Mineyuki Mizuguchi

FT-IR study of the Ca2+-binding to bovine α-lactalbumin: Relationships between the type of coordination and characteristics of the bands due to the Asp COO− groups in the Ca2+-binding site

Fourier-transform infrared spectroscopy (FT-IR) was applied to examine relationships between the type of coordination and the COO- antisymmetric and symmetric stretches of the COO- groups in the Ca2+-binding site of bovine alpha-lactalbumin. The peaks at 1593, 1578, 1425, and 1403 cm(-1) were assigned to the COO- groups of Asp-82, 87, and 88 coordinating to Ca2+ in the pseudo bridging mode, according to the results of X-ray crystallography. The bands due to the COO- groups were quite similar to each other between alpha-lactalbumin and EDTA which is the model compound for the pseudo bridging state.Fourier‐transform infrared spectroscopy (FT‐IR) was applied to examine relationships between the type of coordination and the COO− antisymmetric and symmetric stretches of the COO− groups in the Ca2+‐binding site of bovine α‐lactalbumin. The peaks at 1593, 1578, 1425, and 1403 cm−1 were assigned to the COO− groups of Asp‐82, 87, and 88 coordinating to Ca2+ in the pseudo bridging mode, according to the results of X‐ray crystallography. The bands due to the COO− groups were quite similar to each other between α‐lactalbumin and EDTA which is the model compound for the pseudo bridging state.

Partial molar volumes and adiabatic compressibilities ofN-acetyl-DL-serinamide andN-acetyl-L-threonmamide in dilute aqueous solutions

Densities and sound velocities in dilute aqueous solutions ofN-acetyl-DL-serinamide andN-acetyl-L-threoninamide were measured at 5, 15, 25, 35, and 45°C. Partial molar volumes and partial molar adiabatic compressibilities of these amino acid derivatives at infinite dilution were determined. The partial molar quantities for the parent amino acids, serine and threonine, were also determined and compared with the acetyl amide derivatives. The contribution of the side chain of theN-acetyl amino acid amide or amino acid to the partial molar quantities were estimated from the difference between the partial molar quantities for the solute studied and those for the corresponding species,N-acetyl-glycinamide or glycine, without the side chain.

Effects of a helix substitution on the folding mechanism of bovine α‐lactalbumin

The structure, stability, and unfolding‐refolding kinetics of a chimeric protein, in which the amino acid sequence of the flexible loop region (residues 105–110) comes from equine lysozyme and the remainder of the sequence comes from bovine α‐lactalbumin were studied by circular dichroism spectroscopy and stopped‐flow measurements, and the results were compared with those of bovine α‐lactalbumin. The substitution of the flexible loop in bovine α‐lactalbumin with the helix D of equine lysozyme destabilizes the molten globule state, although the native state is significantly stabilized by substitution of the flexible loop region. The kinetic refolding and unfolding experiments showed that the chimeric protein refolds significantly faster and unfolds substantially slower than bovine α‐lactalbumin. To characterize the transition state between the molten globule and the native states, we investigated the guanidine hydrochloride concentration dependence of the rate constants of refolding and unfolding. Despite the significant differences in the stabilities of both the molten globule and native states between the chimeric protein and bovine α‐lactalbumin, the free energy level of the transition state is not affected by the amino acid substitution in the flexible loop region. Our results suggest that the destabilization in the molten globule state of the chimeric protein is caused by the disruption of the non‐native interaction in the flexible loop region and that the disruption of the non‐native interaction reduces the free energy barrier of refolding. We conclude that the non‐native interaction in the molten globule state may act as a kinetic trap for the folding of α‐lactalbumin. Proteins 2002;49:95–103.