Hydrogen-deuterium exchange in the histidine residues of bovine alpha-lactalbumin.

The proton magnetic resonance spectrum of bovine alpha-lactalbumin has been observed, and three peaks assignable to the position-2 CH protons of the three histidine rpsidues (His 32, 68, and 107) of this protein have been subjected to detailed examination. The assignments of these peaks to His 32, 68, and 107 were made on the basis of the difference in their reactivities with iodoacetic acid. The rate constants of the hydrogen-deuterium exchange reactions were found to be 8.0 X 10(-5), 2.6 X 10(-4), and 8.0 X 10(-5) min-1, respectively, at pH 8.5 and at 35 degrees, while at 62 degrees all three were found to be 0.84 approximately 1.1 X 10(-2) min-1. On the basis of these data, it has been shown that, in the native form of this protein, His 68 is the most exposed to the solvent while His 32 and His 107 are buried slightly deeper in the surface of the molecule. The fluctuation amplitudes gamma, or the effective chances of His 32, 68, and 107 to be fully exposed to the solvent, were found to be 0.4, 1.3, and 0.4, respectively.     

Proton-magnetic-resonance studies of the lysine residues of ribonuclease A.

The amino groups of ribonuclease A (RNase-A) have been methylated with formaldehyde and borohydride to provide observable resonances for proton magnetic resonance (PMR) studies. Although enzymatic activity is lost, PMR difference spectroscopy and PMR studies of thermal denaturation show native conformation is largely preserved in methylated RNase-A. Resonances corresponding to the NH2-terminal alpha-amino and 10 xi-amino N-methyl groups are titrated at 220 MHz to obtain pK values. After correction for the effects of methylation, using values previously derived from model compound studies, a pK of 6.6 is found for the alpha-amino group, a pK of 8.6 for the xi-amino group of lysine-41 and pK values ranging from 10.6 to 11.2 for the other lysine xi-amino groups. Interactions between lysine-7 and lysine-41 or between the alpha-amino and xi-amino groups of lysine-1 have been proposed to account for deviations from simple titration behaviour. The correct continuities for the titration curves of the histidine H-2 proton resonances have been confirmed by selective deuteration of the H-2 protons. Titration curves for the H-2 proton resonances of histidine-12 and histidine-119 of methylated RNase-A show deviations from the titration curves for the native enzyme, indicating some alteration of the active-site conformation. In the presence of phosphate, titration curves for the H-2 proton resonances of histidine-12 and histidine-119 of methylated RNase-A indicate binding of phosphate at the active site, but these curves continue to show deviations from the titration behaviour of native RNase-A. The titration curve for the N-methyl resonance of lysine-41 is perturbed considerably by the presence of phosphate, which indicates a possible catalytic role for lysine-41.     

Application of the pH-jump method to the titration of tyrosine residues in bovine alpha-lactalbumin.

A stopped-flow technique has been developed for the zero-time spectrophotometric titration of tyrosine residues in the purely native or in the purely alkaline denatured state of alpha-lactalbumin that undergoes an alkaline conformational transition in the pH region of tyrosine ionization. The progressive absorption change at 298 nm caused by a pH jump from neutral pH is shown to result from the change in ionization of the tyrosine residues brought about by a first-order process of the conformational transition. Extrapolation to zero time gives the titration curve for purely native alpha-lactalbumin. Similarly, the pH jump from highly alkaline pH gives the titration curve for the purely alkaline denatured protein. The method should be generally applicable to other proteins that contain tyrosines. Analysis of the titration curves suggests that the four tyrosines in native alpha-lactalbumin have pK values of 10.5, 11.8, 11.8, and 12.7, respectively. After the alkaline transconformation, all of them become titrated normally with a pK value of 10.3. A comparison of these results with the ionization behavior of tyrosines in hen egg white and human lysozymes is presented and discussed in terms of differences in the sequences of the proteins.     

Tyrosyl-tRNA-synthetase from bovine liver. Functional role of histidine residues]

A specific chemical modification of histidyl residues in tyrosyl-tRNA synthetase by diethyl pyrocarbonate was performed. It is shown that five of sixteen histidyl residues can react with diethyl pyrocarbonate in the native conditions. Modification of two histidyl residues per dimer results in the inactivation of tyrosyl-tRNA synthetase in both steps of the tRNATyr aminoacylation. All substrates protect tyrosyl-tRNA synthetase against inactivation with diethyl pyrocarbonate, the most effective protector being combination of ATP and tyrosine. Histidyl residues of tyrosyl-tRNA synthetase are suggested to be involved in the catalytic mechanism of aminoacylation of tRNATyr.     

Hydrogen exchange of the tryptophan residues in bovine alpha-lactalbumin studied by UV spectroscopy

The effect of Ca²⁺ ion on structural fluctuation of a milk Ca²⁺-binding protein, α-lactalbumin, under native conditions was investigated by comparing hydrogen-exchange reactions of tryptophan residues in the apo-form without Ca²⁺ and in the holo-form at 1 mM CaCl₂ at pH 7.0 in the presence of 0.1M Na⁺. The reactions were followed by measuring time-dependent absorption changes at 298–300 nm due to the ²H-¹H exchange of the tryptophan imino protons and were found to be biphasic under all the conditions examined. Two of the four tryptophan protons are insensitive to Ca²⁺ concentration and show a relatively fast exchange rate. The other two protons are much more extensively protected (a protection degree of 10³–10⁵) and are markedly affected by the presence of Ca²⁺. Examinations of the temperature dependence and pH dependence of the individual exchange rates have been utilized for elucidating the exchange mechanism. The fast protons show a low activation energy reaction with so-called EX₂ kinetics. The exchange reaction of the slow protons is accompanied by a high activation energy, and the exchange mechanism of the protons depended on the presence or absence of stabilizing Ca²⁺ ions—the EX₁ kinetics for the apo-protein and the EX₂ kinetics for the holo-protein at 1 mM Ca²⁺. The exchange reaction in the thermally unfolded state was also found to be biphasic, but the fast phase, which has an exchange rate in the fully exposed state, becomes predominant with decreasing temperature. By taking this fact and using a structural unfolding model of hydrogen exchange, the present results are fully consistent with thermodynamic parameters of the thermal transition and kinetic parameters of refolding reactions induced by concentration jumps of guanidine hydrochloride obtained in previous studies. It is demonstrated that the reaction of the slow protons in the native state is mediated by a transient global unfolding equivalent to the “thermal” unfolding under a native condition and that switching of the exchange mechanism from the EX₁ to EX₂ kinetics results from acceleration of the refolding rate with an increase in Ca²⁺ concentration. The transient global unfolding takes place even under a strongly native condition, e.g., at a temperature 20° below the beginning of the thermal transition.     

A comparative study on bovine alpha-lactalbumin and lysozyme by nanosecond fluorometry.

Analysis of the time decay of fluorescence anisotropy of 1-dimethylaminoaphthalene-5-sulfonyl (DNS) and fluorescamine derivatives of bovine alpha-lactalbumin and lysozyme reveals that no significant differences in mean rotational relaxation times are present. While fluorescamine molecules appear to orient randomly on these proteins, DNS is bound with a preferential orientation. Other fluorescence characteristics of the labels are also cited.     

The low-temperature luminescence properties of bovine alpha-lactalbumin.

The luminescence of bovine alpha-lactalbumin at 77 K has been studied and compared with that of lysozyme. Alpha-Lactalbumin has several unusual properties, including a fluorescence spectrum showing vibrational fine structure, an abnormal phosphorescence spectrum, a high fluorescence: phosphorescence ratio and an abnormal phosphorescence decay. These properties are largely due to the proximity of tryptophan residues to disulphide bonds. Reduction of all these bonds causes considerable changes in alpha-lactalbumin luminescence, as does denaturation in acid solution. Reduction of a single labile disulphide bond has little effect, and the properties of alpha-lactalbumin III, a variant lacking one disulphide bond and one trypotophan residue, are similar to those of the normal protein. Several differences between alpha-lactalbumin and lysozyme are reported. The results support the suggestion that the two tryptophan residues found in the active site cleft of alpha-lactalbumin may be largely responsible for its luminescence.     

Kinetics of the renaturation of bovine alpha-lactalbumin

Multiparametric kinetic study of bovine alpha-lactalbumin renaturation from the unfolded state has shown the existence of an intermediate which is formed within 10(-2) s with properties close to those of the molten globule. Apart from the fast kinetic phase which results in the intermediate, two slower phases were found with intrinsic times approximately 1 s and approximately 10 s. The slowest one is apparently due to proline isomerization.     

Efficient expression of bovine alpha-lactalbumin in Saccharomyces cerevisiae.

A synthetic gene encoding the mature bovine alpha-lactalbumin fused to the preproregion of the yeast alpha-mating factor has been expressed and secreted at high level in Saccharomyces cerevisiae under the control of the alpha-mating promoter. Growth conditions were found to be critical for the expression: recombinant alpha-lactalbumin could only be detected in the medium provided the culture was grown at neutral pH. The secreted bovine alpha-lactalbumin is enzymatically active and identical to the whey protein, as confirmed by SDS/PAGE, IEF, ultraviolet and CD spectral analysis, and amino-terminal sequence determination.     

Secretion of bovine alpha-lactalbumin into the milk of transgenic rats.

Bovine alpha-lactalbumin (b alpha LA) gene prepared by polymerase chain reaction was introduced into rat genomes by microinjection. Out of 17 transgenic rat lines, 11 secreted b alpha LA into their milk at concentrations higher than 0.2 micrograms/ml. Of these, three lines secreted b alpha LA at concentrations higher than those in bovine milk. The highest concentration of b alpha LA secreted into rat milk was 2,400 micrograms/ml.     

A circular dichroic study of Cu(II) binding to bovine alpha-lactalbumin.

The visible and ultraviolet circular dichroic spectra resulting from the interaction of bovine alpha-lactalbumin with successive Cu(II) ions have been recorded under a variety of conditions. Analysis of the observed change-transfer and d-d band transitions can be made in terms of two kinds of binding sites: at a histidyl group and at the N-terminal amino group, respectively. At basic pH the amide nitrogens of the peptide backbone progressively take part in the coordination. The occupation of the high affinity calcium binding site by Ca(II) and Mn(II) does not influence the Cu(II) binding process, suggesting that there is no direct interaction between this site and the Cu(II) binding sites.     

Hydrogen-tritium exchange titration of the histidine residues in bovine heart cytochrome c and analysis of their microenvironment.

Microenvironments of the three histidine residues located at the positions 18, 26, and 33 from the amino terminus in bovine heart cytochrome c were analysed in solution by the hydrogen-tritium exchange titration method, which has been developed in this laboratory. Histidine-18, which is liganded to the heme iron, and histidine-26 did not incorporate tritium in native state, indicating that the two are located in solvent inaccessible hydrophobic regions. Histidine-33 was labeled with tritium to an appreciable extent and seemed to be partially buried in the molecule. The pKa value estimated for histidine-33 was 6.1 at 37 degrees by the tritium exchange titration, suggesting that the residue interacts very weakly with a neighboring cationic group. These results seem to be compatible with the tertiary structure of the protein deduced from the X-ray crystallographic analysis.     

Hydrogen exchange of the tryptophan residues in bovine, goat, guinea pig, and human alpha-lactalbumin

Hydrogen exchange of the individual tryptophan residues of bovine, goat, guinea pig, and human alpha-lactalbumin has been studied by both ultraviolet and NMR spectra. The assignment of the slowly exchanging imino proton resonances to the tryptophan residues (Trp26 and Trp60) was obtained by comparison of the nuclear Overhauser effect difference spectra of bovine, guinea pig, and human alpha-lactalbumin. Taking account of the thermal unfolding of each alpha-lactalbumin, the hydrogen exchange rates of the individual tryptophan residues are analyzed. The temperature dependence of the exchange rates classified their exchange mechanisms into two exchange processes: the "low activation energy process" and the "high activation energy process" which is associated directly with the global thermal unfolding of the protein. Trp26 of alpha-lactalbumin exchanges through the high activation energy process. The exchange behavior of Trp26 of guinea pig alpha-lactalbumin suggests a difference of the globally unfolded state of the protein from the other species. The exchange mechanism of Trp60 of human alpha-lactalbumin is the low activation energy process in contrast with those of the bovine and goat proteins, although their global thermodynamic properties are similar to each other. Trp104 and Trp118 of alpha-lactalbumin exchange through the low activation energy process, and the reaction rates are affected by the local structural differences around the tryptophan residues among these proteins. The results presented in this paper indicate that the hydrogen exchange rate through the low activation energy process provides the information only about the local nature of a protein while that through the high activation energy process provides the information about the global nature of a protein.     

Compactness of the kinetic molten globule of bovine alpha-lactalbumin: a dynamic light scattering study.

During folding of globular proteins, the molten globule state was observed as an equilibrium intermediate under mildly denaturing conditions as well as a transient intermediate in kinetic refolding experiments. While the high compactness of the equilibrium intermediate of alpha-lactalbumin has been verified, direct measurements of the compactness of the kinetic intermediate have not been reported until now. Our dynamic light scattering measurements provide a complete set of the hydrodynamic dimensions of bovine alpha-lactalbumin in different conformational states, particularly in the kinetic molten globule state. The Stokes radii for the native, kinetic molten globule, equilibrium molten globule, and unfolded states are 1.91, 1.99, 2.08, and 2.46 nm, respectively. Therefore, the kinetic intermediate appears to be even more compact than its equilibrium counterpart. Remarkable differences in the concentration dependence of the Stokes radius exist revealing strong attractive but repulsive intermolecular interactions in the kinetic and equilibrium molten globule states, respectively. This underlines the importance of extrapolation to zero protein concentration in measurements of the molecular compactness.