Cation binding effects on the pH, thermal and urea denaturation transitions in α-lactalbumin

Permyakov, Eugene A.; Morozova, Ludmila A.; Burstein, Edward A.

doi:10.1016/0301-4622(85)85003-1

Cited by 119 publications

(111 citation statements)

References 26 publications

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“…Native a-LA is typically isolated with stoichiometric levels of calcium bound at a unique, strong Ca2+-binding loop that has been observed in all crystal structures of a-LA reported to date, but this structural feature is absent in most C-type lysozymes (Hiraoka et al, 1980;Stuart et al, 1986;Acharya et al, 1989Acharya et al, , 1991. The binding of calcium stabilizes the protein against thermal as well as guanidine HCl and urea denaturation (Hiraoka et al, 1980;Permyakov et al, 1985). The so-called Ca2+-binding "elbow" has been also shown to bind Mg2+, Na+, and K C weakly (Permyakov et al, 1981a(Permyakov et al, , 1981b(Permyakov et al, , 1985.…”

mentioning

confidence: 99%

“…The binding of calcium stabilizes the protein against thermal as well as guanidine HCl and urea denaturation (Hiraoka et al, 1980;Permyakov et al, 1985). The so-called Ca2+-binding "elbow" has been also shown to bind Mg2+, Na+, and K C weakly (Permyakov et al, 1981a(Permyakov et al, , 1981b(Permyakov et al, , 1985. Fluorescence measurements show that the removal of calcium at physiological temperatures induces a conformational change, which is accompanied both by increased emission intensity and a red shift (Permyakov et al, 1981a(Permyakov et al, , 1985Murakami et al, 1982;Ostrovsky et al, 1988), increased affinity for the fluorescent hydrophobic probe, bis-ANS (Musci & Berliner, 1985a), and retardation on phenylSepharose (Lindahl & Vogel, 1984).…”

mentioning

confidence: 99%

“…Two conserved tryptophan residues on a-LA have been implicated in the interaction with GT (Barman, 1972;Barman & Bagshaw, 1972;Bell et ai., 1975;Grobler et al, 1994), although a putative role of tryptophan in the control of protein function in vitro is unproven at present. a-LA conformation is also modulated by other metal ions and pH (Murakami & Berliner, 1983;Permyakov, 1985Permyakov, , 1988bAramini et al, 1996). In particular, native a-LA possesses a relatively strong secondary Zn2+ site (K,,,, = 5 X lo5 M"), which causes subtle conformational changes upon binding to the calcium-loaded protein as monitored by intrinsic fluorescence (Permyakov et al, 1991;Ren et al, 1993) and increased affinity for bis-ANS (Musci & Berliner, 1985a).…”

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confidence: 99%

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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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confidence: 99%

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

1996

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“…This transition in a-LA has been studied by near-UV CD (Robbins & Holmes, 1970; Kuwajima et al, 1975Kuwajima et al, , 1976Kuwajima et al, , 1980, intrinsic tryptophan fluorescence (Sommers & Kronman, 1980;Permyakov et al, 1981Permyakov et al, , 1985Lala & Kaul, 1992), and differential spectrophotometry (Kuwajima et al, 1980) techniques. The single transition in the pH range of 3-4 was detected by each one of these techniques.…”

Section: N + a Transitionmentioning

confidence: 99%

“…Hence, the reduced values of the pK,(gab) in our data evidently correspond to those obtained at the higher Ca2+ concentration. Permyakov et al (1981Permyakov et al ( , 1985 used computer simulation in a study of the protonation transition of a-LA in the pH range of 2.3-5. They suggest that CaZ+ can be exchanged with protons of three carboxylate groups with pK, 5.0 * 0.1.…”

Section: N + a Transitionmentioning

confidence: 99%

Two steps in the transition between the native and acid states of bovine α‐lactalbumin detected by circular polarization of luminescence: Evidence for a premolten globule state?

Gussakovsky

Haas

1995

Protein Science

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A few studies indirectly support the existence of an intermediate in the transition of Ca2+-saturated bovine a-lactalbumin (a-LA) from the native (N) to the acidic (A) state, known as the molten globule state. However, direct experimental evidence for the appearance of this intermediate has not been obtained. The signal of circular polarization of luminescence (CPL) is sensitive to fine conformational transitions because of its susceptibility to changes in the environmental asymmetry of fluorescent chromophores in their excited electronic states. In the present study, CPL measurements were applied using the intrinsic tryptophan fluorescence of a-LA as well as the fluorescence of 8-anilino-1-naphthalenesulfonic acid (ANS) bound to a-LA. CPL of tryptophan and ANS was measured in the pH range of 2.5-6 in order to find direct experimental evidence for the proposed intermediate. CPL (characterized by the emission anisotropy factor, gem) depends on the asymmetry of the protein molecular structure in the environment of the tryptophan and the ANS chromophores in the excited electronic state. The pH dependence of both the g, , , absorption anisotropy factor determined by CD, and the ANS steady state fluorescence, showed a single transition at pH 3-3.7 as already reported elsewhere. This transition was interpreted as being a result of a change of the a-LA tertiary structure, which resulted in a loss of asymmetry of the environment of both the tryptophan residues and the ANS hydrophobic binding sites. The pH dependence of the tryptophan and ANS g, , showed an additional conformational transition at pH 4-5, which coincided with the pK, of

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Fine tuning the N-terminus of a calcium binding protein: ?-lactalbumin

et al. 1999

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The effects of amino acid substitutions in the N-terminus of bovine recombinant alpha-lactalbumin (including enzymatic removal of the N-terminal methionine and deletion of Glu-1) were studied by intrinsic fluorescence, circular dichroism (CD), and differential scanning microcalorimetry (DSC). Wild-type recombinant alpha-lactalbumin has a lower thermostability and calcium affinity compared to the native protein, while the properties of wild-type protein with the N-terminal methionine enzymatically removed are similar to the native protein. Taken together, the fluorescence, CD, and DSC results show that recombinant wild type alpha-lactalbumin in the absence of calcium ion is in a type of molten globule state. The delta-E1 mutant, where the Glu(1)residue of the native sequence is genetically removed, leaving an N-terminal methionine in its place, shows almost one order of magnitude higher affinity for calcium and higher thermostability (both in the absence and presence of calcium) than the native protein isolated from milk. It was concluded that the N-terminus of the protein dramatically affects both stability and function as manifested in calcium affinity. Proteins 1999;37:65-72.

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Cation binding effects on the pH, thermal and urea denaturation transitions in α-lactalbumin

Cited by 119 publications

References 26 publications

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

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

Two steps in the transition between the native and acid states of bovine α‐lactalbumin detected by circular polarization of luminescence: Evidence for a premolten globule state?

Fine tuning the N-terminus of a calcium binding protein: ?-lactalbumin

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