescales, and order parameters with lower than average values for both the GMPPCP and PBD46 constructs. In contrast, the dynamics of Switch II change when PBD46 binds. In the GMPPCP form, Switch II experiences motion on a single (slow) timescale and chemical exchange. However, the chemical exchange component disappears upon binding of PBD46. Preliminary results suggest that the local dynamics of the GDP-bound form of Cdc42Hs are very similar to those of the GMPPCP form, implying that the backbone conformation either undergoes a static change upon activation of the protein, or that the primary conformational change occurs only in the sidechains. These results suggest that activation of Cdc42Hs by an exchange of GTP for GDP would have little effect on the dynamic properties of the protein, but that binding the effector can have important distal dynamic effects in Switch II. This is interesting in light of increasing evidence that the signaling mediated by GTPases requires multiple effector interactions and may occur within multicomponent complexes.

Institute: ETH-Zurich, Switzerland
Keywords:
Abstract:
Bacteriochlorophyll a and bacteriopheophytin a have been employed as test systems to investigate the use of MAS recoupling pulse sequences (RIL,¹ DREAM²) in a molecule of intermediate size using a uniformly labelled sample.

The bacteriochlorophyll a spectrum has sufficient resolution to show the connectivity patterns of the chlorophyll macrocycle and the phytyl side chain. The chlorophyll macrocycle contains one pair of overlapping resonances and the phytyl side chain two groups of five overlapping resonances and one group of four overlapping resonances, due to the repeating C₅ unit in the side chain. In bacteriopheophytin a more overlap exists in the macrocycle: two groups of three resonances and two pairs.

Significant cross peaks that correspond to transfer over 3 carbon-carbon bonds appear at 4 ms mixing using a RIL zero-quantum recoupling sequence. Phase-sensitive spectra (with quadrature detection in both dimensions) are recorded with suppression of cross-peaks between sidebands of the same sideband family to "clean up" the spectrum and avoid misinterpretation of cross peaks.^3,4 Furthermore, the possible benefits of adiabatic pulse sequences like DREAM and through-bond correlation sequences like TOBSY⁵ over the RIL sequence will be discussed.

¹ M. Baldus, M. Tomaselli, B.H. Meier and R.R. Ernst, Chem.Phys.Letters 230, 329, (1994).
² R. Verel, M. Baldus, M. Ernst and B.H. Meier, Chem.Phys.Letters 287, 421, (1998).
³ A. Hagemeyer, K. Schmidt-Rohr and H.W. Spiess, Adv.Mag.Res. 13, 85, (1989).
⁴ A.P.M. Kentgens, E. de Boer, W.S. Veeman, J.Chem.Phys. 87, 6859, (1987).
⁵ M. Baldus, R.J. Iuliucci and B.H. Meier, J.Am.Chem.Soc. 119, 1121, (1997).

069
Session Assigned: M&T P
Title: Solid-State C-13 NMR Studies of Alpha-Elastin

Institute: University of Hawaii at Manoa
Keywords:
Abstract:
Elastin is the major protein component of elastic fibers, such as those found in blood vessels, lungs, and skin. This protein is believed to be responsible for the elastic properties of these structures. Elastin is a multi-domained protein of considerable molecular weight (>70 kDa). Its sequence consists largely of repeating hydrophobic regions interspersed with alanine-rich lysine-containing sequences. To date, there are no high-resolution data that describes the structure of this protein.

C-13 CPMAS spectra will be presented for samples of alpha-elastin prepared from the aorta of normal and copper-deficient pigs. The latter has a decreased number of lysine-derived crosslinks, which are believed to be essential in normal elastic function. The peaks in both spectra have similar chemical shifts, particularly of the backbone carbons, which is indicative of similarities in the overall structures of normal and defective elastin. However, difference spectra reveal that the undercrosslinked sample has more intensity in the aliphatic region. These difference peaks have chemical shifts of methyl groups and a number of alpha-carbons, for example. This result draws attention to the nature of crosslinking in elastin. Results of preliminary solid-state spectral editing experiments on these large peptides will also be discussed.

070
Session Assigned: M&T P
Title: Conformational transitions of human calcitonin in the process of fibril formation as studied by hign resolution solid state ¹³C NMR

Institute: Himeji Institute of Technology, Hyogo, Japan
Keywords: Fibril, Human calcitonin, solid state ¹³C NMR
Abstract:
Conformational transitions of human calcitonin (hCT) during fibril formation in the acidic and neutral conditions were investigated by high resolution solid state ¹³C NMR spectroscopy. In aqueous CH₃COOH solution (pH 3.3), an a-helical form is dominant around Gly10, whereas a random coil is present at Phe22 and Ala26, and a turn structure is formed at Ala31 as viewed from the ¹³C chemical shifts determined from the ¹³C DD-MAS NMR spectra. On the other hand, b-sheet structures at Gly10 and Phe22, both b-sheet and random coil conformations around Ala26 and both b-sheet and turn conformations at Ala31 were detected by ¹³C CP-MAS NMR experiments in the fibril grown in acidic conditions. The results indicate that conformational transitions from a-helix to b-sheet, and from random coil to b-sheet structures occurred in the central and C-terminus regions, respectively, during the fibril formation. The increase of resonance intensities after the delay time observed in the CP-MAS NMR spectra suggests that the fibrillation can be explained by a two step reaction mechanism in which the first step is a homogeneous association and the second step is an autocatalytic heterogeneous fibrillation. In contrast to the fibril at pH 3.3, the fibril at pH 7.5 formed a b-sheet conformation at the central region and exhibited a random coil at the C-terminus region. It is postulated that not only a hydrophobic interaction among the amphiphilic a-helices, but also an electrostatic interaction between charged side-chains plays an important role for the fibril formation at pH 7.5 and 3.3 acting as electrostatically favorable and unfavorable interactions, respectively. These results suggest that hCT fibrils are formed by stacking antiparallel b-sheets at pH 7.5 and a mixture of antiparallel and parallel b-sheets at pH 3.3.

071
Session Assigned: M&T P
Title: Investigation of the proton transfer regulatary role of Histidine-37 in the Influenza A Virus M2 Protein by Solid State NMR

Institute: NHMFL, Florida State University, Tallahassee, FL
Keywords:
Abstract:
The trans-membrane ion channel formed by a tetramer of M2 protein from influenza A plays an important role in viral infection. The low pH in the endosome activates the M2 ion channel so that protons are transfered into the virus particle which leads to viral uncoating. His-37 is the only a.a in the transmembrane domain which is potentially capable of changing its charge state in the range of pH 5.4 ~ 7.5.

The protonation and charge states of N-pi and N-tao in His 37 imidazole in M2 channel transmembrane peptide (M2-TMP) have been investigated from hydrated powder samples by CPMAS. When the pH changes from 5 to 9, deprotonation occurs . At pH 7 there is a mixture of charged and neutral histidines present. Orientational constraints for this histidine sidechain were also aquired using N¹⁵ PISEMA experiments on oriented samples under different pH values . The regulatary function of this histidine residue in proton conductance of M2 will be discusseed in detail.

072
Session Assigned: M&T P
Title: Determination of the Solid-State Conformations of Polyalanine Using Magic-Angle Spinning NMR Spectroscopyrs in Polypeptides Using A Simple One-Dimensional Method

Institute: The University of Michigan
Keywords:
Abstract:
Conformations of the powder samples of poly-L-alanine with molecular weights 356Da (tetraalanine), 15000Da (PLA-200) and 23600Da (PLA-333) were characterized by the 13C cross-polarization magic-angle spinning (CPMAS) and 1H combined rotation and multiple pulse (CRAMPS) solid-state NMR spectroscopy. From the 13C and 1H isotropic chemical shift values, it is predicted that the main chain conformations of tetraalanine and PLA-200 are mainly beta-sheet while the conformation of PLA-333 is mainly an alpha-helix. It is unusual and interesting that a high molecular weight homopolypeptide, PLA-200, has a beta-sheet conformation rather than an alpha-helix conformation. The effect of dichloroacetic acid (DCA) solvent on the backbone conformation of these peptides was also studied. It is inferred from solid-state NMR results that conformations of tetraalanine and PLA-333 are similar before and after crystallization from DCA. On the other hand, the backbone conformation of PLA-200 is 60% alpha-helix and 40% beta-sheet after crystallization from the DCA solvent.

073
Session Assigned: M&T P
Title: Structure Determination of Selectively and Extensively ¹³C-Labeled Proteins by Multidimensional Solid-State NMR

Institute: University of Massachusetts
Keywords:
Abstract:
The efficient structure determination of noncrystalline proteins by solid-state NMR requires the ability to measure multiple structural constraints in a single experiment on a single sample. To achieve this goal, we have developed a new approach combining 2D and 3D magic-angle-spinning NMR with novel isotopic labeling schemes. Selective and extensive ¹³C labeling simplifies the NMR spectra, reduces line broadening, and facilitates the measurement of long-range couplings. We demonstrate this labeling pproach on a model globular protein (M_r 8500) and a membrane protein (M_r 22000). Based on this labeling approach, we have simultaneously measured multiple backbone torsion angles phi on the model protein, designed and implemented several 2D and 3D resonance assignment experiments, and developed a diagnostic method for secondary structure determination based on ¹³C chemical shift anisotropy. The high spectral resolution and sensitivity achieved in these experiments indicate that the new isotope-edited NMR approach is a promising route to the efficient structure determination of membrane proteins and protein aggregates. (NSF grant MCB-9870373)

075
Session Assigned: M&T P
Title: Orientational Constraints for the Influenza Viral M2 Protein from 2D-PISEMA in Solid-State 15N NMR

Institute: National High Magnetic Field Lab,Tallahassee,FL,US
Keywords:
Abstract:
The integral membrane protein, M2 (97 amino acids), functions as a tetramer forming a proton channel in the influenza A virion. Its single transmembrane portion, M2-TMP, is a 25 amino acid peptide showing analogous channel activity. M2-TMP adopts primarily an a-helical structure, and a four-helix bundle in lipid environment.

We have used solid-state 15N NMR to investigate this M2 / M2-TMP system. Two-dimensional PISEMA has been applied to selectively and multiply 15N labeled M2-TMP samples well oriented in DMPC or DOPC lipid bilayers. The orientation dependent 15N chemical shifts and 15N-1H dipolar splittings from high resolution 2D spectra are correlated and analyzed for defining the polypeptide backbone structure.

The orientation of the transmembrane helix is primarily independent of lipid choice and hydrophobic thickness suggesting that the helix tilt is an integral property of the four-helix bundle held together by relatively specific interactions. The backbone structure also appears to be unaffected by the binding of the antiviral drug, amantadine, in the channel pore. We are now extending the solid-state 2D PISEMA experiments to uniformly 15N labeled, biosynthetic M2 samples.

076
Session Assigned: M&T P
Title: Structural elucidation of biologically active peptides in the magnetically oriented lipid bilayers

Institute: Himeji Institute of Technology, Hyogo, Japan
Keywords: Biologically active peptide, lipid bilayer
Abstract:
Magnetically oriented lipid bilayer systems have been recognized as a powerful means to elucidate the structure and dynamics of biological molecules in biomembranes. Although diamagnetic molecule are not normally oriented spontaneously to the magnetic field because of a very small diamagnetic anisotropy, assembly of lipid bilayers in the liquid crystalline state can give enough diamagnetic anisotropy to align to the magnetic field such as bicelles. We have found that dimyristoylphosphatidylcholin (DMPC) containing melittin or dynorphin exhibits a new class of magnetic ordering of lipid bilayers by forming a elongated liposome as a result of induced fragmentation and fusion of the membranes. It was clearly proved that the lipid bilayers form vesicle rather than a disc type bilayers such as bicelles by measuring potassium leakage using an ion selective electload. The shape of the elongated liposome was investigated by analyzing the ³¹P NMR lineshape. Actually it turned out that the longer axis of the elongated liposome was 5 times longer than the shorter axis. We also found that acidic membranes such as dimyristoylphosphatidylgrycerol (DMPG) did not show magnetic ordering in the neutral condition, while magnetic alignment was seen in the acidic condition. This fact indicate that the surface charge plays an essential role in the interaction between the lipid and the peptide.

We have developed the method to elucidate the structure and orientation of a-helical peptide bound to magnetically oriented membranes by inspecting ¹³C chemical shift tensor of the carbonyl carbon in the case where the a-helix is freely rotating in the magnetically oriented membranes about the helical axis. Because the C=O bond in the helix is nearly parallel to the d₂₂ axis of the ¹³C chemical shift tensor, it is shown that the orientation of peptide in the membrane can be determined by inspecting the ¹³C chemical shifts in the oriented state together with the ¹³C anisotropic and isotropic chemical shifts determined under a slow MAS and a fast MAS conditions, respectively. Using this strategy, we have examined the structure of melittin and alamethicin bound to the magnetically oriented membranes. The result indicated that melittin is located in the surface of membrane, although the center region is inserted into the membrane. On the other hand, the helical axis of alamethicin is aligned perpendicular to the lipid surface.

It is clearly demonstrated that the new class of magnetically oriented lipid bilayers is useful to obtain the information of the molecular orientation of biologically active membrane peptides.

077
Session Assigned: M&T P
Title: The structure and dynamics of a membrane anchored amphipathic peptide on a bilayer surface

Institute: CCRC, University of Georgia, Athens, GA
Keywords:
Abstract:
Field oriented phospholipid bilayer disks, or bicelles, can be used to mimic the environment of biological membranes while allowing unique spectroscopic measurements of structural and dynamic properties for molecules that are partially oriented through their association with the bilayer. The degree of order for bicelle associated molecules is too high to allow conventional proton detected experiments but through the use of heteronuclear observe, solid-state, NMR techniques it is possible to measure dipolar coupling and CSA data for various sites in the peptide. These anisotropic spin interactions yield information on the conformational and motional properties of the studied peptide on the bilayer surface. Moreover, these parameters can be measured with adequate resolution to work with multiple isotope labels in single molecular entities.

Site specifically 13C and 15N labeled peptides that are based on the N-terminal helix of ARF1, a myristoylated protein important for membrane trafficking, were synthesized with and without a lipid anchor. Dipolar couplings and CSA data from both the backbone and the aromatic side chains were measured by carbon and nitrogen observe 1D and 2D NMR experiments including proton detected separated local field spectroscopy. Order-matrix calculations were used to analyze the data.

Through the use of solid-sate NMR techniques the orientation of a helical, membrane anchored peptide with respect to the surface of a bilayer has been determined. Effects of the lipid anchor and the behavior of the hydrophobic aromatic side chains will also be discussed.

078
Session Assigned: M&T P
Title: ²³Na NMR in DNA Quadruplexes

Institute: MIT/Harvard Center for Magnetic Resonance
Keywords:
Abstract:
Many recent developments in solid-state NMR of quadrupolar nuclei allow for the direct study of quadrupolar nuclei in biological macromolecules. We present a ²³Na NMR study of guanine rich DNA oligonucleotides which associate to form G-quadruplexes. DNA quadruplexes have been proposed to form at the ends of telomeres, the physical end of chromosomes. DNA quadruplexes have also recently attracted interest as potential targets for anti-cancer drugs. The guanines in the quadruplexes form G-quartets, and the structures are stabilized by coordination of monovalent cations such as sodium and potassium to the carbonyl oxygens in the center of the quartets. We have performed ²³Na NMR on the tetrameric and dimeric quadruplexes formed by [d(TG₄T)]₄ and [d(G₄T₄G₄)]₂, respectively, to identify and assign the multiple cation binding sites in the center of the quadruplexes. We use high field strengths (17.6T) and several 2D methods to obtain valuable resolution, connectivity information, and assignments. Issues of sample preparation and temperature control will also be discussed. Our goal is to clarify the mechanism of competitive exchange of sodium with other cations to better understand the stabilization of DNA quadruplexes.

079
Session Assigned: M&T P
Title: Structure Elucidation of Organic Molecules with 15N-13C,15N-2H and 13C-2H REDOR NMR Spectroscopy

Institute: FU Berlin, Dept. of org. Chemistry
Keywords:
Abstract:
N-1-octyl-D-gluconamide is a medium sized amphiphile which exhibits structural polymorphism in the solid state. Two different modifications with unknown X-ray structure have been studied by ¹⁵N labeled, natural abundance ¹³C Rotational Echo DOuble Resonance (REDOR) NMR spectroscopy. From these experiments ¹³C-¹⁵N magnetic dipolar couplings in the range from 45Hz to 1220Hz have been elucidated and the corresponding CN distances varying from 4.1&Aring; down to 1.35 &Aring; have been determined. These dipolar couplings have been employed together with chemical shift data and ab initio calculations to elucidate the molecular conformation of these modifications. The resulting structures have been compared to X-ray data of a crystalline modification of gluconamide with know X-ray structure.

The application of composite pulse schemes to rotational echo double resonance (REDOR) spectroscopy of X- ²H (X : spin 1/2, observed) systems with strong quadrupolar interaction (.omega._Q >> .omega.₁ ) has been studied experimentally and theoretically and compared to simple .pi. pulses. The basic properties of the composite pulses have been elucidated using average hamiltonian theory and the exact simulation of the experiments were performed by stepwise integration of the equation of motion of the density matrix. As experimental example REDOR experiments on doubly ¹⁵N-²H labeled acetanilide and singly ²H labeled natural abundance ¹³C acetanilide have been performed. The most efficient dephasing has been observed for the 90_x-180_y-90_x composite pulse, followed by the 90_x-90_y-90_x. The dephasing of the simple 180_x pulse is about a factor of 2 less efficient than the other sequences. From the experiments the ²H-¹⁵N magnetic dipolar couplings between the amino deuteron and the amino nitrogen and the ²H-¹³C dipolar couplings between the amino deuteron and the .alpha. and .beta. carbons have been elucidated and the corresponding distances have been determined. From these data the secondary structure of the acetanilide can be determined.

As an application towards larger bioorganic molecules experiments on two different modifications of polyglycine and SH3 are presented.

Institute: Department of Chemistry, Colorado State University
Keywords: Solid State NMR; Multiple Pulse Refocusing;
Abstract:
There are many applications in which the isotropic chemical shifts of solid samples are of main interest, but magic angle spinning (MAS) is difficult to apply. Renewed interest in using magic angle hopping (MAH) [1] as an alternative to MAS has been stimulated by applications such as in-situ monitoring of heterogeneous chemical reactions under flowing conditions [2], and characterizing samples with highly conductive materials [3]. We have developed a new MAH pulse sequence for obtaining one-dimensional high resolution NMR spectra of solids and demonstrated it using a recently built MAH probe with a 120 degree hopping time less than 30 ms. The new pulse sequence employs two hops instead of the three hops used in the original pulse sequence[4]. The signal-to-noise ratio is increased by more than a factor of two. As the minimal number of steps for phase cycling is reduced form 8 to 4, 50% of the total experiment time can be saved, if S/N considerations permit, which can be important, especially for chemical kinetics studies. The key for realizing this two-hop approach is to employ newly developed 8-pulse refocusing sequences to obtain balanced refocusing on both x and y components of magnetization with high efficiency. The balanced refocusing efficiency of various pulse sequences will be discussed on the basis of both simulations and experimental results. The resolution of MAH spectra has been improved to a level that is very close to that of MAS spectra. The sensitivity is still inferior to that of a MAS experiment for the same amount of sample. Methods for increasing S/N and reducing the MAH experiment time will be discussed in detail. However, even at the current stage of development, the lower sensitivity of MAH can be substantially compensated by its large sample volume at very high magnetic fields, where very small samples have to be used in MAS experiments to achieve high spinning speeds. We are currently exploring various novel applications of the MAH approach.

[1] A.D. Bax, N.M. Szeverenyi, and G.E. Maciel, J. Magn. Reson., 52, 147, 1983.
[2] C. Keeler, J. Xiong, H. Lock, S. Dec, T. Tao, and G. E. Maciel, Catalysis Today, in press, 1998.
[3] H. Lock, T. Tao, J. Xiong, C. Keeler, and G.E. Maciel, Solid State NMR, submitted, 1998.
[4] N.M. Szeverenyi, A. D. Bax,and G.E. Maciel, J. Magn. Reson., 61, 440, 1985.

081
Session Assigned: M&T P
Title: Variable Angle Sample Spinning (VASS) of Field-Oriented Bicelles and a Membrane-associated Peptide

Institute: CCRC, The University of Georgia
Keywords:
Abstract:
In the structural study of membrane proteins and membrane-associated molecules by NMR, oriented phospholipid bilayer disks or bicelles are used as membrane mimics that can yield useful information through residual dipolar couplings. However, there is a need to resolve the sign ambiguities of the dipolar interaction, as well as a need to convert second order NMR spectra to first order. It is well known that the orientation of the nematic liquid crystal director to the magnetic field can be tuned using the variable angle spinning technique (1). The tilting of the director from the magnetic field depends on the magnetic susceptibility of the sample, the angle between the spinning axis and the magnetic field (spinning angle), and the spinning rate. With appropriate combination of these parameters, various anisotropic interactions can be scaled down.

The liquid crystal director behavior of field-oriented bicelles (DMPC/DHPC, DMPC/CHAPSO) in VASS has been probed by 31P NMR. A stable configuration occurs when the spinning angle is smaller than the magic angle, 54.7 degree, and the director or bilayer normal of the disks is mainly distributed in a plane perpendicular to the rotation axis. As the spinning angle approaches the magic angle, however, significant deviation of the director from the plane is evident from the patterns and intensities of the spinning sidebands. Using VASS in these angular range and in combination with multiple-pulse techniques, the dipolar interaction can be scaled to the same order as the J-coupling. Thus, the sign of the N-H and C-H dipolar interactions of a membrane-associated peptide can be determined.

(1) J. Courtieu, J. P. Bayle and B. M. Fung, Progress in NMR Spectroscopy, 26, P. 141-169 (1994).

082
Session Assigned: M&T P
Title: High Field Dynamic Nuclear Polarization with a Resonant Cavity and Low Microwave Power

Institute: Francis Bitter Magnet Lab, MIT, Cambridge, MA02139
Keywords:
Abstract:
Dynamic nuclear polarization (DNP) experiments, that transfer the large polarization available from electron to nuclear spins, can be used to increase signal-to-noise ratios in solid state NMR experiments by one to two orders of magnitude. In previous experiments we have utilized a high power (~50 watts) cyclotron resonance maser (gyrotron) operating at 2 mm wavelengths, together with a low-Q horn/reflector resonance structure, to achieve enhancements of ~185. Recently, we have significantly increased the enhancement to a value of ~300. More importantly, this results was achieved with a newly designed triple resonance cavity (e^-, ¹H, ¹³C) with Q~10³ and only ~20 mW of 140 GHz microwave power. We describe the new double resonance cavity, evaluate its microwave and r.f. performance and discuss new applications it enables.

083
Session Assigned: M&T P
Title: Solid, Liquid and Supercritical Fluid DNP On a (0-40 w) 40 GHz DNP/ESR Spectrometer

Institute: Department of Chemistry, University of Utah
Keywords:
Abstract:
A microwave frequency synthesizer driven 40 GHz DNP/ESR spectrometer with variable power (0-40 w) is described. A quick in-situ ESR measurement can be conveniently used to set up the optimum DNP condition. Polystyrene doped with BDPA free radicals, where a solid state effect is dominant, is utilized to demonstrate the higher enhancement factor associated with higher microwave power. DNP results on supercritical ethylene + BDPA will be presented where a positive Overhauser DNP enhancement arising from the scaler interaction together with noticeable contributions from solid state and thermal mixing effects are observed. These results are contrasted with the pure negative Overhauser DNP enhancement of liquid toluene + BDPA where a dipolar contribution is dominant. Preliminary 15N MAS/DNP results at natural abundance obtained on pitch will also be demonstrated.

084
Session Assigned: M&T P
Title: Advances in (MQ)DOR

Institute: National Institute of Chemical Physics & Biophys.
Keywords: DOR MQ quadrupole nuclei
Abstract:
Recent development of Double Rotation technology has introduced a considerable extension to analytical possibilities in solid state NMR of quadrupole nuclei. Improved design, very accurate mashining of rotors and automated intelligent rotation control provide for long time 2D data acquisition and measurements in magnets with a reduced bore size. Spectra of various zeolites, glasses and oxides, demonstrating also advantages of method compared to e.g. MQMAS, are presented.

085
Session Assigned: M&T P
Title: CP transfers between spin-1/2 and quadrupolar nuclei under fast MAS

Institute: University of Lille
Keywords: Cross Polarisation; interatomic connectivities
Abstract:
The process of cross polarization (CP) between a group of strongly coupled spin-1/2 nuclei I and a half integer quadrupolar spin S is analyzed under the condition of very fast MAS. When the second-order quadrupole interaction is neglected, the polarization transfer can be described by a simple formula, which includes the dipolar couplings, the spinning speed, the S spin value, the strengths of RF-fields involved and the off-resonance effects. When second-order quadrupole interactions are introduced, many of the "resonances" in the Hartmann-Hahn matching curve become less efficient. The cross polarization processes that involve multiple quantum transitions are also analyzed and explained. The analysis of interatomic connectivities in solids via CP-MQMAS and HETCOR-MQMAS is discussed.

086
Session Assigned: M&T P
Title: Manipulation of Nuclear Spin Interactions in Solid State MAS NMR: Simultaneous Frequency and Amplitude Modulation and Applications

Institute: NHMFL, Tallahassee, FLorida, USA
Keywords:
Abstract:
A significant feature of solid state MAS NMR is that although the anisotropic interactions containing rich structural information (for example, chemical shift anisotropiy represents the distribution of electrons around nuclei, and dipolar interaction are related to internucler distance) are suppressed by MAS, thus leading to high resolution solid state NMR spectra, they can be selectively recovered by spectroscopic manipulations. It has been demonstrated [1] that the simultaneous frequency and amplitude modulation (SFAM) allows one to interfere with the effect of sample spinning on heteronuclear dipolar interaction preventing the dipolar interaction from being removed by MAS. In this presentation, the features of the sophisticated SFAM will be disccussed in detail and applications to polypeptides will be addressed.

[1] R. Fu, S. Smith, and G. Bodenhausen, Chem. Phys. Lett. <b>272</b>, 361 (1997).

087
Session Assigned: M&T P
Title: Spin Dynamics of Polarization Transfer in PISEMA Experiment of Multiple-Spin Systems

Institute: National High Magnetic Field Laboratory
Keywords:
Abstract:
The spin dynamics of polarization transfer in the PISEMA experiment (Wu, Ramamoothy and Opella, J. Magn. Reson. A109 270 (1994)) of a spin-1/2 (¹³C, ¹⁵N, etc.) coupled to a group of proton spins is studied theoretically, numerically and experimentally, and the origin of resolution enhancement by PISEMA technique is investigated. The analytical solution of polarization transfer in two- and three-spin systems along with numerical calculation of more complicated spin systems show that in the dipolar spectrum of a directly bonded spin-pair the broadening caused by long-range coupling is second-order therefore the resolution of PISEMA experiment is far better than that of the conventional separated local-field experiment. The effects from the ¹H resonance offset and the Hartmann-Hahn mismatch in the PISEMA experiment are also studied and it is found that the ¹H resonance offset affects the dipolar splitting while the Hartmann-Hahn mismatch is compensated by the pulse sequence. Single crystal samples of pentaerythritol and methyl-a-D-glycopyranoside were used to demonstrate these results and effects.

088
Session Assigned: M&T P
Title: Influence of Chemical Shift Anisotropy on DQ MAS Spectra of Dipolar Solids

Institute: Universite H. Poincare, Nancy 1, France
Keywords:
Abstract:
We have shown recently that the proton chemical-shift anisotropy of hydrate crystals affects dipolar single-quantum powder pattern at 7.0 T (Tekely,P.,Palmas,P.,Mutzenhardt,P.,J. Magn.Reson.127,1997,238).

In this study, we present an experimental and theoretical evidence of the effect of chemical shielding interaction on the double-quantum spinning sideband patterns. The NMR double-quantum response to a five-pulse sequence of dipolar

coupled spin-1/2 pairs in powder sample of barium chlorate monohydrate spinning at the magic angle is investigated for different spinning frequencies and different duration of excitation and reconversion periods. The basic understanding of the effect of chemical shielding interaction on MQ spinning sideband patterns can be exploited to determine with good precision the internuclear distances and mutual orientation of dipolar and shielding tensors.

089
Session Assigned: M&T P
Title: Investigation of Hydrogen Bonding and Aromatic <FONT FACE="Symbol">p Packing Effects in Solids by¹H Double Quantum MAS NMR Spectroscopy

Institute: Max-Planck-Inst. f. Polymer Research,Mainz,Germany
Keywords: fast magic angle spinning, abundant spin systems, dipolar connectivities
Abstract:
Solution-state ¹H NMR is today one of the most widely used and powerful spectroscopic methods. In comparison, the use of solid-state ¹H NMR has to date been restricted on account of the substantial dipolar line broadening, which hinders the spectral resolution of chemically distinct sites. Recently, new MAS probes capable of spinning frequencies up to 35 kHz have become available. Such spinning frequencies lead to significantly enhanced resolution, making structure determination by ¹H NMR in the solid state feasible[1]. <BR>

Since the dipolar interaction provides valuable information about the structure and dynamics of the spin system, the spectroscopist desires a method which achieves spectral resolution without losing the information inherent to the dipolar coupling. In this context quantitative ¹H double-quantum (DQ) MAS spectroscopy has recently been introduced [2]. The intensities of the peaks in 2D DQ MAS spectra reflect the strength of the dipolar interaction and the number of involved spin pairs. Information about through-space proton-proton proximities, both intra- and intermolecular, is thus provided - in this way, the method can be considered to be analogous to the solution-state NOESY experiment. The inherent high sensitivity of ¹H NMR means that typical DQ spectra can be obtained in under 2 hours.

The principal method for structure determination in solids is X-ray scattering - however the method has problems in accurately determining proton positions and neutron scattering of deuterated species is required. In this regard, ¹H DQ MAS spectroscopy, which is fast, and requires neither isotopic labelling nor the preparation of single crystals, provides information which is both unique and complimentary to scattering. As a first example, we present investigations of hydrogen bonding in N-alkyl-benzoxazine dimers which serve as model compounds for polymerised phenolic resin systems. Both 1D MAS and 2D DQ MAS spectra of these dimers provide evidence for the presence of both intramolecular O--H--N and intermolecular O--H-O hydrogen bonds, and further indicate clear changes in topology on changing the alkyl substituent [3]. In order to demonstrate the versatility of our new approach, ¹H spectra of substituted hexabenzocoronenes (HBC) are presented, where the stacking of the extended aromatic p systems leads to the resolution in standard MAS spectra of different aromatic ¹H resonances, with the proximities identified in the DQ spectrum inferring information about the stacking arrangement. An analysis of DQ spinning sideband patterns further provides information about the dynamical changes which occur on the formation of liquid-crystal phases.

[1] C. Filip, S. Hafner, I. Schnell, D.E. Demco and H.W. Spiess, J. Chem. Phys. 110 (1999) in press.
[2] J. Gottwald, D.E. Demco, R. Graf, and H.W. Spiess,Chem. Phys. Lett. 243, 314 (1995).
[3] I. Schnell, S. P. Brown, H. Y. Low, H. Ishida, and H. W. Spiess, J. Am. Chem. Soc.120, 11784 (1998).

We thank H. Y. Low and H. Ishida, and J. D. Brand and C. Kayser for providing the benzoxazine and HBC samples, respectively.

090
Session Assigned: M&T P
Title: An MQMAS, CP/MQMAS, HETCOR NMR study of Crystal Formation of AlPO4-CJ2

Institute: University Louis Pasteur, Strasbourg
Keywords: MQMAS, CPMQMAS, HETCOR
Abstract:
AlPO4-CJ2 has been investigated using MAS NMR of all the available nuclei and it has been shown that its diffraction structure and NMR are in agreement with a tetrameric unit composed of two aluminum and two phosphorus. The aluminum s are in five and six coordination and a hydroxide is bridging the two aluminum. This hydroxide may be replaced by a fluorine. It has been shown by in-situ NMR, that this bridging is formed during nucleation and growth of the solid. It is the aim of this contribution to show the different NMR methods that allow to analyse the structure of CJ2. Al-F, Al-P and P-F CP/Hetcor, MQMAS and CPMQMAS have been employed.

The CPMQMAS allows edition of the different coexisting phases, amorphous fluorinated and the titled compound, obtaining therefore on microporous compounds an information of a pure crystal, on a mixture of phases. This coexistence of phases is very general in microporous compounds characterization and can be propose as a general edition method.

Optimization of CP Hetcor experiments is discussed. Cross-polarization steps are first discussed in both cases of Al-P and Al-F. The special case of Al-F allows a more detailed discussion of cross-polarization at 30 kHz.

Dipolar proximity information is emerging from the three different 2D Hetcor nuclei combination, Al-P, Al-F and P-F. It allows to state that the condensation steps, networking and unit bridging are realized in a single stage.

Complete list of authors:

David Lang,(b) Alain Bailly,(a,b) Marek Pruski,(b) Jean Paul Amoureux,(a) Clarisse Huguenard,(c) Mohamed Haouas,(c) Corine Gerardin,(c) Francis Taulelle,(c)Thierry Loiseau,(d) Gerard Ferey (d)