L18 - Biophysical Chemistry Chemistry 24a Winter Term...

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Biophysical Chemistry Chemistry 24a Winter Term 2009-10 Instructor: Sunney I. Chan Lecture 18 March 8, 2010 Magnetic Resonance II. Nuclear Magnetic Resonance
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NMR II. Multi-Dimensional NMR
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Milestones in the development of NMR that culminated in a method for “Protein Structure Determination” in solution 1. Fourier Transform NMR (1960s) 2. The development of high-field superconduct- ing magnets with ultra-high homogeneity (better than 1 part in 10 8 over ~1 ml volume) [since mid- 1960s: 220, 270, 300, 400, 500, 600, 700, 800, 900 MHz, and 1 GHz (in the works)] 3. Overhauser Enhancement (transfer of magnetization between nearby spins via dipolar interactions)
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Milestones in the development of NMR that culminated in a method for “Protein Structure Determination” in solution 4. 2D NMR (COSY:automated spectral assignments; NOESY: distance constraints) 5. The development of water-suppression methods (selection saturation; pulse gradient) 5. 3D and 4D NMR (introduction of 13 C and 15 N) 6. Cyro-probes (suppression of Johnson noise in detector)
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Milestones in the development of NMR that culminated in a method for “Protein Structure Determination” in solution S/N improvement : 1, 2, and 6 FT NMR: a factor of 10 Higher field: a factor of 100 Cryo- probes: a factor of 2-3 Spectral dispersion : 2 A factor of 3-4 from 220 to 800 MHz New techniques exploiting spin physics: 3, 4, and 5 Improving noise figure in detection of signal: 6 Another factor of 2-3 improvement in S/N.
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Solution Protein Structure Determination To accomplish this task, we need to come up with ways to identify the spins associated with different residues of the protein; and to develop a molecular ruler to measure inter-spin distances.
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NMR Sample (1 mM, 0.4 ml) 2 H, 13C, 15 N-label Obtain NMR spectra (3 weeks) Assign resonances Automation? Obtain restraints (Distances, angles, Orientations etc) Calculate structures Determination of Protein Structure by NMR NMR structures (Ensemble of 20 structures)
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Cytotoxic ribonuclease from the oocytes of Rana calesbeiana (bull frog) 12.5 kDa Sidechain NH & COOH Resonance Assignments 1D spectrum very complex!
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Spectral assignments facilitated by NMR correlation experiments. These are 2-dimensional (2D) or multi-dimensional (3D and 4D) NMR experiments.
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Correlation Spectroscopy: Assignment of Coupled Spin-Systems Most nD experiments are correlation experiments, correlating chemical shifts (or resonance positions) of pairs of spins which are related by e.g., J-coupling, dipole-dipole interaction, or chemical exchange. They may be homonuclear (all 1 H s ), or heteronuclear ( 13 C 1 H or 15 N 1 H).
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Two-Dimensional NMR
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1 H/ 1 H homonuclear correlation experiments COSY = Co rrelated S pectroscop y A 2-pulse experiment Collect a set of FIDs S(t 1 ,t 2 ) by varying t 1 . Double FT of the S(t1,t2) yields the 2D spectrum F( ω 1 , ω 2 ) t 2
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COSY Spectrum Spectrum of spin systems coupled by scalar spin-spin interactions (J I A .I S )
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Total Correlation Spectroscopy CH 3 -CH 2 -CH 2 -OH A M X A 3-pulse experiment t 2
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This note was uploaded on 10/27/2010 for the course BI 110 taught by Professor Richards,j during the Winter '08 term at Caltech.

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L18 - Biophysical Chemistry Chemistry 24a Winter Term...

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