SYED MASOOD HASSAN AKBARIQuestion 1: Describe theory of heteronuclear NMR spectroscopy and its use in pharmaceutical analysis.Current strategies for determining the structures of membrane proteins in lipid environments by NMR spectroscopy rely on the anisotropy of nuclear spin interactions, which are experimentally accessible through experiments performed on weakly and completely aligned samples. Importantly, the anisotropy of nuclear spin interactions results in a mapping of structure to the resonance frequencies and splatting’s observed in NMR spectra. Distinctive wheel-like patterns are observed in two-dimensional 1H–15N heteronuclear dipolar/15N chemical shift PISEMA (polarization inversion spin-exchange at the magic angle) spectra of helical membrane proteins in highly aligned lipid bilayer samples (Marassi and Opella, 2000; Wang et al., 2000). One dimensional dipolar waves are an extension of two-dimensional PISA (polarity index slant angle) wheels that map protein structures in NMR spectra of both weakly and completely aligned samples (Marassi and Opella, 2000). Dipolar waves describe the periodic wave-like variations of the magnitudes of the heteronuclear dipolar couplings as a function of residue number in the absence of chemical shift effects. Since weakly aligned samples of proteins display these same effects, primarily as residual dipolar couplings, in solution NMR spectra, this represents a convergence of solid-state and solution NMR approaches to structure determination (Marassi and Opella, 2000).NMR structural studies of proteinsThere are three principal spectroscopic considerations for NMR structural studies of proteins: the overall rotational correlation time of the protein, the extent of alignment of the protein in the sample, and the strategy for assignment of the resonances to sites in the protein. Each of these considerations needs to be taken into account in the development of NMR for structural studies of membrane proteins (Opella, 1997). For relatively small globular proteins, the sample conditions, instrumentation, experiments, and calculations that lead to structure determination are well established (Cavanagh et al., 1996). The chief requirement for structure determination of globular proteins is that samples can be prepared 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 likely that this can be done for thousands more of the polypeptide sequences found in genomes (Wuthrich, 1998). This is not yet the case for membrane proteins.Resonance assignmentsThe traditional approach to protein structure determination is based on the same overall principles, whether solution NMR or solid-state NMR methods are used and whether the sample is aligned or not. This involves the resolution of resonances through the use of isotopic labels and multidimensional NMR experiments, the measurement of spectral parameters associated with individual resonances, for example, NOEs, J couplings, dipolar couplings, or chemical shift frequencies, the assignment of all resonance to specific sites in the protein, and then 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 means that it is now possible to make effective use of limited amounts of assignment information, for example, some residue-type assignments or a few sequential assignments. It may also be feasible 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 spectroscopy, 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.Dipole–dipole interactionThe local field, which results from the interaction between two nearby nuclei, is a direct source of structural information. Pake’s (1948) seminal paper demonstrated that the dipole–dipole interaction between two spin S = 1/2 nuclei is manifested as a doublet in NMR spectra, with the frequency difference a function of not only the distance between the two nuclei but also the angle between the internuclear vector and the direction of the applied magnetic field. The dipole–dipole interaction provides 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 generally easier to deal with heteronuclear rather than homonuclear dipolar couplings. Heteronuclear dipolar couplings are used extensively to determine the structures of proteins, in particular the 1H–15N interaction at the amide sites in the protein backbone. Uniform labelling with 15N is particularly valuable in proteins because the properties of a “dilute spin” are retained, since the next nearest amide nitrogen is separated by two carbon 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 1H–15N heteronuclear dipolar coupling between the two directly bonded nuclei. The dipole–dipole interaction is anisotropic; therefore, the value of the splitting varies with molecular orientation. It is maximal for an N–H bond parallel 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 1H–15N heteronuclear dipolar interaction has the dual roles of providing a mechanism for resolving among resonances with N–H 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 additional 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 broadening 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 two-dimensional separated local field spectra is intrinsically high resolution because it is obtained while decoupling the hydrogens to remove the broadening due to heteronuclear dipolar couplings. Homonuclear dipolar couplings are minimal 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 original versions of separated local field spectroscopy have more than adequate resolution for studies of peptides or specifically or selectively labelled proteins. However, further 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 conventional separated local field experiment. The combination of narrow lines and favourable scaling factor has such a dramatic effect on the appearance 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 dependent 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 1H–15N 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 N–H 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 and 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 turn periodicity characteristic of an ?-helix results in an arc of 100° 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 N–H 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 orientation 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 N–H 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 showing 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 representative of a C=O functional group.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 showing up as 5H. This means that the benzene 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 useful 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 benzene 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. 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