triple-resonance

triple-resonance - Triple Resonance Experiments For...

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Triple Resonance Experiments For Proteins
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Limitations of homonuclear ( 1 H) experiments for proteins -the utility of homonuclear methods drops quickly with mass (~10 kDa) -severe spectral degeneracy -decreased magnetization transfer efficiency via the small 3 J ( 1 H- 1 H) couplings cytochrome c , 12.5 kDa DQF COSY NOESY
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tutorial: http://web.chem.queensu.ca/FACILITIES/NMR/nmr/webcourse/tocsynoesy.htm Three-dimensional homonuclear experiments 1 H 2D experiments can be combined to create 3D experiments -increased dimensionality can increase resolution and reduce spectral overlap…. -….however, these methods are mostly non-selective and the numbers of signals (peaks) in the resulting spectra are very large -the resulting spectra can be very informative 90 t 1 TOCSY 90 90 90 90 90 τ m t 1 NOESY-TOCSY t 2 90 90 90 90 90 τ m t 1 NOESY t 2 t 2 t 3
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Three-dimensional homonuclear experiments • Example: 3D NOESY-TOCSY of parvalbumin (108 amino acids) -8.7 mM -170 hours (~ 7 days) -50,000 cross peaks !
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Heteronuclear resolved homonuclear experiments -pulse sequences: same idea (combine 2D sequences) -but selective: get increased dimensionality and increased resolution without an increase in the number of signals (peaks) 90 90 90 τ m t 1 NOESY t 2 HSQC τ 180 90 180 90 1 H 15 N decouple 90 180 180 180 90 τ ττ t 1 /2 t 1 90 t 2 90 90 τ m t 1 NOESY-HSQC τ 180 90 180 90 15 N decouple 90 180 180 180 90 τ t 2 t 2 90 t 3 1 H
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Heteronuclear resolved homonuclear experiments Left: 2D NOESY Far left: 2D plane of 3D NOESY- HMQC ( 1 H, 13 C) - 1 H signals resolved by C chemical shifts of bound C atoms
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Triple resonance approach • based on magnetization transfer via (mostly) one bond J couplings -most of these couplings are large compared to linewidths for moderate sized proteins (~20 kDa) -magnetization transfer is efficient -indirect ( 1 H) detection • applicable to uniformly isotopically enriched proteins -uniform 13 C and 15 N labeling: spin 1/2 • provides selective chemical shift correlation -spectral degeneracy minimized
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1 J and 2 J couplings in polypeptides - these 1 J and 2 J couplings are uniform throughout polypeptides/proteins - these 1 J and 2 J couplings are virtually conformation independent
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This note was uploaded on 11/07/2011 for the course CHEM 8853R taught by Professor Gelbaum during the Fall '11 term at Georgia Tech.

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triple-resonance - Triple Resonance Experiments For...

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