Environment of tryptophan residues in various conformational states of α-lactalbumin studied by time-resolved and steady-state fluorescence spectrosc

Ostrovsky, Alexander V.; Kalinichenko, Lina P.; Emelyanenko, V. I.; Klimanov, Alexander V.; Permyakov, Eugene A.

doi:10.1016/0301-4622(88)85008-7

Cited by 28 publications

(17 citation statements)

References 31 publications

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“…For native BLA it is suggested that energy transfer occurs from Trp 26 (27). It has also been observed that unfolding of BLA results in a red shift of the fluorescence emission maximum and an accompanied increase in fluorescence intensity (28) one or more Trp residues in the hydrophobic core of BLA.…”

Section: Resultsmentioning

confidence: 93%

Kinetic and Structural Characterization of Adsorption-induced Unfolding of Bovine α-Lactalbumin

Engel

Mierlo²,

Visser

2002

Journal of Biological Chemistry

102

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Conformational changes of bovine ␣-lactalbumin induced by adsorption on a hydrophobic interface are studied by fluorescence and circular dichroism spectroscopy. Adsorption of bovine ␣-lactalbumin on hydrophobic polystyrene nanospheres induces a non-native state of the protein, which is characterized by preserved secondary structure, lost tertiary structure, and release of calcium. This partially denatured state therefore resembles a molten globule state, which is an intermediate in the folding of bovine ␣-lactalbumin. Stopped-flow fluorescence spectroscopy reveals two kinetic phases during adsorption with rate constants k 1 ϳ 50 s ؊1 and k 2 ϳ 8 s ؊1 . The rate of partial unfolding is remarkably fast and even faster than unfolding induced by the addition of 5.4 M guanidinium hydrochloride to native ␣-lactalbumin. The large unfolding rates exclude the possibility that unfolding of bovine ␣-lactalbumin to the intermediate state occurs before adsorption takes place. Stoppedflow fluorescence anisotropy experiments show that adsorption of bovine ␣-lactalbumin on polystyrene nanospheres occurs within the dead time (15 ms) of the experiment. This shows that the kinetic processes as determined by stopped-flow fluorescence spectroscopy are not affected by diffusion or association processes but are solely caused by unfolding of bovine ␣-lactalbumin induced by adsorption on the polystyrene surface. A scheme is presented that incorporates the results obtained and describes the adsorption of bovine ␣-lactalbumin.Adsorption of proteins on solid/liquid interfaces is a generally occurring phenomenon both in nature and in man-made systems. It is well recognized that proteins undergo conformational changes upon adsorption on solid/liquid interfaces (1-3). Because unfolding of proteins can have a large effect on their function or properties, it is important to increase the knowledge about protein adsorption and about the resulting conformational changes. It is necessary to study not only the conformation of a protein in the adsorbed state but also the kinetics of the adsorption-induced conformational changes. This knowledge will help in controlling wanted or unwanted conformational changes of adsorbed proteins.In this work we focus on the kinetics of adsorption-induced conformational changes of bovine ␣-lactalbumin (BLA) 1 upon interaction with colloidal polystyrene nanospheres. Detailed information on adsorption-induced conformational changes and especially on the kinetics of adsorption-induced conformational changes is sparse. An important reason for this is the experimental difficulty of the presence of an adsorbent, which is usually a solid phase. A few routes have been designed to remove this difficulty. A macroscopic solid/water interface can be used in combination with a reflective technique like ellipsometry or total internal reflection fluorescence (4, 5). In other cases a microscopic system has been used that consists of small particles with diameters ranging from nanometers to micrometers in size (6, 7). The latter syst...

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Section: Resultsmentioning

confidence: 93%

Kinetic and Structural Characterization of Adsorption-induced Unfolding of Bovine α-Lactalbumin

Engel

Mierlo²,

Visser

2002

Journal of Biological Chemistry

102

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“…We observed no lactose synthase activity for vesiclebound a-LA in the presence of GT, suggesting either a disruption of this second hydrophobic cluster upon vesicle binding, or steric limitations to productive GT a-LA association on the bilayer. The most exposed tryptophan was shown to be residue I18 from NMR (Alexandrescu et al, 1992), lifetime (Ostrovsky et al, 1988), and steady-state fluorescence measurements (Sommers & Kronman, 1980). The fluorescence results suggested that this residue accounts for almost 50% of the total fluorescence emission in both N-and H-state a-LA, and that it is substantially red-shifted during thermal melting (e.g., the emission maximum shifts from 326 to 343 nm).…”

Section: Results At Neutral P Hmentioning

confidence: 99%

“…This parameter reported significant reduction in exposure for the SUV-bound protein. This apparent contradiction here in some of the quenching results for acid a-LA will require extensive lifetime studies, which are more complicated for multi-tryptophan proteins (Ostrovsky et al, 1988). If this transition can be verified in the future by other techniques as well, such as mutagenesis to single or double tryptophan mutants, it seriously calls into question the hypothesis that MG intermediates are present in proteins bound to the membrane (Kuwajima, 1989).…”

Section: The Mg State On the Liposome?mentioning

confidence: 89%

“…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). The apo-form also shows a higher maximal activity than Ca2+-LA in the lactose synthase complex (Musci & Berliner, 1985b).…”

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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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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“…This was due to the low tyrosine contribution to total protein fluorescence in this range (Burstein, 1977;Lakowicz, 1983;Permyakov, 1993). Ostrovsky et al (1988) showed that at least two tryptophan residues contribute a significant share of the total emission of a-LA. Sommers and Kronman (1980) concluded that three of the four tryptophan residues in a-LA contribute significantly to the total emission (Trp 28, Trp 108, and Trp 123 contribute about 20, 20, and 50%, respectively).…”

Section: Discussionmentioning

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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Environment of tryptophan residues in various conformational states of α-lactalbumin studied by time-resolved and steady-state fluorescence spectrosc

Cited by 28 publications

References 31 publications

Kinetic and Structural Characterization of Adsorption-induced Unfolding of Bovine α-Lactalbumin

Kinetic and Structural Characterization of Adsorption-induced Unfolding of Bovine α-Lactalbumin

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?

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