L19 - Biophysical Chemistry Chemistry 24a Winter Term...

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Biophysical Chemistry Chemistry 24a Winter Term 2009-10 Instructor: Sunney I. Chan Lecture 19 March 10, 2010 Magnetic Resonance II. Nuclear Magnetic Resonance
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NMR III. Determination of 3D Structure of a Small Protein in Solution
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Step 1: Sequence-specific resonance assignments
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2: Distances from NOESY spectroscopy (Distance constraints) R ij NOESY Spectrum
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1 H – 1 H NOESY of RC-RNase I S
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For more crowded NOESY spectrum, can use isotopic filtering to simplify analysis (by introducing 13 C or 15 N labeled amino acids). Only 1 Hs that are coupled to 13 C or 15 N will appear in NOESY spectrum .
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Step 3. 3D-structure from conformational constraints BPTI, Bovine pancreatic trypsin inhibitor
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NMR Structural Constraints 1. Internuclear distances (Nuclear Overhauser Effect) NOE R -6 2. Dihedral angles (J-coupling): 3 J NH α = 6.4 cos 2 ( Φ -60) – 1.4cos( Φ - 60) + 1.9 3. Chem ical Shift Index (CSI): Chemical shift difference between observed and random coil chemical shift values 2 nd structure determination 4. Residual dipolar coupling: Partial orientation of protein molecules in liquid crystal media permits observation of residual dipolar coupling for assessing long range orientations of dipolar coupled bonds. Building a protein structure based on the NMR structural constraints
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Protein structure (Cartesian space) Peptide plane representation (Angular space) Strings of Peptide planes
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Tertiary structure (Backbone rmsd: 1.72 Å /1.0Å ) (CYANA with extensive manual assignments)
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(a) (a) Chemical Shift Indices: ( Chemical Shift Indices: ( 1 H α , , 13 13 C α , , 13 13 C β , , 13 13 CO) CO) - Change in chemical shift due to secondary Change in chemical shift due to secondary structure differences
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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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L19 - Biophysical Chemistry Chemistry 24a Winter Term...

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