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lecture14 - Today's Lecture 14 Wed Nov 1 Assignments II...

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A. S. Edison University of Florida 2006 Today’s Lecture 14) Wed, Nov 1: Assignments: II a.Important 2D heteronuclear experiments b.3D NMR c.Assignment strategies in proteins d.Intro to protein structure determination
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A. S. Edison University of Florida 2006 TOCSY/COSY NOESY/ROESY 1 H-based Peptide Resonance Assignments N C α C’ N C α C’ H O H O H CH 3 H CH CH 3 CH 3 N H
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A. S. Edison University of Florida 2006 1 H-based Resonance Assignments F2 G3 E5 G10 M6 M8 F14 V11 D4 L12 R13 "NH" 2 8.7 8.4 8.1 7.8 ppm 4.8 4.5 4.2 3.9 GFGDEMSMPGVLRFamide S7
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A. S. Edison University of Florida 2006 Some Heteronuclear 2D Experiments Name Type of correlation Notes HMQC H eteronuclear M ultiple Q uantum C orrelation 1 H atoms directly bonded to “X” (usually 13 C or 15 N) mediated by J-coupling. Starts with and detects 1 H and very sensitive. One of the simplest heteronuclear NMR experiment HSQC H eteronuclear S ingle Q uantum C orrelation 1 H atoms directly bonded to “X” (usually 13 C or 15 N) mediated by J-coupling. Similar information to HMQC Sensitive and better relaxation properties for proteins. Very important and common experiment for proteins. HMBC H eternuclear M ultiple B ond C orrelation 1 H atoms 2-3 bonds away from “X” (usually 13 C) mediated by J-coupling. Very important experiment for natural products. One of the best (sometimes only) ways to identify quaternary carbons. INADE QUATE I ncredible N atural A bundance D ouble Qua ntum T ransfer E xperiment 13 C- 13 C directly bonded correlations mediated by J-coupling. Useful for natural products. Not very sensitive, because it starts with and detects 13 C and not proton.
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A. S. Edison University of Florida 2006 Why Heteronuclear NMR? Beyond relatively small peptides, most NMR of proteins or nucleic acids requires isotopically labeled samples. Proteins are usually produced by over-expressing them in bacteria with 15 N-labeled, 13 C-labeled, 15 N+ 13 C-labeled, or 15 N+ 13 C+ 2 H- labeled nutrients. Specific labeling strategies have been developed, but uniform isotopic enrichment is most common. There are several reasons to isotopically label a sample: Overlapping 1 H resonances can be resolved using the frequencies of the attached heteronucleus. The heteronuclei create efficient pathways to correlate atoms along the backbone and side-chains of proteins and nucleic acids.
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