¹H‐NMR assignments and local environments of aromatic residues in bovine, human and guinea pig variants of α‐lactalbumin

Abstract

'H-NMR assignments have been defined for the aromatic-ring protons of the bovine, guinea pig and human variants of a-lactalbumin. Spin-system networks were identified by means of doublequantum-filtered two-dimensional J-correlated spectroscopy and two-dimensional relayed coherence spectroscopy data. Analysis of two-dimensional nuclear-Overhauser-enhancement spectroscopy data of the proteins indicated that in each case two clusters of aromatic residues exist. The two clusters are also evident in the crystal structure of the human protein, and this evidence, in conjunction with sequence differences between the three proteins, permitted sequence-specific assignments to be made for the majority of aromatic residues. Remaining ambiguities in the assignments could be resolved by analysis of photochemically induced dynamic nuclear polarization (PCIDNP) effects. Comparison of the PCIDNP spectra of the three proteins indicated the presence of only minor differences in the surface exposure of conserved aromatic residues. Taken together, these results indicate that the environments of the conserved aromatic residues in bovine, guinea pig and human a-lactalbumin in solution are very similar to each other, and that the solution and the crystal forms of at least the human protein are similar.

Probing Protein Structure by Solvent Perturbation of NMR Spectra

“…The molten globule state of bovine a-lactalbumin has been most extensively characterized at pH 2 (Dolgikh et al, 1985;Baum et al, 1989;Alexandrescu et al, 1992;Chyan et al, 1993;Shimizu et al, 1993). However, several observations have dissuaded us from studying bovine a-lactalbumin under these conditions.…”

“…Furthermore, the presence of fifteen aromatic residues almost evenly distributed in the sequence makes the use of these residues as probes of the folding of the entire protein relatively easy. In the native state, some of the aromatic residues are spatially close and form two aromatic clusters (Acharya et al, 1989;Alexandrescu et al, 1992). The first comprises Phe31, His32, Tyr36 and Trpll8 while the second contains Trp26, Phe53, Trp60, Tyrl03 and Trpl04.…”

“…The present study exploits both techniques to study the molten globule state of a-lactalbumin, one of the best characterised stable folding intermediates (Dolgikh et al, 1981(Dolgikh et al, , 1985Baum et al, 1989;Ewbank and Creighton, 1991 ;Alexandrescu et al, 1992). a-Lactalbumins are known to form molten globule states under a variety of denaturing conditions (extreme pH, removal of bound calcium, reduction of disulphide bonds, high temperature (Kuwajima, 1989).…”

Probing Protein Structure by Solvent Perturbation of NMR Spectra

“…Assignments were obtained on the basis of spectra acquired at 500 MHz, pH 10.0 and 310 K, and were compared with the values published by Alexandrescu et al (1992) under similar conditions. The extrapolation of the assignment to neutral conditions was straightforward for most of the resonances since little dependence of the spin patterns on pH and temperature was observed.…”

“…First, a-lactalbumins are relatively small globular proteins of approximately 14kDa molecular mass and therefore well suited for NMR studies. The crystallographic structure of baboon a-lactalbumin has been determined (Acharya et al, 1989) and partial NMR assignment of the native state of a-lactalbumins from other species has been reported (Alexandrescu et al, 1992(Alexandrescu et al, , 1993. Secondly, this protein provides one of the first recognized stable protein folding intermediates (Dolgikh et al, 1981).…”