Katsutoshi Nitta

Three-State denaturation of α-lactalbumin by guanidine-hydrochloride

Abstract The reversible unfolding of α-lactalbumin by guanidine hydrochloride has been studied at 25.0 °C by means of ultraviolet circular dichroism measurements. The non-coincidence of the apparent transition curves obtained from the ellipticity changes at far (222 nm) and at near (270 nm and 296 nm) ultraviolet wave-lengths demonstrates the presence of at least one intermediate in the denaturation process. The aromatic residues which contribute to the Cotton effects at 270 nm and at 296 nm appear to be exposed to solvent in the first stage of a two-stage process, while the helical regions of the polypeptide chain appear to be destroyed in the second stage. Earlier work has demonstrated an acid transition between two compact forms of α-lactalbumin, a native (neutral pH) form and an acid form. Results presented here suggest that the acid form is produced as an intermediate in the first stage of total unfolding at neutral pH. Lysozyme and α-lactalbumin are known to have similar primary structures and are expected to have similar tertiary structures, but several differences in their properties have been described. The comparison of the unfolding transitions of α-lactalbumin and lysozyme provides a result compatible with similar tertiary structures, although the free energy of stabilization of the native state is 3 to 5 kcal/mol smaller for α-lactalbumin than for lysozyme. The pH dependence of the unfolding reaction can be described in terms of abnormal histidyl and carboxyl residues. The presence of a stable intermediate in the denaturation process may cause a difference in dynamic character in the native state between the two proteins and thus provide a reasonable interpretation for their known differences in chemical reactivity.

The calcium-binding property of equine lysozyme

It was found that equine lysozyme binds one Ca2+. It was eluted with equimolar Ca2+ from a Bio‐Gel P‐4 column. Human lysozyme did not behave similarly. Equine lysozyme is concluded to be a calcium metalloprotein like α‐lactalbumin, which is a homologue of hen egg white lysozyme.

Equilibrium and kinetics of the unfolding of α-lactalbumin by guanidine hydrochloride (II)

Abstract The reversible unfolding of α-lactalbumin by guanidine hydrochloride has been studied at 25.0°C by means of difference spectra and optical rotation. At about pH 5.50, two tryptophanyl residues buried in the interior of the native protein were considered to be exposed on its surface in the denaturated state. The dependence of the equilibrium constant of the unfolding reaction in aqueous solutions of guanidine hydrochloride on pH can be described in terms of abnormal histidyl residues. The dependence on guanidine hydrochloride concentration results in a smaller intrinsic binding constant of the denaturant with the protein, a smaller difference between the numbers of the sites on the denaturated and the native protein molecules, and a more unstable structure in the native state, than for lysozyme. On the other hand, from kinetic measurements, the unfolding was considered to be an apparent two-state transition. Apparent rate constants and half-times of the transition suggest a faster transition than in the case of lysozyme.

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.

Expression and purification of a small cytokine growth-blocking peptide from armyworm Pseudaletia separata by an optimized fermentation method using the methylotrophic yeast Pichia pastoris

A small multifunctional cytokine, growth-blocking peptide (GBP), from the armyworm Pseudaletia separata larvae was expressed as a soluble and active recombinant peptide in the methylotrophic yeast Pichia pastoris. An expression vector for GBP secretion was constructed using vector pPIC9, and GBP was expressed under the control of the alcohol oxidase (AOX1) promoter. Although we first tried to cultivate GBP in shake flask cultures, the yield was low, probably due to proteolysis of the recombinant protein. To overcome this problem, we utilized a high-density fermentation method. The pH of the medium in the fermenter was kept at 3.0, and the medium was collected within 48h post methanol shift to minimize exposure of the target peptide to proteases. Recombinant GBP was purified through three reverse-phase HPLC columns. We characterized the 25 amino acid GBP by molecular mass spectrometry and amino acid sequencing. Plasmatocyte spreading, one of the activities of GBP, was similar between chemically synthesized GBP and purified recombinant GBP. Up to 50mg GBP was recovered per 1L of yeast culture supernatant.

Structure of the antimicrobial peptide tachystatin A

The solution structure of antimicrobial peptide tachystatin A from the Japanese horseshoe crab (Tachypleus tridentatus) was determined by two-dimensional nuclear magnetic resonance measurements and distance-restrained simulated annealing calculations. The correct pairs of disulfide bonds were also confirmed in this study. The obtained structure has a cysteine-stabilized triple-stranded β-sheet as a dominant secondary structure and shows an amphiphilic folding observed in many membrane-interactive peptides. Interestingly, tachystatin A shares structural similarities with the calcium channel antagonist ω-agatoxin IVA isolated from spider toxin and mammalian defensins, and we predicted that ω-agatoxin IVA also have the antifungal activity. These structural comparisons and functional correspondences suggest that tachystatin A and ω-agatoxin IVA may exert the antimicrobial activity in a manner similar to defensins, and we have confirmed such activity using fungal culture assays. Furthermore, tachystatin A is a chitin-binding peptide, and ω-agatoxin IVA also showed chitin-binding activities in this study. Tachystatin A and ω-agatoxin IVA showed no structural homology with well known chitin-binding motifs, suggesting that their structures belong to a novel family of chitin-binding peptides. Comparison of their structures with those of cellulose-binding proteins indicated that Phe9 of tachystatin A might be an essential residue for binding to chitin.

Equilibrium and kinetic folding of hen egg‐white lysozyme under acidic conditions

The equilibrium and kinetic folding of hen egg‐white lysozyme was studied by means of circular dichroism spectra in the far‐ and near‐ultraviolet (UV) regions at 25°C under the acidic pH conditions. In equilibrium condition at pH 2.2, hen lysozyme shows a single cooperative transition in the GdnCl‐induced unfolding experiment. However, in the GdnCl‐induced unfolding process at lower pH 0.9, a distinct intermediate state with molten globule characteristics was observed. The time‐dependent unfolding and refolding of the protein were induced by concentration jumps of the denaturant and measured by using stopped‐flow circular dichroism at pH 2.2. Immediately after the dilution of denaturant, the kinetics of refolding shows evidence of a major unresolved far‐UV CD change during the dead time (<10 ms) of the stopped‐flow experiment (burst phase). The observed refolding and unfolding curves were both fitted well to a single‐exponential function, and the rate constants obtained in the far‐ and near‐UV regions coincided with each other. The dependence on denaturant concentration of amplitudes of burst phase and both rate constants was modeled quantitatively by a sequential three‐state mechanism, U↔I↔N, in which the burst‐phase intermediate (I) in rapid equilibrium with the unfolded state (U) precedes the rate‐determining formation of the native state (N). The role of folding intermediate state of hen lysozyme was discussed. Proteins 2002;49:472–482.

Adsorption of human lysozyme onto hydroxyapatite. Identification of its adsorbing site using site-directed mutagenesis.

To elucidate hydroxyapatite‐protein interaction, mutant human lysozymes in which the surface charge was modified by site‐directed mutagenesis were used. Five mutant human lysozymes (K1A, K13A, K33A, R10A, R14A) were expressed in yeast. The chromatographic behavior of these lysozymes was studied with a HPLC hydroxyapatite column. Elution molarities of K1A and R14A mutants were greatly lowered. While Lys‐13 and Arg‐10 are located around Lys‐1 and Arg‐14, K13A and R10A mutants bound onto hydroxyapatite stronger than K1A and R14A mutants. In combination with an X‐ray crystal structure of human lysozyme, it is concluded that the adsorbing site of human lysozyme is at the back of the active site and that Arg‐14, Lys‐1, Arg‐10 and Lys‐13 play important roles in binding.

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.

Comparison of the binding of Ca2+ and Mn2+ to bovine α-lactalbumin and equine lysozyme

Abstract The enthalpy change of the binding of Ca 2+ and Mn 2+ to equine lysozyme was measured at 25°C and pH 7.5 by batch microcalorimetry: ΔH° Ca 2+ = −76 ± 5 kJ mol −1 , ΔH° Mn 2+ = −21 ± 10 kJ mol −1 . Binding constants, log K Ca 2+ = 6.5 ± 0.2 and log K Mn 2+ = 4.1 ± 0.5, were calculated from the calorimetric data. Therefore, ΔS Ca2+ ° = −131 ± 20 JK −1 mol −1 and ΔS° Mn 2+ = 8 ± 44 JK −1 mol −1 . Removal of Ca 2+ induces small but significant changes in the circular dichroism spectrum, indicating the existence of a partially unfolded apo-conformation, comparable with, but different from, the apo-conformation of bovine α-lactalbumin.