Nuclear magnetic resonance spectroscopy large proteins

However, other perspectives suggest that there are likely to be as yet unidentified protein folds. As of 2004, the Pfam database (version 10.0) contained 6190 domains only about a third were associated with a protein of known structure. Some of these may be cases in which highly divergent or completely unrelated sequences adopt an already discovered fold. Some may not be amenable to structural characterization, either because they are too large for characterization by nuclear magnetic resonance spectroscopy (NMR), or contain disordered regions that interfere with crystallization. However, there may indeed be unidentified protein domain structures remaining to be discovered. 

Aside from the direct techniques of X-ray or electron diffraction, the major possible routes to knowledge of three-dimensional protein structure are prediction from the amino acid sequence and analysis of spectroscopic measurements such as circular dichroism, laser Raman spectroscopy, and nuclear magnetic resonance. With the large data base now available of known three-dimensional protein structures, all of these approaches are making considerable progress, and it seems possible that within a few years some combination of noncrystallo-graphic techniques may be capable of correctly determining new protein structures. Because the problem is inherently quite difficult, it will undoubtedly be essential to make the best possible use of all hints available from the known structures. 

A way to test these hypotheses directly would be to examine by X-ray crystallography or high resolution nuclear magnetic resonance (NMR) spectroscopy, the three-dimensional structure of the peptide-protein complexes. However, examination of the high resolution structure of integral membrane proteins has turned out to be very difficult, not only because of their reasonably large size (40-80... 

To determine these complicated structures the only general method available is X-ray diffraction of the single crystals of these materials. Although the structure of small proteins (molecular weight (MW) less than about 10000 daltons (D)) can be determined in solution with nuclear magnetic resonance (NMR) spectroscopy and the assembly of proteins in a complex can be studied with electron microscopy, only X-ray diffraction can give the three-dimensional structure of small as well as large proteins with a precision of about 0.1-0.2 A. 

The large number of histidine residues associated with the active site of bovine SOD facilitates the study of its chemical and structural properties by nuclear magnetic resonance (NMR) spectroscopy. Mainly the downfield histidine imidazole N-proton resonances were subject of a large quantity of investigations The deuteration of histidine residues is dependent only on the absence of co-ordinated metal ions and not changes in the protein structure Therefore, in apo-, zinc- and holo-super-oxide dismutase, only those histidine residues not co-ordinated to metal ions will be deuterated at the C-2 position. This can be used as a simple method for the identification of co-ordinated histidine residues in metalloproteins employing only minimal chemical modifications. 

---