19 - 3D NMR - 5 Making it 3-dimensional: 2D to 3D to 4D 6 A...

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1 Beyond infinity: 3D and 4D spectroscopy • When the size of your molecule becomes large, spectral overlap is more common and analysis is problematic if not impossible. • Adding a dimension or two helps (other methods, like adding some deuterium to remove dipolar broadening can also be used). • X-ray crystallography is the method of choice with very large systems. NMR solves small (< 50 kDa), uncrystallizable molecules. • Sometimes the two techniques are used together and they do complement each other. Problems with overlap in the 1 H spectrum For proteins, label the molecule with 15 N and 13 C (both spin=1/2).
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2 The 1D 1 H spectra of some proteins How do we make use of those labels? N N H HR O RH O H n all N= 15 N all C= 13 C
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3 Making a 3D experiment from 2 2D experiments: • Combine two basic experiments: a NOESY and an HSQC, for example (or a TOCSY and an HSQC). • First, what does experiment look like on its own NOESY of a 98 residue protein:
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4 TOCSY of the 98 residue protein: HSQC of another protein (69 residues, in this case)
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Unformatted text preview: 5 Making it 3-dimensional: 2D to 3D to 4D 6 A 3D experiment: NOESY-HSQC TOCSY-HSQC 7 What these data look like: ( 1 H-15 N NOESY-HSQC) 1 H-13 C NOESY-HSQC 8 15 N-separated NOESY (800 MHz, pH 7.2, 25 ) plane at ( 15 N) = 120 ppm Too many black dots 9 How do we make assignments ( 1 H, 15 N, 13 C) How to get those backbone assignments: triple resonance experiments. 10 The basic experiments The HNCA 11 Fe(III) S6803 Hb ~ 2 mM, pH 7.5, 298 K 14.1 T HNCA spectrum Chemical shifts of the amino acids: 12 Getting to structure The global fold through long-range NOEs 13 The 1 H-15 N HSQC for those slices: 38 and 37 are near 60 and 26, for example. Likewise, V24 is near the NH of 6 and 26. What is looks like in 2-dimensions: 14 Flow chart for structure determination: After many iterations of assigning NOEs and calculating, we come up with: 15 In a ribbon respresentation: Another protein: a truncated hemoglobin 16 A ribbon diagram of the truncated hemoglobin...
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19 - 3D NMR - 5 Making it 3-dimensional: 2D to 3D to 4D 6 A...

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