Interaction of α-lactalbumin with dimyristoyl phosphatidylcholine vesicles. I. A microcalorimetric and fluorescence study

Hanssens, Ignace; Houthuys, Catherine; Herreman, W.; Cauwelaert, F.H. Van

doi:10.1016/0005-2736(80)90333-8

Cited by 45 publications

(26 citation statements)

References 32 publications

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“…220:l molar ratio of lipid to protein. For these ratios, no change in morphology of the vesicles is induced by the protein at neutral pH (Hanssens et al, 1980). Only freshly isolated a-LA-lipid complexes were used, with fluorescence studies being completed within 2 h of isolation (Berliner & Koga, 1987).…”

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

confidence: 99%

“…for the acid a-LA on liposomes Figure 7 depicts results of temperature-dependence fluorescence studies for free and DMPC-bound acid-state a-LA, incubated at pH 2 (150 mM KC1/50 mM glycine) for 1 min in order to avoid morphology changes in the liposomes (Hanssens et al, 1980). The free (unbound) protein fluorescence parameters (Fig.…”

Section: Temperature Dependence and Quenchingmentioning

confidence: 99%

“…This interest has lead to substantial progress in defining the effects of lipid on the physical and functional state of a-LA. Previous physical studies of a-LAhesicle mixtures have provided some valuable information on the nature and extent of interactions of various CY-LA conformations with the lipid (Hanssens et al, 1980(Hanssens et al, , 1983(Hanssens et al, , 1985Herreman et al, 1981aHerreman et al, , 1981bAmeloot et al, 1984;Berliner & Koga, 1987;Permyakov et al, 1988a). Fluorescent results with probes placed in the bilayer suggested that the lipid was not strongly perturbed upon interaction with a-LA at neutral pH, and that the association was largely electrostatic with the vesicle surface (Hanssens et al, 1980(Hanssens et al, , 1983(Hanssens et al, , 1985Herreman et al, 1981aHerreman et al, , 1981bAmeloot et al, 1984), as evidenced by the absence of an energy transfer band with a protein tryptophan (Brown, 1984).…”

mentioning

confidence: 99%

“…Previous physical studies of a-LAhesicle mixtures have provided some valuable information on the nature and extent of interactions of various CY-LA conformations with the lipid (Hanssens et al, 1980(Hanssens et al, , 1983(Hanssens et al, , 1985Herreman et al, 1981aHerreman et al, , 1981bAmeloot et al, 1984;Berliner & Koga, 1987;Permyakov et al, 1988a). Fluorescent results with probes placed in the bilayer suggested that the lipid was not strongly perturbed upon interaction with a-LA at neutral pH, and that the association was largely electrostatic with the vesicle surface (Hanssens et al, 1980(Hanssens et al, , 1983(Hanssens et al, , 1985Herreman et al, 1981aHerreman et al, , 1981bAmeloot et al, 1984), as evidenced by the absence of an energy transfer band with a protein tryptophan (Brown, 1984). On the other hand, at acidic pH (<4), it was found that protein insertion into the bilayer is pronounced, exhibiting strong protein-probe energy transfer (Herreman et al, 1981a(Herreman et al, , 1981b) and a marked tendency to solubilize the lipid into micellar complexes (Hanssens et al, 1980(Hanssens et al, , 1983Dangreau et al, 1982), suggesting that, at low pH, a-LA is an intrinsic membrane protein (Brown, 1984).…”

mentioning

confidence: 99%

“…For example, apo-a-LA is known to interact more strongly with PC liposomes at neutral pH than does the calcium protein (Berliner & Koga, 1987). It was suggested that partial insertion of tryptophan residues into the bilayer can account for the limited red shifts observed in the intrinsic protein fluorescence (Hanssens et al, 1980(Hanssens et al, , 1985. Such questions have been addressed here by probing intrinsic fluorescence in the presence of soluble quenchers (e.g., Lakey et al, 1991;Meers & Mealy, 1993;Volwerk et al, 1994).…”

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

confidence: 99%

Section: Temperature Dependence and Quenchingmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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

1996

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pH responsive microdomain formation in a De Novo polypeptide

1997

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Recombinant DNA technology has been employed to produce a polypeptide capable of forming pH responsive hydrophobic microdomains. The design of this peptide is based upon an idealized conceptual model in which electrostatic, hydrophobic, and hydration forces are responsible for the association of amphipathic α‐helical elements. Reduction in solution pH is responsible for reducing electrostatic repulsions between similarly charged residues, promoting the hydrophobic collapse of helical elements. A polymerizable synthetic element (dn31) has been synthesized and inserted into an appropriate expression vector. A clone containing a single copy of the dn31 gene (designated dn31x1) was isolated and the corresponding gene product DN3Lx1 isolated. The physical properties of DN3Lx1 were examined in solution by gel filtration chromatography, CD, and fluorescence probe analysis. It was determined that DN3Lx1 self‐associates in solution with the degree of aggregation dependent on pH and ionic strength. An initial objective of this work was to examine domain organization in higher molecular weight species containing ten or more repetitive sequences. However, attempts to express multiple repeats of DN3Lxn from concatemers were unsuccessful. © 1997 John Wiley & Sons, Inc. Biopoly 41: 521–532, 1997.

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Elucidating the binding thermodynamics of 8‐anilino‐1‐naphthalene sulfonic acid with the A‐state of α‐lactalbumin: An isothermal titration calorimetric investigation

Singh

Kishore

2006

Biopolymers

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Isothermal titration calorimetry has been used to demonstrate that the heat profile associated with the binding of 8-anilino-1-naphthalene sulfonic acid (ANS) with the acid induced molten globule state (A-state) of alpha-lactalbumin (alpha-LA) is different from that with the native and denatured states of the protein. The results corroborate the spectroscopic observations that ANS binds more strongly to the partially folded states of the protein compared to that with the native and denatured states. ANS binds to the A-state of alpha-LA at two independent binding sites that remain nearly the same in the temperature range of 10-35 degrees C. The number of moles of ANS binding at site 1 at 10 degrees C is 14.0+/-0.2 and remains nearly the same with rise in temperature. However, the number of moles of ANS molecules binding at site 2 show an increase from 1.6+/-0.2 at 10 degrees C to 4.1+/-0.1 at 35 degrees C. The deviation of the slope of enthalpy-entropy compensation plot from unity and nonadherence to van't Hoff dictates implies that the binding sites on the A-state of alpha-LA for ANS are not well defined and specific; rather, these binding sites are formed due to greater exposure of hydrophobic clusters in the A-state of the protein. The results for the first time demonstrate the use of isothermal titration calorimetry in characterizing the A-state of alpha-LA both qualitatively and quantitatively.

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Interaction of α-lactalbumin with dimyristoyl phosphatidylcholine vesicles. I. A microcalorimetric and fluorescence study

Cited by 45 publications

References 32 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

pH responsive microdomain formation in a De Novo polypeptide

Elucidating the binding thermodynamics of 8‐anilino‐1‐naphthalene sulfonic acid with the A‐state of α‐lactalbumin: An isothermal titration calorimetric investigation

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