NMR of Proteins

NMR of ProteinsNMR of ProteinsDetermining Protein Structures by NMRD etermining Protein Structures by NMR the process of determining a solution structure by NMR is one of measuring many (hundreds/thousands) of short proton- proton distances and angles, and restraining the protein structure with these computationallyH H ..}d1H NMR Spectra of Proteins1H NMR Spectra of Proteins 1D, 1H NMR spectra of even small Proteins are impossible to interpret in any comprehensive manner -normally, only gross statements about secondary structure, tertiary structure, etc. can be madeubiquitin (76 amino acids, kDa)simple 1D 1H experiment9090t1 COSYt22D 1H COSY experimentcytochrome c, kDa for even moderate sized Proteins , addition of a second dimension still does not alleviate spectral crowding and overlap in 1H spectranD, heteronuclear NMR Spectra of ProteinsnD, heteronuclear NMR Spectra of Proteins Modern NMR spectroscopic studies of Proteins rely on multidimensional experiments involving 1H, 13C, and 15N nuclei in isotopically labeled Proteins These methods provide for signal selection (selectivity) and a means to reduce signal overlap ubiquitin (76 amino acids, kDa)simple 2D 1H, 15N HSQC experiment In order to measure the distances between protons, we need to find out what protons give rise to the signals in the spectra, we have to assign the protein (f)

expression using defined media-bacterial expression most common-also yeast, and cell-free systems are being developed-minimal media using 13C 6 glucose as the sole carbon source and 15NH 4 Cl (or -SO 4) as the sole nitrogen source-normally >98% atom excess-also labeled “rich” media ($$)-for larger proteins, uniform or fractional 2H labeling ...

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