Magnetic resonance spectroscopy correlation with histological analysis in gliomas and structure determination of a hypothetical protein

Magnetic resonance spectroscopy correlation with histological analysis in gliomas and structure determination of a hypothetical protein Xu Ying National University of Singapore 2004 Magnetic resonance spectroscopy correlation with histological analysis in gliomas and structure determination of a hypothetical protein Xu Ying A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE CHEMISTRY DEPARTMENT NATIONAL UNIVERSITY OF SINGAPORE 2004 ACKNOWLEDGEMENTS I would like to thank my supervisor, assistant professor Yang Daiwen for his encouragement, patience and guidance during the course of my research project I would like to thank Dr Lim Tchoyoson from National Neuroscience Institute (Singapore) for providing brain tissue samples Special thanks go out to Mr Li Kai and Mr Zheng Yu for their useful scripts, Mr Xu Xingfu and Mr Lin Zhi for their kindly help on protein structure project Many thanks to post-doctors and students from NMR structural biological lab and my friends in Department of Chemistry and other department or institutes, who made my stay in NUS a pleasant and memorable journey Finally, I wish to thank the National University of Singapore for granting me a Research Scholarship i Contents Acknowledgements i Contents ii Summary iv List of figures vi List of tables viii Symbols and abbreviations ix Introduction 1.1 Introduction to brain metabolites studies 1.2 Introduction to NMR structural studies of proteins 11 1.3 Objective 19 1.3.1 Objective of brain metabolites study 19 1.3.2 Objective of NMR structural studies of protein ec314 19 NMR studies of brain metabolites 20 2.1 Materials and methods 20 2.1.1 Sample preparation 20 2.1.2 NMR experiments 20 20 2.1.2.1 1D HRMAS HMRS experiment 13 21 2.1.2.2 2D H- C HSQC experiment 1 21 2.1.2.3 2D H- H COSY experiment 2.1.3 Chemical shift database 21 2.1.4 Quantitative analysis of brain metabolites 23 24 2.1.4.1 HRMAS HMRS 2.1.4.2 Matlab program 25 2.1.4.3 Curve fitting and calculating 28 2.2 Results and discussion 31 2.2.1 Identification of brain metabolites 31 2.2.2 Quantitative analysis of metabolites 40 2.3 Conclusion 44 NMR structural studies on protein ec314 45 3.1 Materials and methods 45 3.1.1 NMR experiments 45 15 45 3.1.1.1 2D H- N HSQC spectrum 3.1.1.2 HNCACB and CBCA (CO) NH 45 3.1.1.3 C (CO) NH and H (CCO) NH 47 3.1.1.4 HCCH-TOCSY 49 15 13 49 3.1.1.5 N edited NOESY and C edited NOESY 3.1.2 Chemical shift assignment 52 3.1.2.1 Backbone sequential assignment 52 3.1.2.2 Aliphatic side chain assignment 52 3.1.3 Secondary structure 53 3.1.3.1 Chemical shift index prediction 53 3.1.3.2 Sequential NOE pattern and short, medium-range NOE analysis ii 3.1.4 NOE assignment of 15N edited NOESY and 13C edited NOESY 3.1.5 Structure calculation 3.1.5.1 NOE restraints 3.1.5.2 Dihedral angle restraints 3.1.5.3 Hydrogen bond restraints 3.1.5.4 Structure calculation 3.2 Results 3.2.1 Backbone assignment and aliphatic side chain assignment 3.2.1.1 Backbone assignment 3.2.1.2 Aliphatic side chain assignment 3.2.2 Secondary structure 3.2.2.1 Chemical Shift Index 3.2.2.2 NOE analysis 3.2.3 NOE assignments 3.2.4 Structural statistics 3.3 Discussion 3.3.1 Sequential assignment and secondary structure prediction 3.3.2 Description of structure of protein ec314 3.3.3 Helix regions 3.3.4 Proline conformation 3.3.5 Unstructured region and loops 3.3.6 Comparison to similar structures Conclusion and future work Reference 53 54 54 54 55 55 56 56 56 59 61 61 63 64 64 65 65 66 68 68 68 68 71 73 iii Summary Human and mouse brain tissues obtained through National Neuroscience Institute (Singapore) were submitted to record 1D HRMAS 1H NMR and 2D 1H-1H COSY and 1H-13C HSQC spectra Using 2D COSY and HSQC spectra to identify metabolites, comparing two human brain samples, three significant differences were observed It suggests the possibility of differentiating brain tumors by analyzing metabolites using NMR technique 1D HRMAS 1H NMR spectra of both human and mouse brain tissue were used to calculate the concentration of major brain metabolites The absolute amounts of major metabolites in each sample were calculated for 20 brain tissues New method using Matlab program to fit the curves of metabolites was demonstrated HRMAS 1H NMR technique has been proved that it is suitable for quantitative analysis of brain tissues Furthermore the calculation becomes straightforward by using Matlab To determine the 3D structures of protein ec314, various 2D and 3D NMR spectra were recorded Using 2D H-15N HSQC spectrum as reference, 3D hetero-nuclear experiments HNCACB and CACB(CO)NH were combined to sequentially assign backbone atoms Aliphatic side chain carbon and proton spin system were assigned and connected to the sequentially assigned backbone resonances by using C(CO)NH, H(CCO)NH and HCCH-TOSCY spectral Chemical Shift Index and NOE patterns were used to identify secondary structures The 3D structure of protein ec314 was calculated using NOE restraints, iv hydrogen bond restraints and dihedral angle restraints The final structure shows ec314 is composed by helices It is similar to the C-terminal domain of E coli RecA protein v List of Figures Figure 2.1 HRMAS 1HMRS spectrum of brain metabolites 24 Figure 2.2 Matlab program 27 Figure 2.3 Result of curve simulation by Matlab 29 Figure 2.4A COSY spectrum of human brain tissue sample NNI1 32 Figure 2.4B HSQC spectrum of human brain tissue sample NNI1 34 Figure 2.5A COSY spectrum of human brain tissue sample NNI2 36 Figure 2.5B HSQC spectrum of human brain tissue sample NNI2 37 Figure 3.1 Schematic show of coherence transfer pathways employed and 46 the correlations made in CBCA(CO)NH and HNCACB experiments Figure 3.2 Overlook of NOESYCN spectrum 51 Figure 3.3 TCL script for conversion between 13C and 15N chemical shifts 51 Figure 3.4 CACB connectivity from CBCA(CO)NH (①) and HNCACB 57 (②) for a stretch of residues from E24 to G29 Figure 3.5 The 1H-15N HSQC spectrum of 15N labeled ec314 58 Figure 3.6 Aliphatic side chain assignments 60 Figure 3.7 Selected H(F3) and H(F1) planes at different 13 C(F2) 61 chemical shifts of the HCCH-TOCSY spectrum illustrating connectivity vi Figure 3.8 13 62 Figure 3.9 The secondary structure analysis based on short- and 63 Cα-Cβ Chemical Shift Index plot of ec314 medium-range NOEs Figure 3.10 Stereo-view of the superposition of backbone atoms for 20 66 structures Figure 3.11 Ribbon diagram representation of 3D structure of protein 67 ec314 Figure 3.12 Ribbon diagram representation of 3D structure of protein 70 RecA vii List of Tables Table 2.1 Chemical shift table of metabolites 23 Table 2.2 Structure formula and total proton numbers of TSP and 30 metabolites Table 2.3a Amount of metabolites of mouse brain tissues 41 Table 2.3b Amount of metabolites of human brain tissues submitted on 27th 41 March Table 2.3c Amount of metabolites of human brain tissues submitted on 31st 42 March Table 2.3d Amount of metabolites of human brain tissues submitted on 19th 42 May Table 3.1 Summary of the restraints used to calculate the structures 64 Table 3.2 Structural statistics for the CYANA calculation and final 65 ensemble viii Furthermore, the secondary structure prediction based on Chemical Shift Index also gives some clues at the initial stage of structure determination Moreover, short- and medium-range NOE assignment gives more information on the secondary structure of protein ec314 Based on CSI and NOE analysis, protein ec314 is composed of helical structure 3.3.2 Description of structure of protein ec314 The structure of ec314 contains helical regions The long helix1 is alpha helix and connected to helix2 by a long loop The helix2 is a short 3-10 helix only contains three residues Helix3 is an alpha helix and almost parallel to helix1 Helix2 and helix3 connected each other by a short turn formed by residues K38 and V39 Figure 4.1 shows the superposition of backbone atoms for the calculated structures of ec314, while Figure 4.2 shows the ribbon diagram representation of the protein Figure 3.10 Stereo-view of the superposition of backbone atoms for 20 structures The helices are in green 66 A B Figure 3.11 Ribbon diagram representation of 3D structure of protein ec314 A Side view B Top view 67 3.3.3 Helix Regions The helix1 region from P10 to E24 is an alpha-helix The backbone RMSD for this region is 0.33 The helix2 region from L35 to Q37 is a 3-10 helix and the helix3 region from A40 to Q45 is an alpha-helix The backbone RMSD for these two helices region from L35 to L46 is 0.41 All these RMSD values suggest that the helical regions are very well defined 3.3.4 Proline conformation All three Pro residues of ec314 adopt trans conformation This is consistent with the prediction from the chemical shift values of Cβ values of these proline residues (Grathwohl, 1976) Pro is in the beginning of the first loop of the structure Pro 10 is the first residue of the long alpha-helix1 Pro 66 is in the last loop of the structure Therefore, the three prolines not affect the structure calculation 3.3.5 Unstructured region and loops There are several regions of the structure are poorly defined The flexible loop regions include loops from residue W28 to E32, from V54 to H59 and from P66 to Q69 It is due to lack of NOE signals in these regions 3.3.6 Comparison to similar structures There are six sequences show sequence identity more than 70% through protein-protein blast provided by NCBI The bacterium Escherichia coli O157:H7 EDL933 has the exact same sequence as our protein Hypothetical protein Salmonella enterica serovar Typhi (str CT18) which is the aetiological agent of typhoid fever 68 shows 87% sequence identity and putative cytoplasmic protein Salmonella tyhimrium LT2 which is a leading cause of human gastroenteritis shows 89% sequence identity to protein ec314 Hypothetical protein Yersinia pestis which is the causative agent of the systemic invasive infectious disease shows 76% sequence identity, Erwinia carotovora atroseptica SCRI1043 show 75% sequence identity and hypothetical protein photorhabdus luminescens laumondii TT01 shows 70% sequence identity However, for all these six proteins, no structure has been published and deposited into Protein Data Bank Compare to the three-dimensional structure of protein 1AA3, C-terminal domain of the escherichia coli RecA protein has similar structure to protein ec314 The bacterial RecA protein plays a central role in the repair of stalled replication forks, double-strand break repair, general recombination, induction of the SOS response and SOS mutagenesis The major activity of RecA in DNA metabolism is the promotion of DNA strand exchange reactions RecA is the prototype for a ubiquitous family of proteins but exhibits a few activities that some of its eukaryotic, archaeal, and viral homologous appear to lack (Cox, 2003) Protein RecA contains 63 amino acids and has a molecular weight around kDa It is composed of α-helices and β-sheets The only difference between these two structures is β-sheets connect the two short α-helices in protein RecA (Aihara, 1997) while a short turn in protein ec314 The ribbon diagram of 3D structure of protein RecA is shown below 69 Figure 3.12 RecA Ribbon diagram representation of 3D structure of protein 70 Conclusions The study of brain tumor metabolites using NMR techniques shows the possibility of applying NMR techniques in clinical research and medical diagnosis and the power of NMR techniques in this area Combining two dimensional experiments HSQC and COSY, the brain metabolites can be identified according to both their 1H and 13 C chemical shifts and the correlations between protons within a compound Furthermore, the high-resolution magic-angle spinning proton NMR spectrum can be used to calculate the concentrations of major metabolites in brain tumors With the wide application of various relatively simple NMR techniques and improvement on the understanding of metabolites, it will become possible to use NMR as a tool for clinical diagnosis on brain tumor by analyzing spectra of brain metabolites based on the correlations between brain metabolites and brain tumors The other part of study on NMR structural solution of protein is achieved by three steps Firstly, near complete 1H, 13C and 15N backbone and side chain chemical shift assignments were obtained by pairs of three-dimensional NMR spectroscopy The chemical shift gives important clues on structure study of the protein Secondly, the secondary structure analysis based on the chemical shift index and short- and medium-range NOE assignment provides the initial impression of the three dimensional structure Thirdly, the three dimensional structure of protein ec314 have been solved Based on distance constraints from NOE assignments, hydrogen bond and dihedral angle, a helical structure represents protein ec314 has been calculated by CYANA 71 The results show the first two helices in the structure are connected by long loop, and the helix2 and helix3 are connected to each other by a short turn Comparing to the known structures of proteins, ec314 is similar to protein RecA which is a cyanobacterial gene product It may help us predict the function of protein ec314 based on its 3D structure Future Work On the study of brain tumor metabolites, more samples from various patients will be collected and studies by NMR By analyzing the spectra, the correlation between metabolites and type of brain tumors will be discovered Structure refinement of protein ec314 can be achieved by completing the aromatic proton assignment as more long range NOE can be assigned Based on 3D structure, biological activity test may apply to confirm predicted functions of protein ec314 72 References Abraham, R.J., Fisher, J and Loftus, P (1978) Introduction to NMR spectroscopy Chichester, Wiley Aihara, H., Ito, Y., Kurumizaka, H., Terada, T., Yokoyama, S., Shibata, T (1997) An interaction between a specified surface of the C-terminal domain of RecA protein and double-stranded DNA for homologous pairing J Mol Biol 274:213 Akihiro, I., Tetsuhiko, A and Toshiro, F (2000) MRS of the Brain and Neurological Disorders Allerhand, A., Doddrell, D., Glushko, V., Cochran, D W., Wenkert, E., Lawson, P J., and Curd, F R N (1971) Conformation and segmental motion of native and denatured ribonuclease A in solution Application of natural-aboundance carbon-13 partially relaxed Fourier transform nuclear magnetic resonance J Am Chem Soc 93, 544-546 Arrowsmith, C.H., Christendat, D., Yee, A., Dharamsi, A., Kluger, Y., Savchenko, A., Cort, J.R., Booth, V., Mackereth, C.D., Saridakis, V., Ekiel, I., Kozlov, G., Maxwell, K., Wu, N., McIntosh, L.P., Gehring, K., Kennedy, M.A., Davidson, A.R., Pai, E.F., Gerstein, M., Edwards, A.M (2000) Structural proteomics of an archaeon Nature Structural Biology 7(10):903-909 Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., and Struhl, K (editors) (1993) Current Protocols in Molecular Biology 4th edition, John Wiley & Sons, New York Baxter, N J., and Williamson, M P (1997) Temperature dependence of 1H chemical 73 shifts in proteins J Biomol NMR 9, 359-69 Birken, D.L., Oldendorf, K.H (1989) N-Acetyl-L-aspartic acid: a literature review of a compound prominent in 1H-NMR spectroscopic studies of brain Neurosci Biobehav Rev 13:23-31 Bloch, F (1946) Phys Rev 70:460 Burtscher, I.M and Holtas, S (2001) Proton magnetic resonance spectroscopy in brain tumours: clinical application Neuroradiology 43:345-352 Carlos A.M., Dimitris S., Adam M., Maurice G., Thomas F.B and Alexander P (2004) High-resolution NMR of static samples by rotation of the magnetic field Journal of Magnetic Resonance 169:13–18 Cavanagh et al (1996) Protein NMR Spectroscopy, Academic Press, San Diego USA Cerdan, S., Parrilla, R., Santoro, J and Rico, M (1985) 1H NMR detection of cerebral myo-inositol FEBS Lett 187:167-172 Cheng, L.L., Ma, M.J., Becerra, L., Ptak, T., Tracey, I., Lackner, A., and Gonzalez, R.G (1997) Quantitative neuropathology by high resolution magic angle spinning proton magnetic resonance spectroscopy Proc Natl Acad Sci USA 94:6408-6413 Clore, G M., and Gronenborn, A M (1998) New methods of structure refinement for macromolecular structure determination by NMR Proc Natl Acad Sci U S A 95, 5891-8 Clore, G.M., Gronenborn, A.M (2000) Protein NMR spectroscopy in structural genomics Nat Struct Biol Suppl: 982-985 74 Cornilescu, G., Delaglio, F & Bax, A (1999) Protein backbone angle restraints from searching a database for chemical shift and sequence homology J Biomol NMR 13: 289-302 Cox, M.M (2003) The bacterial RecA protein as a motor protein Annu Rev Microbiol 57:551-577 Delaglio, F et al (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes J Biomol NMR 6: 277-293 Dempsey, C.E (1986) pH dependence of hydrogen exchange from backbone peptide amides in apamin Biochemistry 25:3904-3911 Dimitris, S., Carlos, A.M., Rachel, W.M and Alexander, P (2003) High-resolution NMR of anisotropic samples with spinning away from the magic angle Chemical Physics Letters 377:333-339 Ernst, R R., and Anderson, W A (1966) Rev Sci Instrum 37: 93 Francis, D., Laetitia, H., Franck, C and Corinne, C.C (2004) Function and regulation of the cyanobacterial genes lexA, recA and ruvB: LexA is critical to the survival of cells facing inorganic carbon starvation Molecular Microbiology 53(1):65-80 Garg, M., Gupta, R.K., Husain, M., Chawla, S., Chawla, J., Kumar, R., Rao, S.B., Misra M.K and Prasad, K.N (2004) Brain abscesses: etiologic categorization with in vivo proton MR spectroscopy Radiology 230(2):519-527 Gerhard, W (2000) Structure determination of biological macromolecules in solution using NMR spectroscopy BioTechniques 29:1278-1294 Govindaraju, V., Young, K and Maudaley, A.A (2000) Proton NMR chemical shifts 75 and coupling constants for brain metabolites NMR Biomed 13:129-153 Grathwohl, C., Wuthrich, K (1976) Nmr studies of the molecular conformations in the linear oligopeptides H-(L-Ala)n-L-Pro-OH Biopolymers 15:2043-2057 Grzesiek, S & Bax, A (1993A) Amino acid type determination in the sequential assignment procedure of uniformly 13C/15N enriched proteins J Biomol NMR 3: 185-204 Grzesiek, S., Anglister, J & Bax, A (1993B) Correlation of backbone amide and aliphatic side-chain resonances in 13C/15N-enriched proteins by isotropic mixing of 13C magnetization J Magn Res B 101: 114-119 Güntert, P., Mumenthaler, C & Wüthrich, K (1997) Torsion angle dynamics for NMR structure calculation with the new program DYANA J Mol Biol 273: 283-298 Güntert, P (2003) Automated NMR protein structure calculation Prog In NMR Spec 43:105-125 Hausser, K.H and Kalbitzer, H.R (1991) NMR in Medicine and Biology Herrmann, T., Güntert, P & Wüthrich, K (2002) Protein NMR structure determination with automated NOE assignment using the new software CANDID and the torsion angle dynamics algorithm DYANA J Mol Biol 319: 209-227 Jardestzky, O (1965) Proc Int Symp Nuclear Magnetic Res., Tokyo, Japan, N-3-14 Johnson, B.A & Blevins, R.A (1994) NMRView: A computer program for the visualization and analysis of NMR data J Biomol NMR 4: 603-614 Kay, L.E., Xu, G.Y., Singer, A.U., Muhandiram, D.R & Forman-Kay, J.D (1993) A 76 gradient-enhanced HCCH-TOCSY experiment for recording side-chain 1H and 13C correlation in H2O sample of proteins J Magn Res B 101: 333-337 Knight, W.D (1949) Nuclear Magnetic Resonance Shift in Metals Phys Rev 76:1259-1260 Koradi, R., Billeter, M & Wüthrich, K (1996) MOLMOL: a program for display and analysis of macromolecular structures J Mol Graphics 14: 51-55 Leyden, D.E and Cox, R.H (1977) Analytical applications of NMR Lowe, I.J (1959) Nuclear-Magnetic-Resonance Line Narrowing by a Rotating rf Field Phys Rev 140(A):1261-1271 Martin, M., Merle, M., Labouesse, J., Canioni, P (1994) Postnatal decrease of acetate concentration in rat cerebellum J Neurosci Res 37:103-107 Montelione, G T., Lyons, B A., Emerson, S D., and Tashiro, M (1992) An efficient triple resonance experiment using carbon-13 isotropic mixing for determining sequence-specific resonance assignments of isotopically-enriched proteins J Am Chem Soc 114: 10974-10975 Montelione, G.T., Zheng, D., Huang, Y.J., Gunsalus, K.C., Szyperski, T (2000) Protein NMR spectroscopy in structural genomics Nat Struct Biol Suppl: 982-985 Moseley, H., and Montelione, G.T (1999) Automated analysis of NMR assignments and structures for proteins Curr Opin in Stru Bio 9:635-642 Pace CN, Vajdos V, Fee L, Grimsley G, Gray T (1995) How to measure and predict the molar absorption coefficient of protein Protein Sci;4:2411-2423 77 Palmer, A.G (2001) Nmr probes of molecular dynamics: overview and comparison with other techniques Annu Rev Biophys Biomol Struct 30:129-155 Pervushin, K., (2000) Impact of transverse relaxation optimized spectroscopy (TROSY) on NMR as a technique in structural biology Q Rev Biophys 33:161-197 Pervushin, K., Riek, R., Wider, G., Wuthrich, K (1997) Attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution Proc Natl Acad Sci U S A 94:12366-12371 Pouwels, P.J and Frahm, J (1998) Regional metabolite concentrations in human brain as determined by quantitative localized proton MRS Magn Reson Med 39:53-60 Provencher, S.W (1993) Estimation of metabolite concentrations from localized in vivo proton NMR spectra Magn Reson Med 30:672-679 Purchell, E M., Torrey, H C., and Pound, R V (1946) Phys Rev 69: 37 Ratmond, A.E., Bradbury, A and Eades, R.G (1958) Nature (London) 182:1695 Ravi, D., Rakesh, K., Gupta, M., Harish, P., Raja, R., Sanjay, S., Mazhar, H (1998) Role of in vivo proton magnetic resonance spectroscopy in the diagnosis and management of brain abscesses Neurosurgery 42:37-43 Reid, D.G (1997) Protein NMR Techniques Humana press, US Roca, A.I., Singleton, S.F (2003) Direct evaluation of a mechanism for activation of the RecA nucleoprotein filament J Am Chem Soc 125(50):15366-15375 Ross, B.D (1991) Biochemical considerations in 1H spectroscopy Glutamate and 78 glutamine; myo-inositol and related metabolites NMR Biomed 4:59-63 Schwarzinger, S., Kroon, G.J., Foss, T.R., Chung, J., Wright, P.E., Dyson, H.J (2001) Sequence-dependent correction of random coil NMR chemical shifts J Am Chem Soc 123:2970-2978 Szyperski, T., Neri, D., Leiting, B., Otting, G & Wüthrich, K (1992) Support of 1H NMR assignments in proteins by biosynthetically directed fractional 13C-labeling J Biomol NMR 2, 323-334 Thompson, C.C., Thompson, F.L., Vandemeulebroecke, K., Hoste, B., Dawyndt, P., Swings, J (2004) Use of recA as an alternative phylogenetic marker in the family Vibrionaceae Int J Syst Evol Microbiol 54(3):919-924 Torchia, D.A., Sparks, S.W., Bax A (1989) Staphylococcal nuclease: sequential assignments and solution structure Biochemistry 28:5509-5524 Wang, Y., Li, S.J (1998) Differentiation of metabolic concentrations between gray matter and white matter of human brain by in vivo 1H magnetic resonance spectroscopy Magn Reson Med 39:28-33 Wieland, W., Jorn, E., Annette, B and Dieter, L (1996) Metabolite identification in cell extracts and culture media by proton-detected 2D-H,C-NMR spectroscopy J Magn Res Anal 2:21-32 Wishart, D.S., Bigam, C.G., Yao, J., Abildgaard, F., Dyson, H.J., Oldfield, E., Markley, J.L., Sykes, B.D (1995) 1H, 13C and 15 N chemical shift referencing in biomolecular NMR J Biomol NMR 6:135-140 Wishart, D.S., Nip, A.M (1998) Protein chemical shift analysis: a practical guide 79 Biochem Cell Biol 76(2-3):153-163 Wishart, D.S., Sykes, B.D and Richards, F.M (1992) The chemical shift index: a fast and simple method for the assignment of protein secondary structure through NMR spectroscopy Biochemistry 31(6):1647-1651 Wittekind, M and Mueller, L (1993) HNCACB, a high-sensitivity 3D NMR experiment to correlate amide-proton and nitrogen resonances with the alphaand beta-carbon resonances in proteins J Magn Res B 101, 201-205 Wüthrich, K (1986) NMR of Proteins and Nucleic Acids Wiley, New York Zarembinski, T.L et al (1998) Structure-based assignment of the biochemical function of a hypothetical protein: a test case of structural genomics Proc Natl Acad Sci USA 95:15189-15193 Zhang, O., Kay, L E., Olivier, J P., and Forman-Kay, J D (1994) Backbone 1H and 15 N resonance assignments of the N-terminal SH3 domain of drk in folded and unfolded states using enhanced-sensitivity pulsed field gradient NMR techniques J Biomol NMR 4: 845-858 80 ... N-Acetyl aspartate (NAA), N-Acetylaspartylglutamate (NAAG), Alanine, Choline, Creatine, Glutamate, Glycine, Myo-inositol, Lactate, Pyruvate, Serine, Threonine, Tyrosine and Valine Acetate is an.. .Magnetic resonance spectroscopy correlation with histological analysis in gliomas and structure determination of a hypothetical protein Xu Ying A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF. .. and fatty acid energy reserves, and compounds associated with neuronal function including glutamate, glutamine, γ-aminobutyric acid (GABA), and N-acetylaspartate, a putative neuronal maker, are