Theory of Heteronuclear NMR Spectroscopy and its Application

Theory of Heteronuclear NMR Spectroscopy and its Application SYED MASOOD HASSAN AKBARI Question 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 t he 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 proteins There 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 sequence s found in genomes (Wuthrich, 1998). This is not yet the case for membrane proteins. Resonance assignments The 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 al so be feasible to implement an â€Å"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 interaction The 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 protein s, 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 â€Å"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 â€Å"magic angle†. All of these possibilities are observed in experimental data from aligned proteins. The 1H–15N het eronuclear 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) wheels The 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 â€Å"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 whee l 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 ÃŽ ±-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 ÏÆ'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 ÃŽ ±-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 ÃŽ ±-helix; (D) dipolar wave for an ideal ÃŽ ±-helix. Question 2: Structure Elucidation for C11H15NO.HCl Mw = 213.70 FT-IR Shows 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 NMR Shows 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 group 2.97-3.03ppm are the 2CH ­Ã‚ ­3 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 NMR The 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-MS Shows 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+(CH ­3)2 ion. Chemical Structure Figure 1: Shows the structure of C11H15NO.HCl. References Almeida 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.1997;270:481–495.[PubMed] Bak M, Schultz R, Vosegaard T, Nielsen NC. Specification and visualization of anisotropic interaction tensors in polypeptides and numerical simulations in biological solid-state NMR.J. Magn. Reson.2002;154:28–45.[PubMed] Bax A, Kontaxis G, Tjandra N. Dipolar couplings in macromolecular structure determination.Methods Enzymol.2001;330:127–172.[PubMed] Cavanagh J, Fairbrother WJ, Palmer AG, Skelton NS.Protein NMR spectroscopy.New York: Academic 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.2002;124:2450–2451.[PubMed] Cross TA, DiVerdi JA, Opella SJ. Strategy for nitrogen NMR of biopolymers.J. Am. Chem. Soc.1982;104:1759–1761. Griffin RG. Dipolar recoupling in MAS spectra of biological solids.Nat. Struct. Biol. NMR Suppl.1998;II:508–512.[PubMed] Howard KP, Opella SJ. High resolution solid-state NMR spectra of integral membrane proteins reconstituted into magnetically oriented phospholic bilayers.J. Magn. Reson.1996;112:91–94.[PubMed] Ma C, Opella SJ. Lanthanide ions bind specifically to an added â€Å"EF-hand† and orient a membrane protein in micelles for solution NMR spectroscopy.J. Magn. Reson.2000;146:381–384.[PubMed] Marassi FM, Opella SJ. A solid-state NMR index of helical membrane protein structure and topology.J. Magn. Reson.2000;144:150–155.[PMC free article][PubMed] Marassi 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.1997;94:8551–8556.[PMC free article][PubMed] McDonnell PA, Opella SJ. Effect of detergent concentration on multidimensional solution NMR spectra of membrane proteins in micelles.J. Magn. Reson.1993;B102:120–125. Mesleh MF, Veglia G, DeSilva TM, Marassi FM, Opella SJ. Dipolar waves as NMR maps of protein structure.J. Am. Chem. Soc.2002;124:4206–4207.[PMC free article][PubMed] Opella SJ. NMR and membrane proteins.Nat. Struct. Biol. NMR Suppl.1997;I:845–848.[PubMed] Opella SJ, Stewart PL, Valentine KG. Structural analysis of solid-state NMR measurement of peptides and proteins.Q. Rev. Biophys.1987;19:7–49.[PubMed] Opella 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.1999;6:374–379.[PMC free article][PubMed] Pake GE. Nuclear resonance absorption in hydrated crystals: fine structure of proton line.J. Chem. Phys.1948;16:327–336. 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.1993;26:421–444. Veglia G, Opella SJ. Lanthanide ion binding to adventitious sites aligns membrane proteins in micelles for solution NMR spectroscopy.J. Am. Chem. Soc.2000;122:11 733–11 734. 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.2000;144:162–167.[PubMed] Waugh JS. Uncoupling of local field spectra in nuclear magnetic resonance: determination of atomic positions in solids.Proc. Natl. Acad. Sci. U.S. A.1976;78:1894–1897.[PMC free article][PubMed] Wu CH, Ramamoorthy A, Opella SJ. High-resolution heteronuclear dipolae solid-state NMR spectroscopy.J. Magn. Reson.1994;A109:270–272. Wuthrich K. The second decade-into the third millennium.Nat. Struct. Biol. NMR. Suppl.1998;II:492–495.[PubMed]

Gregory Crewdson

Crescendo's photographs draw on Gothic Romantic and he as an artist's focuses on dramatic surrealists. Known for overtly cinematic photographs that use tricks of light to convey their mystery. Photographs: often of suburban scenes that exude the kind of eerie terror of Hitchcock films. His photography advocates unanswered questions that the viewer can than answer them Correspond tryst to create transparency, a â€Å"perfect representation† and a â€Å"perfect world. (he does not want grain, pixels, His photos shift focus in the series of away from the strangeness of ordinary life Into the heightened surrealists of dream and fantasy Example o Figurative interior o Subject matter: A figure sitting on the bed, surrounded by roses and twigs, there is a further trail of twigs scattered on the ground leading from the living room to the omens bed, there are two doorways, one gives you a glimpse of the bathroom, the other leads too living room. Large depth of feel because we are able to see into the background Socrates atmosphere using all this light o The color pallet Is balanced by the lighting (subdued warm brown)o (contrast between Interior, night and roses, making the figure stand out) o Taken at levelly and It Is wide angled. O Domestic in time voyeur – looking from outside in o Draws on fears and anxieties. O Ugliness has been made beautiful

Work Sheet

* * Scavenger Hunt Worksheet Assignment Background: The point of this assignment is to get you to search and become familiar with the GCU Learning Management System (LMS) LoudCloud and the GCU web site. These two sites have many resources to help you be a successful student. Assignment Instructions: Answer the following questions that require you to search the LoudCloud classroom and GCU web site. Feel free to search around the sites until you find the things you are looking for. LoudCloud Questions 1.After logging into the LoudCloud classroom, list the tabs and other items on the screen when you first enter a course in LoudCloud. 2. From the course home page in LoudCloud, click on the â€Å"Tasks† tab and then click on â€Å"Forums†. Make notes on the various forums and their purposes. * * 3. Find the Student Success Center under the Resources Tab. Click on this link. List some of the services provided to students at this link. * * 4. Inside the Student Success Center, click on the Writing Center link. Click on LoudCloud Courses.What writing style is required for 100- and 200-level courses at GCU? 5. Inside the Student Success Center, click on the Support Services link. List the services available at this link. 6. Inside the Student Success Center, click on the Succeed at GCU link. Click on the GCU Tutorials link at the bottom of the page. What tutorials are available for students at this link? 7. Click on the Resources Tab in the LoudCloud classroom. List each section. Review each section and list what you find in it. 8.Click on the Course Home link the left-hand corner of the screen. List some of the components found on this page. 9. Go to http://library. gcu. edu. Click on the Frequently Asked Questions (on the left) link. List how a GCU student finds a Book, DVD, streaming video, or other multimedia at the GCU library: * GCU Web site Questions (http://www. gcu. edu) 1. Locate and click the Spiritual Life link on the right side of the screen. You will notice a navigation pane on the left side of the screen. Click the â€Å"+† sign next to Chapel and the Gathering.What information is listed under Chapel and the Gathering? 2. On the Home page, locate and click the Current Students button on the right side of the screen. Scroll down the page. What links are listed under the Student Resources section? 3. On the Home page, locate and click on the Resources tab at the top of the screen. Click the Technical Support link listed under the Support Services section. What information can be found under Technical Support? 4. On the Home page, click on the Contact Us tab which is located just above the home page footer.This is where you can locate phone numbers and addresses of the colleges, the main GCU switchboard, and the Student Services offices. List the phone numbers of the following departments located in the Student Contact Information section: * Career Services- * Center for Learning Advancement- * Academic Advising- * Finance Counselors- * Office of Academic Records- * Technical Support 5. On the Home page, notice the icons on the top right header area. What are the other icons listed next to theâ€Å"† for the GCU Facebook page? 6. On the Home page, click the About Us tab located at the top.What is the vision and mission statement for Grand Canyon University? 7. Also under the About Us tab, locate the four pillars of Grand Canyon University. 8. Navigate from the About Us tab to the Academics tab. Read about Accreditation near the bottom of the page. What did you learn? 9. Navigate from the Academics tab to the Student Life tab. Scroll down the page to locate and click on Bookstore. Click the Online Bookstores link and then through to the Grand Canyon Online Students section. What sort of materials can a student purchase from this site?

Compare and Contrast the American and French Revolutions

Both the American and French Revolutions were focused around liberty and equality. Both countries were trying to gain freedom. America was trying to gain freedom from the rules and taxes put upon them by Great Britain. Whereas the French wanted to abolish the French monarchy and create a better government in which the people could have more of a say in society. Although the revolutions of both started for very similar reasons, and both countries fought for the same thing, the outcomes of the two were very different. The American Revolution was mainly focused on gaining independence. After the British victory during the Seven Year War, America was tied down from the British rules. America was obligated to pay off the war, and to pay the†¦show more content†¦The king did nothing to help the peasants in their times of trouble, which caused even more uproar. The French were rapidly losing trust in the King, in fact many nobles began moving out of the palace of Versailles. 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