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

Abstract

The native state 1H, 15N 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 α‐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 α‐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 α‐lactalbumin but with extreme stability. Study of the molten globules of α‐lactalbumin and equine milk lysozyme showed that the stabilities of their α‐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 α‐helices are more stabilized than in α‐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. Proteins 2000;40:579–589. © 2000 Wiley‐Liss, Inc.

Different Folding Pathways Taken by Highly Homologous Proteins, Goat α-Lactalbumin and Canine Milk Lysozyme

“…The Figure 4b shows the protection profile for the MG state of CML previously obtained at pH⁎ 1.6 and 25°C. 12 In contrast to the results for GLA, the A-and B-helices were much more strongly protected than the C-helix, and the protection factors, which ranged from 5 to 500, were higher than those for the GLA MG state. The higher protection factors may reflect the higher stability of the MG or the I state of CML (Table 1).…”

“…12 We have successfully assigned 102 signals out of 120 peptide NH groups of GLA (Supplementary Table S3). The H/Dexchange reaction was carried out in 90% D 2 O at pH⁎ 1.7 and 25°C, where the protein was in the MG state; pH⁎ denotes the pH-meter reading.…”

“…[4][5][6][7][8] Our previous studies on the equilibria and kinetics of the folding/unfolding of GLA and CML induced by a denaturant, guanidine hydrochloride (GdnHCl), employed circular dichroism (CD) and fluorescence spectroscopy. 5,[9][10][11][12][13][14][15][16][17] Those studies showed that the two proteins exhibit a stable equilibrium unfolding intermediate that is equivalent to an early kinetic folding intermediate formed during the dead time (a few milliseconds) of a stopped-flow experiment. The intermediate has been well characterized experimentally and has characteristics of the molten globule (MG) state.…”

“…The MG state of CML is more stable and more native-like than that of GLA. 5,11,12 Our recent studies on the effect of the Ca 2+ binding on the folding of CML have further shown that neither the MG state nor the transition state of folding has any capacity to bind Ca 2+ , 17 whereas the MG state of α-lactalbumin is known to bind Ca 2+ . [18][19][20] The origin of the difference between these highly homologous proteins, however, remains to be clarified.…”

Molecular Mechanisms of the Cytotoxicity of Human α-Lactalbumin Made Lethal to Tumor Cells (HAMLET) and Other Protein-Oleic Acid Complexes

“…We chose canine milk lysozyme, apo-myoglobin, and ␤ 2 -microglobulin. Canine milk lysozyme, a Ca 2ϩ -binding lysozyme, is homologous to ␣-LA and forms the MG state at pH 2 (26,27). Apo-myoglobin and ␤ 2 -microglobulin form the MG state at pH 4 (15,28,29).…”