Kinetics of folding and unfolding of goat α‐lactalbumin

Abstract

The equilibrium unfolding and the kinetic folding and unfolding of goat alpha-lactalbumin (GLA) were studied by near- and far-ultraviolet circular dichroism (CD) and by stopped-flow fluorescence spectroscopy. Specifically, the influence of environmental conditions such as pH and Ca2+ binding was examined. Compared to the apo-form, the Ca2+-bound form was found to be strongly stabilized in equilibrium conditions at pH 7.5 and 25 degrees C. The kinetics of the refolding of apo-GLA show a major change of fluorescence intensity during the experimental dead-time, but this unresolved effect is strongly diminished in holo-GLA. In both cases, however, the chevron plots can adequately be fitted to a three-state model. Moreover, double-mix stopped-flow experiments showed that the native state (N) is reached through one major pathway without the occurrence of alternative tracks. In contrast to the homologous bovine alpha-lactalbumin (BLA), the compactness of GLA is strongly influenced by the presence of Ca2+ ions. Unlike the two-state transition observed in guanidine hydrochloride (GdnHCl)-induced equilibrium denaturation experiments at higher pH, an equilibrium intermediate state (I) is involved in denaturation at pH 4.5. In the latter case, analysis of the kinetic data makes clear that the intermediate and the unfolded states (U) show practically no Gibbs free energy difference and that they are in rapid equilibrium with each other. A possible explanation for these variations in stability and in folding characteristics with pH could be the degree of protonation of His107 that directly influences non-native interactions. Variation of environmental conditions and even small differences in sequence, therefore, can result in important effects on thermodynamic and folding parameters.

α-Lactalbumin species variation, HAMLET formation, and tumor cell death

“…At 270 nm, the bovine and caprine proteins showed the lowest minimum, while the equine and porcine spectra gave a weaker signal than human α-lactalbumin. The spectra of equine and porcine α-lactalbumin resembled the other species variants, as previously reported [8,12,[15][16][17][18][19]. The HAMLET-like complexes showed a loss of signal compared to each holo protein.…”

“…Near UV CD spectra were recorded in 5 mM Tris, pH 7.0 on (A) human, equine, bovine, caprine and porcine α-lactalbumin and (B) HAMLET and HAMLETlike complexes. The spectra of human, bovine and caprine α-lactalbumins have previously been described [8,12,[15][16][17][18][19] but spectra of equine and porcine α-lactalbumin have not previously been reported. The L1210 mouse lymphoma cell line was exposed to (C) HAMLET and HAMLET-like complexes (0.2 mg/ml) or to (D) the corresponding native α-lactalbumin (0.5 mg/ml) for 5h.…”

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

“…Previous studies have well established that I is equivalent to the equilibrium unfolding intermediate that is populated at an intermediate concentration (2.5-3 M) of GdnHCl in the apo form. 9,10,14,15,19 The CML refolding also exhibited the presence of I as shown in our previous study. 17 Kinetic unfolding reactions for the holo and apo forms of GLA were investigated by stopped-flow CD ( Fig.…”

“…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.…”

OLIGAMI: OLIGomer Architecture and Molecular Interface

“…For example, we manually verified chain formulas and coordinates for two PDB entries of goat alpha-lactalbumin (PDB ID: 1HFY, 1HMK), which is classified as a member of the C-type lysozyme family. Alpha-lactalbumin is a monomeric protein in solution, and it is frequently used as a model protein in protein folding studies [13][14][15][16]. When comparing the two PDB entries for goat alpha-lactalbumin, one PDB entry contained the chain formula [A2] and the other entry contained the chain formula [A].…”