A set of three experiments is described which correlate aromatic resonances of histidine and tryptophan residues with amide resonances in 13C/15N-labelled proteins. Provided that backbone 1H and 15N positions of the sequentially following residues are known, this results in sequence-specific assignment of histidine 1H(delta2)/13C(delta2) and 1H(epsilon1)/13C(epsilon1) as well as tryptophan 1H(delta1)/13C(delta1), 1H(zeta2)/13C(zeta2), 1H(eta2)/13C(eta2), 1H(epsilon3)/13C(epsilon3), 1H(zeta3)/13C(zeta3) and 1H(epsilon1)/15N(epsilon1) chemical shifts. In the reverse situation, these residues can be located in the 1H-(15)N correlation map to facilitate backbone assignments. It may be chosen between selective versions for either of the two amino acid types or simultaneous detection of both with complete discrimination against phenylalanine or tyrosine residues in each case. The linkages between delta-proton/carbon and the remaining aromatic as well as backbone resonances do not rely on through-space interactions, which may be ambiguous, but exclusively employ one-bond scalar couplings for magnetization transfer instead. Knowledge of these aromatic chemical shifts is the prerequisite for the analysis of NOESY spectra, the study of protein-ligand interactions involving histidine and tryptophan residues and the monitoring of imidazole protonation states during pH titrations. The new methods are demonstrated with five different proteins with molecular weights ranging from 11 to 28 kDa.

Megalin, an approx. 600 kDa transmembrane glycoprotein that acts as multi-ligand transporter, is a member of the low density lipoprotein receptor gene family. Several cysteine-rich repeats, each consisting of about 40 residues, are responsible for the multispecific binding of ligands. The solution structure of the twelfth cysteine-rich ligand-binding repeat with class A motif found in megalin features two short beta-strands and two helical turns, yielding the typical fold with a I-III, II-V and IV-VI disulfide bridge connectivity pattern and a calcium coordination site at the C-terminal end. The resulting differences in electrostatic surface potential compared to other ligand-binding modules of this gene family, however, may be responsible for the functional divergence.