Proton nuclear magnetic resonance assignments.
The procedures outlined here have been used successfully for more than 30 proteins to date, and are nearly routine for molecules up to a molecular weight of 10,000. Some of the proteins assigned have a molecular weight greater than 10,000. For these larger proteins, relayed-COSY and TOCSY experiments have been essential for the identification of spin systems, although for thioredoxin these experiments could not be used. In this case, assignments were accomplished using nonspecific deuteration to the level of 75% and specific, nearly complete, deuteration of certain kinds of residues (see LeMaster [2], this volume). Nonspecific deuteration reduces the cross-relaxation rates of each proton to the rest of the molecule, thus reducing the linewidths. The cross-peak patterns were also narrowed due to simplification of the coupling patterns. Such a laborious procedure of nonspecific deuteration may not be necessary for complete proton assignments of proteins in this size range, as evidenced by the fact that this method was not used for the other two molecules mentioned above. It may prove, however, to be quite valuable in the study of larger molecules, where linewidths are expected to increase due to longer rotational correlation times. Overlap problems in the NH chemical shifts can be dealt with by making use of the differential temperature dependence of these shifts. Another technique is to take advantage of the wide range of exchange rates between these protons and the solvent. Spectra containing only the slowly exchanging NH protons can be obtained by acquiring spectra of the protein soon after dilution in D2O, and spectra of only the rapidly exchanging protons can be obtained by obtaining spectra in a freshly prepared H2O solution of the protein after having completely exchanged all the NH protons with deuterium. Variation of the pH will resolve problems of overlap in all regions of the spectrum, although many chemical shifts may be unaffected by pH. In some cases, pH variation may change the conformation of the molecule. This may, in fact, assist in the sequential assignment if the chemical shifts can be followed with pH. Finally, the relayed-NOESY experiments can resolve overlap problems with the alpha-proton chemical shifts. Thus, it is very likely that the assignment methods outlined here will be successful for the assignment of the proton spectra of even larger molecules if there is significant secondary structure and significant variety of residues to provide enough dispersion of the chemical shifts.(ABSTRACT TRUNCATED AT 400 WORDS)
Basus
V J
VJ
D000596:Amino Acids; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D010455:Peptides; D011487:Protein Conformation; D011506:Proteins; D013329:Structure-Activity Relationship
D000596:Amino Acids; D010455:Peptides; D011506:Proteins