Studies on the solution conformation of human thioredoxin using heteronuclear nitrogen-15-proton nuclear magnetic resonance spectroscopy

Abstract

The solution conformation of uniformly labeled 15N human thioredoxin has been studied by two-dimensional heteronuclear 15N-1H nuclear magnetic resonance spectroscopy. Assignments of the 15N resonances of the protein are obtained in a sequential manner using heteronuclear multiple quantum coherence (HMQC), relayed HMQC-correlated (COSY), and relayed HMQC-nuclear Overhauser (NOESY) spectroscopy. Values of the 3JHN.alpha. splittings for 87 of the 105 residues of thioredoxin are extracted from a variant of the HMQC-COSY experiment, known as HMQC-J, and analyzed to give accurate 3JHN.alpha. coupling constants. In addition, long-range C.alpha.H(i)-15N(i+1) scalar connectivities are identified by heteronuclear multiple bond correlation (HMBC) spectroscopy. The presence of these three-bond scalar connectivities in predominantly .alpha.-helical regions correlates well with the secondary structure determined previously from a qualitative analysis of homonuclear nuclear Overhauser data [Forman-Day, J.D., Clore, G. M., Driscoll, P. C., Wingfield, P.T., Richards, F. M., and Gronenborn, A.M. (1989) Biochemistry 28, 7088-7097], suggesting that this technique may provide additional information for secondary structure determination a priori. The accuracy with which 3JHN.alpha. coupling constants can be obtained from the HMQC-J experiment permits a more precise delineation of the beginnings and ends of secondary structural elements of human thioredoxin and of irregularities in these elements.