Masaaki Hirose

Molten globule state of food proteins

Abstract The occurrence of the molten globule state, a unique state with a partially folded conformation that can be distinguished from either the native or the fully denatured forms, has been demonstrated in many globular proteins. The molten globule state is formed as a kinetic intermediate for reversible denaturation, in equilibrium under certain mild denaturing conditions, and upon insertion into a cell membrane during cellular translocation. Several food proteins from animal sources take on the molten globule state, which may be involved in the functional properties of these food proteins.

Alternative structural state of transferrin. The crystallographic analysis of iron-loaded but domain-opened ovotransferrin N-lobe.

Transferrins bind Fe3+ very tightly in a closed interdomain cleft by the coordination of four protein ligands (Asp60, Tyr92, Tyr191, and His250 in ovotransferrin N-lobe) and of a synergistic anion, physiologically bidentate CO3 2−. Upon Fe3+uptake, transferrins undergo a large scale conformational transition: the apo structure with an opening of the interdomain cleft is transformed into the closed holo structure, implying initial Fe3+ binding in the open form. To solve the Fe3+-loaded, domain-opened structure, an ovotransferrin N-lobe crystal that had been grown as the apo form was soaked with Fe3+-nitrilotriacetate, and its structure was solved at 2.1 Å resolution. The Fe3+-soaked form showed almost exactly the same overall open structure as the iron-free apo form. The electron density map unequivocally proved the presence of an iron atom with the coordination by the two protein ligands of Tyr92-OH and Tyr191-OH. Other Fe3+ coordination sites are occupied by a nitrilotriacetate anion, which is stabilized through the hydrogen bonds with the peptide NH groups of Ser122, Ala123, and Gly124 and a side chain group of Thr117. There is, however, no clear interaction between the nitrilotriacetate anion and the synergistic anion binding site, Arg121.

Binding of ligands by proteins: a simple method with sephadex gel

A simple procedure for the measurement of ligand binding by proteins was devised with Sephadex gel; this procedure differs from the more common method of gel filtration.

The structural mechanism for iron uptake and release by transferrins.

Transferrins are a group of iron-binding proteins that control the levels of iron in the body fluids of vertebrates by their ability to bind two Fe3+ and two CO3 2-. The transferrin molecule, with a molecular mass of about 80 kDa, is folded into two similarly sized homologous N- and C-lobes that are stabilized by many intrachain disulfides. As observed by X-ray crystallography, each lobe is further divided into two similarly sized domains, domain 1 and domain 2, and an Fe3+-binding site is within the interdomain cleft. Four of the six Fe3+ coordination sites are occupied by protein ligands (2 Tyr residues, 1 Asp, and 1 His) and the other two by a bidentate CO3 2-. Upon uptake and release of Fe3+, transferrins undergo a large-scale conformational change dependng on a common structural mechanism: domains 1 and 2 rotate as rigid bodies around a rotation axis that passes through the two antiparallel β-strands linking the domains. The extent of the rotaion is, however, variable for different transferrin species and lobes. As a Fe3+ release mechanisms at low pH from the N-lobes of serum transferrin and ovotransferrin, the structral evidence for ‘dilysine trigger mechanism’ is shown. A structural mechanism for the Fe3+ release in presence of a non-synergistic anion is proposed on the basis of the sulfate-bound apo crystal structure of the ovotransferrin N-lobe. Domain-opened structures with the coordinated Fe3+ by the two tyrosine residues are demonstrated in fragment and intact forms, and their functional implications as a possible intermediate for iron uptake and release are discussed.

Structure of aluminium-bound ovotransferrin at 2.15 Å resolution

Transferrin, well known as an iron-transport protein, can bind other metal ions, including toxic ones, and is considered to play an important role in the transportation of such metal ions. Here, a crystal structure of aluminium-bound transferrin is described for the first time. Colourless needle-shaped crystals of aluminium-bound ovotransferrin were obtained in PEG 400 solution. Structural determination was performed by molecular replacement using diferric (iron-bound) ovotransferrin as a model and the structural refinement was performed in the 50-2.15 Angstroms resolution range. The overall organization of the aluminium-bound form is almost the same as the iron-bound form: the protein is folded into two homologous lobes (N- and C-lobes) with two domains; two metal-binding sites are located within the inter-domain clefts of each lobe. Four residues (one Asp, two Tyr and one His) and one bicarbonate anion were found to bind an aluminium ion in either lobe, as in the iron-bound form. The highly similar domain-closed structure of the Al(3+)-bound form may permit the binding of Al(3+)-bound transferrin to the transferrin receptor. An unusual interaction, the dilysine trigger, which facilitates iron release at low pH in the endosome, was also found in the Al(3+)-bound form. These findings support the participation of transferrin in the transport of Al(3+) ions in vivo.

Refolding Process of Ovalbumin from Urea-denatured State EVIDENCE FOR THE INVOLVEMENT OF NONPRODUCTIVE SIDE CHAIN INTERACTIONS IN AN EARLY INTERMEDIATE

Ovalbumin contains one cystine disulfide (Cys73-Cys120) and four cysteine sulfhydryls (Cys11, Cys30, Cys367, and Cys382) in a single polypeptide chain of 385 amino acid residues. The refolding mechanism of ovalbumin was investigated under disulfide-bonded and disulfide-reduced conditions using the denatured protein state, DA, as the starting protein sample. For the preparation of DA, the disulfide-intact and disulfide-reduced forms of ovalbumin were denatured by protein incubation in 9 M urea at pH 2.2. When DA was placed in a refolding buffer, pH 8.2, an intermediate state IN was produced in either the disulfide-bonded or the disulfide-reduced condition; IN showed about 60% of the native CD ellipticity at 222 nm and the intrinsic tryptophan fluorescence with the native spectrum peak but with decreased intensity. The formation of IN as detected by far UV CD ellipticity was quite rapid and finished within a mixing dead time of 20 ms. When DA was diluted with an acidic buffer, pH 2.2, a partially folded equilibrium intermediate IA with the structural characteristics equivalent to those of IN was formed. After the formations of IN and IA, the regains in CD ellipticity and tryptophan fluorescence at pH 8.2 followed biphasic kinetics in the disulfide-bonded condition but monophasic kinetics in the disulfide-reduced condition. As unexpected findings, the native disulfide in DA and IA underwent nonproductive disulfide rearrangements in the disulfide-bonded condition at an early refolding stage and then was recovered during the subsequent refolding. The integrity of overall refolding was confirmed by the observation that the proteins refolded for 20 h in the disulfide-bonded and disulfide-reduced conditions showed, on differential scanning calorimetry analyses, almost exactly the same denaturation temperatures as their native protein counterparts. These results were consistent with a refolding process for ovalbumin which includes nonproductive side chain-side chain interactions in the early intermediate IN, which requires subsequent reorganization for the correct refolding.

Determination of sulfhydryl groups and disulfide bonds in a protein by polyacrylamide gel electrophoresis.

A general method by polyacrylamide gel electrophoresis for the determination of sulfhydryls and disulfides in a protein was developed. The method included a two-step alkylation procedure: the first step consisted of alkylation of the sulfhydryl groups with iodoacetic acid in the presence and absence of 8 M urea; the second step consisted of alkylation of the disulfide groups with iodoacetamide after reduction with a thiol. By high-pH urea gel electrophoresis, all the half-cystine residues in a protein could be categorized into three states: reactive sulfhydryls, nonreactive sulfhydryls, and disulfide bonded. The particular advantage of the method is that the states of half-cystines in different protein species can be analyzed independently both in isolated protein and in biological translation systems.

Anion-mediated Fe3+ release mechanism in ovotransferrin C-lobe: a structurally identified SO4(2-) binding site and its implications for the kinetic pathway.

The differential properties of anion-mediated Fe3+ release between the N- and C-lobes of transferrins have been a focus in transferrin biochemistry. The structural and kinetic characteristics for isolated lobe have, however, been documented with the N-lobe only. Here we demonstrate for the first time the quantitative Fe3+ release kinetics and the anion-binding structure for the isolated C-lobe of ovotransferrin. In the presence of pyrophosphate, sulfate, and nitrilotriacetate anions, the C-lobe released Fe3+ with a decelerated rate in a single exponential progress curve, and the observed first order rate constants displayed a hyperbolic profile as a function of the anion concentration. The profile was consistent with a newly derived single-pathway Fe3+ release model in which the holo form is converted depending on the anion concentration into a “mixed ligand” intermediate that releases Fe3+. The apo C-lobe was crystallized in ammonium sulfate solution, and the structure determined at 2.3 Å resolution demonstrated the existence of a single bound SO 4 2 − in the interdomain cleft, which interacts directly with Thr461-OG1, Tyr431-OH, and His592-NE2 and indirectly with Tyr524-OH. The latter three groups are Fe3+-coordinating ligands, strongly suggesting the facilitated Fe3+ release upon the anion occupation at this site. The SO 4 2 − binding structure supported the single-pathway kinetic model.

Studies on a role of the 2,3-diphosphoglycerate phosphatase activity in the yeast phosphoglycerate mutase reaction.

Abstract 1. 1. In order to clarify a role of the 2,3-diphosphoglycerate phosphatase activity catalyzed by yeast phosphoglycerate mutase (2,3-diphospho- d -glycerate:2-phospho- d -glycerate phosphotransferase, EC 2.7.5.3), chemical modification experiments and kinetic studies on the phosphatase activity have been performed. The results indicated that the substrate site of the mutase was also required for the phosphatase activity. 2. 2. It was found that such compounds as phosphoglycolate, phosphohydroxypyruvate and phosphoenolpyruvate stimulated the phosphatase activity, although they inhibited the mutase activity. A kinetic pattern of their stimulatory effects was consistent with an equation derived from the ideas that a ternary complex (enzyme-2,3-diphosphoglycerate-2,3-diphosphoglycerate) was an active intermediate in the phosphatase reaction and that the activators interacted with the enzyme at the substrate site to stimulate the hydrolysis of a phosphoester bond in 2,3-diphosphoglycerate at the coenzyme site. 3. 3. It was found that the phosphatase activity of component I (the native enzyme) was stimulated in higher extent than that of component V (the final product of the enzymic modification of component I). This observation offered a significant clue to explain a cause of the decrease in the mutase activity by the enzymic modification of component I to component V. 4. 4. From these results, a hypothetical model for a role of the phosphatase activity on the mutase reaction mechanism is presented in this paper.

Analyses of intramolecular disulfide bonds in proteins by polyacrylamide gel electrophoresis following two-step alkylation

A method that makes use of polyacrylamide gel electrophoresis was developed for the analysis of intramolecular disulfide bonds in proteins. Proteins with different numbers of cleaved disulfide bonds are alkylated with iodoacetic acid or iodoacetamide as the first step. The disulfide bonds remaining were reduced by excess dithiothreitol, and the newly generated free sulfhydryl groups were alkylated with the reagent not yet used (iodoacetamide, iodoacetic acid, or vinyl-pyridine) as the second step. This treatment made it possible for lysozyme (Mr, 14,000; 4 disulfides), the N-terminal half-molecule of conalbumin (Mr, 36,000; 6 disulfides), the C-terminal half-molecule of conalbumin (Mr, 40,000; 9 disulfides), and whole conalbumin (Mr, 78,000; 15 disulfides) to be separated by acid-urea polyacrylamide gel electrophoresis into distinct bands depending on the number of disulfide bonds cleaved. The method allowed us to determine the total number of disulfide bonds in native proteins and to assess the cleaved levels of disulfide bonds in partially reduced proteins. Two-step alkylation used in combination with radioautography was especially useful for the analysis of disulfide bonds in proteins synthesized in complex biological systems.