Theory of Heteronuclear NMR Spectroscopy and its Application

Theory of Heteronuclear proton charismatic sonorousness Spectroscopy and its ApplicationSYED MASOOD HASSAN AKBARIQuestion 1 pull back theory of heteronuclear proton magnetic resonance spectroscopic abbreviation and its use in pharmaceutical analysis. authoritative strategies for determining the organises of tissue layer proteins in lipid environments by proton magnetic resonance spectrometry rely on the anisotropy of nuclear plait interactions, which ar experimentally affable through experiments performed on weakly and completely adjust warnings. Importantly, the anisotropy of nuclear spin interactions results in a partping of building to the resonance frequencies and splattings discover in nuclear magnetic resonance spectra. Distinctive wheel-like patterns atomic number 18 observed in matted 1H15N heteronuclear dipolar/15N chemical substance shift PISEMA (polarization inversion spin-exchange at the dissembling angle) spectra of coiled tissue layer proteins in hi ghly re place lipid bilayer samples (Marassi and Opella, 2000 Wang et al., 2000). One dimensional dipolar waves are an extension of flavourless PISA (polarity index peddle angle) wheels that map protein structures in nuclear magnetic resonance spectra of twain weakly and completely aligned samples (Marassi and Opella, 2000). dipolar waves describe the periodic wave-like variations of the magnitudes of the heteronuclear dipolar bring togethers as a function of counterweight number in the absence of chemical shift effects. Since weakly aligned samples of proteins display these same effects, primarily as residual dipolar marriages, in solution nuclear magnetic resonance spectra, this represents a convergence of solidness and solution NMR approaches to structure finis (Marassi and Opella, 2000).NMR structural studies of proteinsThere are threesome bargainer spectroscopic considerations for NMR structural studies of proteins the overall rotational coefficient of correlation time of the protein, the extent of alignment of the protein in the sample, and the strategy for appointee of the resonances to websites in the protein. Each of these considerations needs to be taken into account in the knowledge of NMR for structural studies of membrane proteins (Opella, 1997). For comparatively small globular proteins, the sample conditions, instrumentation, experiments, and calculations that lead to structure decisiveness are well established (Cavanagh et al., 1996). The promontory requirement for structure determination of globular proteins is that samples send packing be alert of isotopically labelled polypeptides that are folded in their native conformation and reorient relatively rapidly in solution. Such samples have been prepared for many hundreds of proteins, and it is plausibly that this can be done for thousands more of the polypeptide sequences found in genomes (Wuthrich, 1998). This is non yet the case for membrane proteins.Resonance assignme ntsThe traditional approach to protein structure determination is based on the same overall principles, whether solution NMR or solidness NMR methods are used and whether the sample is aligned or not. This involves the village of resonances through the use of isotopic labels and multidimensional NMR experiments, the measurement of spectral parameters associated with singular resonances, for example, NOEs, J couplings, dipolar couplings, or chemical shift frequencies, the assignment of all resonance to specific sites in the protein, and so the calculation of structures. There are examples of the application of this approach to membrane proteins in micelles (Almeida and Opella, 1997) and bilayers (Opella et al., 1999). The availability of orientation information associated with individual resonances path that it is now mathematical to make effective use of limited amounts of assignment information, for example, some resi callable-type assignments or a few sequential assignments. It may also be executable to implement an assignment-free approach. The use of either limited or no assignment information prior to calculating structures would greatly speed the process of structure determination by NMR spectroscopic analysis, especially in the case of membrane proteins where assignments are difficult to make in nearly all situations due to overlap of resonances and unfavourable relaxation parameters.Dipoledipole antenna interactionThe topical anesthetic anesthetic orbit, which results from the interaction among two nearby nuclei, is a indicate cum of structural information. Pakes (1948) seminal paper demonstrated that the dipoledipole interaction mingled with two spin S = 1/2 nuclei is manifested as a doublet in NMR spectra, with the frequency residual a function of not only the distance between the two nuclei nevertheless also the angle between the internuclear vector and the direction of the utilise magnetic field. The dipoledipole interaction provid es direct access to geometrical parameters that can be translated into molecular structures. Moreover, it is important for many aspects of solid-state NMR spectroscopy for example, it is essential to minimize its influence through decoupling to obtain well-resolved spectra. In this regard, it is largely easier to deal with heteronuclear rather than homonuclear dipolar couplings. Heteronuclear dipolar couplings are used extensively to stop the structures of proteins, in particular the 1H15N interaction at the amide sites in the protein key. resembling labelling with 15N is particularly valuable in proteins because the properties of a dilute spin are retained, since the next nearest amide north is separated by two carbon copy atoms in the polypeptide backbone (Cross et al., 1982). In addition, each 15N label in an amide site provides three spin interactions for analysis the 15N chemical shift, the 1H chemical shift, and, of course, the 1H15N heteronuclear dipolar coupling betwee n the two directly bring togethered nuclei. The dipoledipole interaction is anisotropic therefore, the value of the splitting varies with molecular orientation. It is maximal for an NH impound duplicate to the field, half-maximal when the bond is perpendicular to the field, and zero when the bond is at the magic angle. All of these possibilities are observed in experimental data from aligned proteins. The 1H15N heteronuclear dipolar interaction has the dual roles of providing a mechanism for resolving among resonances with NH bonds at different orientations and of providing the input for structure determination in the form of frequency measurements that can be translated into angles between individual bonds and the external axis imposed by the magnetic field. The angular information can then be used in conjunction with the well-established geometry of peptide planes to determine the three-dimensional structure of the polypeptide backbone (Opella et al., 1987). These methods can be extended to redundant nitrogen and carbon sites for characterization of side chain conformations. Separated local field spectroscopy (Waugh 1976) combines several of the elements of high-resolution solid-state NMR spectroscopy to average out the unwanted enormousening influences of homonuclear dipolar couplings and double resonance and multidimensional spectroscopy to average out and separate the heteronuclear dipolar couplings in different parts of the experiment. The chemical shift dimension in mat separated local field spectra is intrinsically high resolution because it is obtained small-arm decoupling the hydrogens to remove the broadening due to heteronuclear dipolar couplings. Homonuclear dipolar couplings are nominal among the dilute nuclei and generally do not require attention. This enables the dipolar couplings between bonded pairs of 1H and 15N nuclei to be measured for individual 15N sites with different chemical shift frequencies. The captain versions of separ ated local field spectroscopy have more than adequate resolution for studies of peptides or specifically or selectively labelled proteins. However, elevate improvements in resolution were needed for studies of uniformly 15N labelled proteins.PISEMA (polarization inversion spin-exchange at the magic angle) (Wu et al., 1994) is a high-resolution version of separated local field spectroscopy. Line widths in the key dipolar frequency dimension are reduced by more than one order of magnitude compared with the stuffy separated local field experiment. The combination of narrow lines and favourable scale factor has such a dramatic effect on the expression of the spectra that it is now feasible to formulate solid-state NMR experiments where heteronuclear dipolar coupling frequencies complement chemical shifts as a mechanism for spectroscopic resolution as well as the measurement of readily interpretable orientationally strung-out frequencies.PISA (polarity index slant angle) wheelsThe secondary structure and topology of a membrane protein can be described by the patterns of resonances observed in two-dimensional PISEMA spectra of uniformly 15N labelled polypeptides in aligned bilayers (Marassi and Opella, 2000 Wang et al., 2000). The characteristic wheel-like patterns observed in these spectra reflect helical wheel projections of residues in both transmembrane and in-plane helices. Therefore, PISA wheels provide direct indices of both secondary structure and topology. The resonance frequencies in both the 1H15N heteronuclear dipolar and 15N chemical shift dimensions in PISEMA spectra of aligned samples of membrane proteins depend on helix orientation as well as on backbone dihedral angles, the magnitudes and orientations of the principal elements of the amide 15N chemical shift tensor, and the NH bond length. It is possible to calculate spectra for any protein structure (Bak et al., 2002). The principals involved in the PISA wheel analysis of helices (Marassi an d Opella, 2000) are illustrated in Fig. 2. In Fig. 2A, the projection down the axis of a helical wheel shows that the 3.6 residues per acidify periodicity characteristic of an -helix results in an venting of light speed between adjacent residues. The drawing of a peptide plane in Fig. 2B shows the orientations of the principal axes of the three operative spin interactions at the 15N-labelled amide site. The 17 difference between the NH bond axis and the 33 principal element of the amide 15N chemical shift tensor is of particular importance because of its impact on the spectral appearance of a PISA wheel. The striking wheel-like pattern of resonances calculated from a two-dimensional PISEMA spectrum of an ideal helix is shown in Fig. 2C. A PISA wheel reflects the slant angle (tilt) of the helix, and the assignment of the resonances reflects the polarity index (rotation) of the helix. When the helix axis is parallel to the bilayer normal, all of the amide sites have an identical or ientation relative to the direction of the applied magnetic field, and therefore, all of the resonances overlap with the same dipolar coupling and chemical shift frequencies. Tilting the helix away from the membrane normal results in variations in the orientations of the amide NH bond vectors relative to the field. This is seen in the spectra as dispersions of both the heteronuclear dipolar coupling and the chemical shift frequencies. Nearly all transmembrane helices are tilted with respect to the bilayer normal, and it is the combination of the tilt and the 17 difference between the tensor orientations in the molecular frame that makes it possible to resolve many resonances from residues in otherwise uniform helices and is responsible for the wheel-like pattern in PISEMA spectra, such as that illustrated in Fig. 2C.Figure 1 Illustrates principles of PISA wheels (Marassi and Opella, 2000). (A) Helical wheel covering the 100 arc between adjacent residues that is a consequence of the periodicity of 3.6 residues per turn in an -helix (B) orientations of the principal elements of the spin interaction tensors associated with 15N in a peptide bond (C) PISA wheel for an ideal -helix (D) dipolar wave for an ideal -helix.Question 2 Structure Elucidation for C11H15NO.HClMw = 213.70FT-IRShows a sharp peak at 1690cm-1 which is illustration of a C=O functional multitude.There is a broad peak turning up at the 3500cm-1 representative of a C-H group.1H NMRShows a cluster of peaks from 7.62-8.02ppm show up as 5H. This means that the benzine ring is branched at one location.5.25ppm shows up as a 1H this is the CH group2.97-3.03ppm are the 2CH3 groups bonded to the Nitrogen.1.64ppm comes up as a doublet with 3H this means that it is a methyl.The strong peak at the 4.80ppm is representative of the amine.13C NMRThe utile information gathered from this spectra is as there are negative peaks showing up so the angle at which this spectra was got was at 1350 clearly showing the CH2 in the ring and the benzol facing down.196.51ppm shows the negative peak of the benzene ring.136.69ppm shows the CH2 groups in the benzene ring.The peaks ranging from 128.54-131.90 are of the symmetrical benzene ring carbons.69.57ppm is the CH3 group close to the ketone.41.29ppm is the CH group which is beside the ketone.14.46ppm is the 2 CH3 groups bonded to the amine.EI-MSShows a small signal at 29 m/z which is representative of a CHO group.And the signal at 72 m/z is representative of a H3CHC=N+(CH3)2 ion. chemical StructureFigure 1 Shows the structure of C11H15NO.HCl.ReferencesAlmeida FCL, Opella SJ. fd coat protein structure in membrane environments structural dynamics of a loop connecting a hydrophobic trans-membrane helix and an amphiapathic helix in a membrane protein.J. Mol. Biol.1997270481495.PubMedBak M, Schultz R, Vosegaard T, Nielsen NC. Specification and visualisation of anisotropic interaction tensors in polypeptides and numerical simulations in biological sol id-state NMR.J. Magn. Reson.20021542845.PubMedBax A, Kontaxis G, Tjandra N. dipolar couplings in macromolecular structure determination.Methods Enzymol.2001330127172.PubMedCavanagh J, Fairbrother WJ, Palmer AG, Skelton NS.Protein NMR spectroscopy.New York pedantic Press 1996.Chou JJ, Kaufman JD, Stahl SJ, Wingfield PT, Bax A. Micelle-induced curvature in a water-insoluble HIV-1 Env peptide revealed by NMR dipolar coupling measurement in stretched polyacrylamide gel.J. Am. Chem. Soc.200212424502451.PubMedCross TA, DiVerdi JA, Opella SJ. Strategy for nitrogen NMR of biopolymers.J. Am. Chem. Soc.198210417591761.Griffin RG. Dipolar recoupling in MAS spectra of biological solids.Nat. Struct. Biol. NMR Suppl.1998II508512.PubMedHoward KP, Opella SJ. High resolution solid-state NMR spectra of integral membrane proteins reconstituted into magnetically oriented phospholic bilayers.J. Magn. Reson.19961129194.PubMedMa C, Opella SJ. Lanthanide ions bind specifically to an added EF-hand and orie nt a membrane protein in micelles for solution NMR spectroscopy.J. Magn. Reson.2000146381384.PubMedMarassi FM, Opella SJ. A solid-state NMR index of helical membrane protein structure and topology.J. Magn. Reson.2000 revenue one hundred fifty155.PMC free articlePubMedMarassi FM, Ramamoorthy A, Opella SJ. Complete resolution of the solid-state NMR spectrum of a uniformly15N-labeled membrane protein in phospholipid bilayers.Proc. Natl. Acad. Sci. U.S. A.19979485518556.PMC free articlePubMedMcDonnell PA, Opella SJ. Effect of detergent concentration on multidimensional solution NMR spectra of membrane proteins in micelles.J. Magn. Reson.1993B102120125.Mesleh MF, Veglia G, DeSilva TM, Marassi FM, Opella SJ. Dipolar waves as NMR maps of protein structure.J. Am. Chem. Soc.200212442064207.PMC free articlePubMedOpella SJ. NMR and membrane proteins.Nat. Struct. Biol. NMR Suppl.1997I845848.PubMedOpella SJ, Stewart PL, Valentine KG. Structural analysis of solid-state NMR measurement of peptides and proteins.Q. Rev. Biophys.198719749.PubMedOpella SJ, Marassi FM, Gesell JJ, Valente AP, Kim Y, Oblatt-Montal M, Montal M. Structures of the M2 channel-lining segments from nicotinic acetylcholine and NMDA receptors by NMR spectroscopy.Nat. Struct. Biol.19996374379.PMC free articlePubMedPake GE. Nuclear resonance density in hydrated crystals fine structure of proton line.J. Chem. Phys.194816327336.Sanders CR, Hare BJ, Howard K, Prestegard JH. Magnetically-oriented phospholipid micelles as a tool for the study of membrane-associated molecules.Prog. NMR Spectrosc.199326421444.Veglia G, Opella SJ. Lanthanide ion masking to adventitious sites aligns membrane proteins in micelles for solution NMR spectroscopy.J. Am. Chem. Soc.200012211 73311 734.Wang J, Denny J, Tian C, Kim S, Mo Y, Kovacs F, Song Z, Nishimura K, Gan Z, Fu R, Quine JR, Cross TA. Imaging membrane protein helical wheels.J. Magn. Reson.2000144162167.PubMedWaugh JS. Uncoupling of local field spectra in nuclear magnetic resonance determination of atomic positions in solids.Proc. Natl. Acad. Sci. U.S. A.19767818941897.PMC free articlePubMedWu CH, Ramamoorthy A, Opella SJ. High-resolution heteronuclear dipolae solid-state NMR spectroscopy.J. Magn. Reson.1994A109270272.Wuthrich K. The second decade-into the third millennium.Nat. Struct. Biol. NMR. Suppl.1998II492495.PubMed