Ca2+ Alteration in the Unfolding Behavior of α-Lactalbumin1

Ikeguchi, Masamichi; Kuwajima, Kunihiro; Sugai, Shintaro

doi:10.1093/oxfordjournals.jbchem.a135582

Cited by 143 publications

(111 citation statements)

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“…deviation of 0.59 Å for C ␣ atoms between residues 81-93). The absence of any cation from the crystallization medium in apo-bLA replacing the calcium of the holo protein, supports the view that stabilization of the native state of the apo protein by higher concentrations of monovalent cations (19,16) results, in this case, from increased ionic strength and not specific binding. Water molecules that interact with the Ca 2ϩ ion in the holo protein are also absent.…”

Section: Resultssupporting

confidence: 62%

Crystal Structures of Apo- and Holo-bovine α-Lactalbumin at 2.2-Å Resolution Reveal an Effect of Calcium on Inter-lobe Interactions

Chrysina¹,

Brew²,

Acharya³

2000

Journal of Biological Chemistry

240

217

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High affinity binding of Ca2؉ to ␣-lactalbumin (LA) stabilizes the native structure and is required for the efficient generation of native protein with correct disulfide bonds from the reduced denatured state. A progressive increase in affinity of LA conformers for Ca 2؉ as they develop increasingly native structures can account for the tendency of the apo form to assume a molten globule state and the large acceleration of folding by Ca 2؉ . To investigate the effect of calcium on structure of bovine LA, x-ray structures have been determined for crystals of the apo and holo forms at 2.2-Å resolution. In both crystal forms, which were grown at high ionic strength, the protein is in a similar global native conformation consisting of ␣-helical and ␤- ␣-Lactalbumin (LA)1 is the regulatory protein of lactose synthase (1-3), modulating the affinity of the catalytic component, UDP-galactose ␤-N-acetylglucosaminide ␤1,4-galactosyltransferase I, for acceptor substrates through a reversible proteinprotein interaction (4). LA is also secreted into milk and aggregates of a partially folded form of the human protein have been found to have selective apoptotic effects on tumor cells (5). LA is homologous with the c-type lysozymes (LZs) (6) and provides an example of extreme functional divergence in homologous proteins with closely similar structures (7-9). LA binds calcium strongly and specifically but the LZs include subgroups that bind Ca 2ϩ at a site corresponding to that in LA and others ("conventional" LZs) that lack a Ca 2ϩ -binding site (10). The K d app for Ca 2ϩ binding to apo-LA under physiological conditions is of the order of 10 Ϫ7 M (11-13). The Ca 2ϩ ion in LA has a structural role, being required for folding and native disulfide bond formation in the reduced denatured protein (14, 15). At ambient temperature and low ionic strength, apo-LA undergoes a transconformation to a molten globule (MG) state, which lacks a fixed tertiary structure but retains much of the native secondary structure. The MG is also stabilized by mildly disruptive treatments such as low pH or intermediate concentrations of denaturants (16,17); it is also identical to a kinetic MG that is highly populated during the refolding of chemically denatured LA (18 -20).Crystal structures of holo-LA from various species in different crystal forms and metal complexes (7,8,(21)(22)(23)(24)(25)(26) are similar to those of the homologous LZs, with two lobes of structure separated by a cleft (Fig. 1A). The larger ␣-helical lobe is formed by the amino-and carboxyl-terminal sections of the polypeptide chain (residues 1-34 and 86 -123) while the smaller lobe, which encompasses a small three-stranded antiparallel ␤-sheet, a small 3 10 helix and some irregular structure, is formed by the central section of the polypeptide chain (residues 35-85). We refer to the lobes as "subdomains" since they lack properties normally expected of true domains such as sequence contiguity and distinct functional properties and origins. The high affinity Ca 2ϩ -binding site is l...

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Section: Resultssupporting

confidence: 62%

Crystal Structures of Apo- and Holo-bovine α-Lactalbumin at 2.2-Å Resolution Reveal an Effect of Calcium on Inter-lobe Interactions

Chrysina¹,

Brew²,

Acharya³

2000

Journal of Biological Chemistry

240

217

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“…␣-Lactalbumin, the well studied protein for its three-state unfolding properties, binds Ca 2ϩ and removal of this ion does affect the stability and the propensity of this protein to adopt the molten-globule state (40,41). The homologous enzyme, lysozyme, which does not bind Ca 2ϩ , has not been shown to exhibit similar equilibrium molten-globule states.…”

Section: ␣-Crystallin Binds To Molten-globulementioning

confidence: 99%

Molten-Globule State of Carbonic Anhydrase Binds to the Chaperone-like α-Crystallin

Krishnan

Raman

Rao

1996

Journal of Biological Chemistry

102

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␣-Crystallin, a multimeric protein, exhibits chaperone-like activity in preventing aggregation of several proteins. We have studied the chaperone-like activity of ␣-crystallin toward heat-induced aggregation of bovine and human carbonic anhydrase. Human carbonic anhydrase aggregates at 60°C, while bovine carbonic anhydrase does not aggregate significantly at this temperature. Removal of the enzyme-bound metal ion, Zn 2؉ , by EDTA modulates the aggregation behavior of bovine carbonic anhydrase. Fluorescence and circular dichroism studies show that removal of the metal ion from the bovine carbonic anhydrase by a chelator such as EDTA enhances the propensity of the enzyme to adopt the molten-globule state. ␣-Crystallin binds to this state of the enzyme and prevents aggregation. Fluorescence and circular dichroism studies on the ␣-crystallin-enzyme complexes show that the enzymes in the complex are in the molten-globule state. These results are of relevance to the interaction of chaperones with the partially unfolded states of target proteins.␣-Crystallin is a multimeric protein present in large quantity in the eye lens. It is made up of acidic (␣A) and basic (␣B) subunits and is also known to be expressed in nonlenticular tissues under stress or disease (1-6). ␣-Crystallin is heatstable (7) and is structurally related to small heat shock proteins (8 -11). Its expression can be induced by thermal (8) or hypertonic stress (12). It interacts with membranes (13) and modulates the intermediate filament assembly (14). Horwitz (15) has shown that ␣-crystallin prevents the aggregation of other crystallins and enzymes. This chaperone-like activity of ␣-crystallin has been further studied in detail by other workers (16 -23). We have earlier investigated the effect of ␣-crystallin on the photoaggregation of ␥-crystallin (19), thermal aggregation of -crystallin, dithiothreitol-induced aggregation of insulin (20), and the rapid refolding of ␤-and ␥-crystallins (21). On the basis of these studies, we postulated that ␣-crystallin prevents aggregation of other proteins by providing appropriately placed hydrophobic surfaces, a structural perturbation above 30°C enhances the chaperone-like activity of ␣-crystallin severalfold.␣-Crystallin from the old human lenses (24) and from the selenite-induced cataractous lenses of an animal model (25) are found to exhibit decreased chaperone-like activity. Our earlier study on refolding of crystallins at high concentrations (21) shows that ␤-or ␥-crystallins aggregate upon refolding while ␣-crystallin does not aggregate. Co-refolding of ␤-crystallin or ␥-crystallin with ␣-crystallin prevents the aggregation under similar conditions. All these studies provide further evidences that the chaperone-like activity of ␣-crystallin is important in the formation and maintenance of the transparency of the eye lens.In the present study, we have observed that human carbonic anhydrase aggregates at 60°C while the aggregation of bovine carbonic anhydrase is negligible at the same temperature. Removal of the...

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“…Figure 2 depicts the temperature dependence of tryptophan fluorescence for Ca2+(1 mM)-loaded a-LA bound to DMPC or lecithin liposomes, and free Ca2+-a-LA in solution. Calcium concentrations in excess of the protein concentration were employed to compensate for possible weak, nonspecific adsorption to the excess lipid, as well as to provide significant inhibition against formation of the apo-state (Ikeguchi et al, 1986). The plots of emission maximum ( Fig.…”

Section: Temperature Dependence Of Fluorescence For Isolated A-la-vesmentioning

confidence: 99%

Membrane‐bound states of α‐lactalbumin: Implications for the protein stability and conformation

1996

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Abstracta-Lactalbumin (a-LA) associates with dimyristoylphosphatidylcholine (DMPC) or egg lecithin (EPC) liposomes. Thermal denaturation of isolated DMPC or EPC a-LA complexes was dependent on the metal bound state of the protein. The intrinsic fluorescence of thermally denatured DMPC-a-LA was sensitive to two thermal transitions: the T,. of the lipid vesicles, and the denaturation of the protein. Quenching experiments suggested that tryptophan accessibility increased upon protein-DMPC association, in contrast with earlier suggestions that the limited emission red shift upon association with the liposome was due to partial insertion of tryptophan into the apolar phase of the bilayer (Hanssens I et al., 1985, Biochim Biophys Acta 817:154-166). On the other hand, above the protein transition (70 "C), the spectral blue shifts and reduced accessibility to quencher suggested that tryptophan interacts significantly with the apolar phase of either DMPC and EPC. At pH 2, where the protein inserts into the bilayer rapidly, the isolated DMPC-a-LA complex showed a distinct fluorescence thermal transition between 40 and 60 "C, consistent with a partially inserted form that possesses some degree of tertiary structure and unfolds cooperatively. This result is significant in light of earlier findings of increased helicity for the acid form, i.e., molten globule state of the protein (Hanssens I et al., 1985, Biochim Biophys Acta 812154-166). These results suggest a model where a limited expansion of conformation occurs upon association with the membrane at neutral pH and physiological temperatures, with a concomitant increase in the exposure of tryptophan to external quenchers; i.e., the current data do not support a model where an apolar, tryptophan-containing surface is covered by the lipid phase of the bilayer.

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Ca2+ Alteration in the Unfolding Behavior of α-Lactalbumin1

Cited by 143 publications

References 0 publications

Crystal Structures of Apo- and Holo-bovine α-Lactalbumin at 2.2-Å Resolution Reveal an Effect of Calcium on Inter-lobe Interactions

Crystal Structures of Apo- and Holo-bovine α-Lactalbumin at 2.2-Å Resolution Reveal an Effect of Calcium on Inter-lobe Interactions

Molten-Globule State of Carbonic Anhydrase Binds to the Chaperone-like α-Crystallin

Membrane‐bound states of α‐lactalbumin: Implications for the protein stability and conformation

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