Canine Milk Lysozyme Research Articles

Folding mechanism of canine milk lysozyme studied by circular dichroism and fluorescence spectroscopy

We have studied the guanidine hydrochloride‒induced equilibrium unfolding and the kinetics of refolding of canine milk lysozyme by circular dichroism and fluorescence spectroscopy. The thermodynamic analysis of the equilibrium unfolding measured by circular dichroism and fluorescence has shown that unfolding is represented by a three‒state mechanism and that the intermediate state of canine milk lysozyme is remarkably more stable than the intermediates observed in other lysozyme and α-lactalbumin. In the kinetic refolding of this protein, there are at least two kinetic intermediates; a burst=phase intermediate accumulated within the dead time (4 ms) of the measurement and an intermediate that has been observed during the kinetics with a rate constant of 10–20 s–1after the burst phase. This result is apparently in contrast with those previously observed in the kinetic refolding of α‒lactalbumin and equine lysozyme that show only the burst‒phase intermediate. The relationship between the extraordinarily stable equilibrium molten globule and the kinetic folding intermediates will be discussed.

3M1045 CDおよび蛍光を用いたイヌミルクリゾチームのフォールディング機構の解析(1.蛋白質(C)物性,一般講演,日本生物物理学会第40回年会)

3M1045 CDおよび蛍光を用いたイヌミルクリゾチームのフォールディング機構の解析(1.蛋白質(C)物性,一般講演,日本生物物理学会第40回年会)

Energetics of three-state unfolding of a protein: canine milk lysozyme.

Thermodynamics of thermal transitions of a calcium-binding lysozyme, canine milk lysozyme (CML), was studied using differential scanning calorimetry and compared with those for homologous proteins, human alpha-lactalbumin (alpha-hLA) and equine milk lysozyme (EML). The results showed that CML and EML exhibit two clear heat absorption peaks in the absence of calcium ions (apo-form), although the cooperative thermal transition of alpha-hLA is apparently absent in this form. The first peak represents the unfolding transition from the native to an unfolding intermediate state (N-I transition) and the second peak represents that from the intermediate to the thermally unfolded state (I-U transition). We interpret that the cooperative thermal transition, which is observed between the intermediate and the thermally unfolded states of CML and EML, comes from the native-like packing interaction in their intermediate states. Furthermore, to examine the role of the stabilization mechanism of CML intermediate, we constructed four variant CMLs (H21G, I56L, A93S and V109K), in which the residues of CML are substituted for those of EML, and also investigated their thermal stability. Especially the His21 and Val109 of CML play a role in stabilization of the intermediate state and their contributions to the unfolding free energy are estimated to be 2.0 and 1.8 kJ/mol, respectively. From the results of the mutational analysis, a few differences in the local helical interactions within the alpha-domain are found to be predominant in stabilizing the intermediate state.

3P023イヌミルクリゾチームおよびヒトα-ラクトアルブミンのフォールディングの中間状態における体積挙動

3P023イヌミルクリゾチームおよびヒトα-ラクトアルブミンのフォールディングの中間状態における体積挙動

Structure and thermodynamics of the extraordinarily stable molten globule state of canine milk lysozyme.

Here, we show that an unfolded intermediate of canine milk lysozyme is extraordinarily stable compared with that of the other members of the lysozyme-alpha-lactalbumin superfamily, which has been studied previously. The stability of the intermediate of this protein was investigated using calorimetry, CD spectroscopy, and NMR spectroscopy, and the results were interpreted in terms of the structure revealed by X-ray crystallography at a resolution of 1.85 A to an R-factor of 17.8%. On the basis of the results of the thermal unfolding, this protein unfolds in two clear cooperative stages, and the melting temperature from the intermediate to the unfolded states is about 20 degrees C higher than that of equine milk lysozyme. Furthermore, the (1)H NMR spectra of canine milk lysozyme at 60 degrees C, essentially 100% of which exists in the intermediate, showed that small resonance peaks that arise from ring-current shifts of aliphatic protons are still present in the upfield region from 0 to -1 ppm. The protein at this temperature (60 degrees C) and pH 4.5 has been found to bind 1-anilino-naphthalene-8-sulfonate (ANS) with enhancement of the fluorescence intensity compared with that of native and thermally unfolded states. We interpret that the extraordinarily stable intermediate is a molten globule state, and the extraordinary stabilization of the molten globule state comes from stronger protection around the C- and D-helix of the aromatic cluster region due to the His-21 residue. The conclusion helps to explain how the molten globule state acquires its structure and stability.

Hydrogen exchange study of canine milk lysozyme: stabilization mechanism of the molten globule.

The native state (1)H, (15)N resonance assignment of 123 of the 128 nonproline residues of canine milk lysozyme has enabled measurements of the amide hydrogen exchange of over 70 amide hydrogens in the molten globule state. To elucidate the mechanism of protein folding, the molten globule state has been studied as a model of the folding intermediate state. Lysozyme and alpha-lactalbumin are homologous to each other, but their equilibrium unfolding mechanisms differ. Generally, the folding mechanism of lysozyme obeys a two-state model, whereas that of alpha-lactalbumin follows a three-state model. Exceptions to this rule are equine and canine milk lysozymes, which exhibit a partially unfolded state during the equilibrium unfolding; this state resembles the molten globule state of alpha-lactalbumin but with extreme stability. Study of the molten globules of alpha-lactalbumin and equine milk lysozyme showed that the stabilities of their alpha-helices are similar, despite the differences in the thermodynamic stability of their molten globule states. On the other hand, our hydrogen exchange study of the molten globule of canine milk lysozyme showed that the alpha-helices are more stabilized than in alpha-lactalbumin or equine milk lysozyme and that this enhanced stability is caused by the strengthened cooperative interaction between secondary structure elements. Thus, our results underscore the importance of the cooperative interaction in the stability of the molten globule state.

3A0915 イヌミルクリゾチームの折り畳み中間体およびモルテングロビュール状態の重水素交換反応による解析

3A0915 イヌミルクリゾチームの折り畳み中間体およびモルテングロビュール状態の重水素交換反応による解析

1A1645 イヌ・ミルク・リゾチームの精製とフォールディング機構の研究

1A1645 イヌ・ミルク・リゾチームの精製とフォールディング機構の研究

Expression of a synthetic gene encoding canine milk lysozyme in Escherichia coli and characterization of the expressed protein.

A high-expression plasmid of the canine milk lysozyme, which belongs to the family of calcium-binding lysozymes, was constructed in order to study its physico-chemical properties. Because the cDNA sequence of the protein has not yet been determined, a 400 base-pair gene encoding canine milk lysozyme was first designed on the basis of the known amino acid sequence. The gene was constructed by an enzymatic assembly of 21 chemically synthesized oligonucleotides and inserted into an Escherichia coli expression vector by stepwise ligation. The expression plasmid thus constructed was transformed into BL21(DE3)/pLysS cells. The gene product accumulated as inclusion bodies in an insoluble fraction. Recombinant canine milk lysozyme was obtained by purification and refolding of the product and showed the same characteristics in terms of bacteriolytic activity and far- and near-UV circular dichroism spectra as the authentic protein. The NMR spectra of refolded lysozyme were also characteristic of a native globular protein. It was concluded that recombinant canine milk lysozyme was folded into the correct native structure. Moreover, the thermal unfolding profiles of the refolded recombinant lysozyme showed a stable equilibrium intermediate, indicating that the molten globule state of this protein was extraordinarily stable. This expression system of canine milk lysozyme will enable biophysical and structural studies of this protein to be extended.

Feline and canine milk lysozymes

1. 1. The electrophoretic characteristics of feline ( Felis catus) and canine ( Canis familiaris) milk lysozymes were studied using starch gel electrophoresis and isoelectric focusing. 2. 2. Feline milk lysozyme was found to be polymorphic (two variants, designated A and B with frequencies of 0.13 and 0.87, respectively). Canine milk lysozyme was not polymorphic. 3. 3. The lytic activities of feline and canine milk lysozymes were examined in buffers of varying pH and ionic strength. Preliminary kinetics studies were done. 4. 4. Maximal lytic activity for both lysozymes was found in imidazole-HCl buffer pH 7.4 and both exhibited second order reaction kinetics. 5. 5. Amino acid compositions of both lysozymes were determined.