1H- and 19F-NMR approaches to the study of the structure of proteins larger than 25 kDa.
The three-dimensional solution structures of proteins larger than about 25 kDa cannot at present be determined by multi-dimensional nuclear magnetic resonance (NMR) methods. However, for proteins that are larger than 25 kDa, for which X-ray structural information is not available, there are a variety of mostly one-dimensional NMR methods that still represent some of the most informative approaches to obtaining structural answers to questions of biochemical interest. This paper provides recent illustrative examples of 1H- and 19F-NMR experiments that describe ways to focus on proteins by region, by amino acid type, or by individual amino acid. Methods to focus on a particular region of a protein include exploiting domain mobility, using transferred nuclear Overhauser enhancements, the use of difference spectroscopy, the use of paramagnetic species, and domain fragmentation. Particular types of amino acid can be identified using selective deuteration, by incorporation of fluorinated amino acid analogues, by using photochemically induced dynamic nuclear polarization, and from the pH dependence of histidine residues. Individual amino acids can be identified by mutagenesis and, in special circumstances, by chemical shift. Many of the examples given are of plasma proteinases and their protein inhibitors, but other classes of protein are also discussed, including antibodies and DNA-binding proteins.
Gettins
P G
PG
Department of Biochemistry, University of Illinois, Chicago 60612.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D003903:Deuterium; D006420:Hemeproteins / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry
D000596:Amino Acids; D006420:Hemeproteins; D010446:Peptide Fragments; D003903:Deuterium; D014568:Urokinase-Type Plasminogen Activator