Kanako Nakagawa

Structural and thermodynamic consequences of removal of a conserved disulfide bond from equine β-lactoglobulin

A disulfide bond between cysteine 66 and cysteine 160 of equine β‐lactoglobulin was removed by substituting cysteine residues with alanine. This disulfide bond is conserved across the lipocalin family. The conformation and stability of the disulfide‐deleted mutant protein was investigated by circular dichroism. The mutant protein assumes a native‐like structure under physiological conditions and assumes a helix‐rich molten globule structure at acid pH or at moderate concentrations of urea as the wild‐type protein does. The urea‐induced unfolding experiment shows that the stability of the native conformation was reduced but that of the molten globule intermediate is not significantly changed at pH 4 by removal of the disulfide bond. On the other hand, the molten globule at acid pH was destabilized by removal of the disulfide bond. This difference in the stabilizing effect of the disulfide bond was interpreted by the effect of the disulfide in keeping the molecule compact against the electrostatic repulsion at acid pH. In contrast to the wild‐type protein, the circular dichroism spectrum in the molten globule state at acid pH depends on anion concentration, suggesting that the expansion of the molecule through electrostatic repulsion induces α‐helices as observed in the cold denatured state of the wild‐type protein. Proteins 2006.

A native disulfide stabilizes non-native helical structures in partially folded states of equine β-lactoglobulin.

Equine β-lactoglobulin (ELG) assumes non-native helices during refolding and in partially folded states. Previously, circular dichroism (CD) combined with site-directed mutagenesis identified helical regions in the acid- and cold-denatured states of ELG. It is also known that a fragment of ELG, CHIBL (residues 88-142), has a structure similar to that of the cold-denatured state. For the study reported herein, the structure of a shorter fragment, CHIBLΔF (residues 97-142), was investigated by CD and nuclear magnetic resonance spectroscopy. The secondary chemical shifts clearly showed that non-native α-helices are present in two different regions, residues 98-107 and 114-135, and are connected by a native disulfide bond. The CD spectra of two peptides that correspond to the helical regions are characterized by weak helical signatures, and the sum of their CD spectra is nearly the same as the spectrum of disulfide-reduced CHIBLΔF. Therefore, the non-native helices are stabilized by the disulfide, and non-native helix formation may occur only during the refolding of the disulfide-intact protein. Supporting this conclusion is the observation that tear lipocalin, a homologue of ELG that lacks the disulfide, does not form non-native helices during folding.

Relationship between chain collapse and secondary structure formation in a partially folded protein

Chain collapse and secondary structure formation are frequently observed during the early stages of protein folding. Is the chain collapse brought about by interactions between secondary structure units or is it due to polymer behavior in a poor solvent (coil-globule transition)? To answer this question, we measured small-angle X-ray scattering for a series of β-lactoglobulin mutants under conditions in which they assume a partially folded state analogous to the folding intermediates. Mutants that were designed to disrupt the secondary structure units showed the gyration radii similar to that of the wild type protein, indicating that chain collapse is due to coil-globule transitions.

Chloride-ion concentration dependence of molecular dimension in the acid-denatured state of equine β-lactoglobulin

The chloride-ion concentration dependence of the molecular dimension in the acid-denatured state of equine β-lactoglobulin (ELG) was investigated by small-angle X-ray scattering. In the presence of chloride ion, ELG has a globular and compact conformation (the A state). The molecular dimension of ELG increases little with decreasing chloride-ion concentration. A remarkable dependence was observed for a mutant protein in which both Cys66 and Cys160 were replaced with Ala (C66A/C160A). In the presence of chloride ion, C66A/C160A has a globular and compact conformation, like the wild type. In the absence of chloride ion, however, the molecular dimension and shape was close to that in the urea-unfolded state. Previously, we have shown that the helix content in the acid-denatured state increases with decreasing chloride-ion concentration [Yamada et al. (2006). Proteins Struct. Funct. Bioinf. 63, 595–602]. These results suggest that the secondary structure in the A state is mainly determined by non-local interactions. When they are absent in an expanded conformation, the local interactions become predominant and the amount of non-native α-helix increases.

Importance of polypeptide chain length for the correct local folding of a β-sheet protein

Equine β-lactoglobulin is a 162-residue β-sheet protein. A partially folded form of equine β-lactoglobulin contains a β-hairpin and an α-helix. The β-hairpin converts into non-native α-helices at temperatures <0°C. CHIBL, a truncated equine β-lactoglobulin (residues 88-142), contains the low-temperature α-helical structure even at room temperature, indicating that the interactions responsible for the stability of the β-hairpin reside in non-CHIBL residues. For the study reported herein, we characterized two truncated mutants and their leucine103 → proline103 variants to identify residues that stabilize the β-hairpin. The dependence of their circular dichroism spectra on chloride ion concentration and temperature revealed that the ability to transition from the non-native α-helices to the β-hairpin depends on the polypeptide chain length and improves as the chain length increases despite the apparent absence of any ordered structure in the extended sequences.

Effect of non-native helix destabilization on the folding of equine β-lactoglobulin

β-lactoglobulin forms a non-native α-helix during an early stage of folding. To address the role of the non-native structure in the folding process, we designed several mutants of equine β-lactoglobulin with reduced helical propensity in the non-native helix region. One of them, A123T, showed a similar structure to that of the wild-type protein; its folding kinetics was investigated by stopped-flow circular dichroism (CD) and fluorescence. Although A123T showed a reduced burst-phase CD intensity, its folding rate was similar to that of the wild-type protein, which indicated that the formation of the non-native helix does not accelerate or decelerate the folding reaction.