The Perception of Hydrophobic Clusters in the Native and Partially Unfolded States
223
nally compact native GLA. The pronounced decrease of the tryptophan fluorescence intensity obtained for apo-GLA in the presence ofbis-ANS at 4°C (Fig. 15.7
A) is accompanied by a fluorescence increase of bis-ANS near 500 nm (not
shown). It indicates that an important part of the tryptophan fluorescence energy
is transferred to the dye. The interaction ofbis-ANS with Ca 2 +-GLA at 37°C does
not result in a comparable energy-transfer (Fig. 15.7 B). Even the tryptophan
fluorescence of the bis-ANS-photolabeled-Ca 2 + -GLA complex is not reduced to
the same degree as for apo-GLA.
4.6
Conclusion
In 10 mM Tris-HCI, pH 7.5 and at 4°C in 2 mM EGTA as well as at 37°C in 2 mM
Ca 2 +, native GLA is close to its thermal transition. Nevertheless, there are a number of interesting differences in the interaction of the dye with the apo- and Ca 2 +form of the protein.
Native apo-GLA binds 2 bis-ANS molecules. The interactions with both probe
molecules can be described by independent equilibria. Therefore, it is likely that
both interacting bis-ANS molecules bind to a different hydrophobic cluster. The
efficient quenching of the tryptophan fluorescence also indicates that the binding
of bis-ANS to the apo-protein occurs at locations in the vicinity of these aromatic
groups. In addition, the far- UV CD spectrum of the complex becomes similar to
that of the protein in its molten globule state. In contrast,S bis-ANS molecules
are bound in a cooperative way to native Ca 2 +-GLA. The far-UV CD spectrum of
native Ca 2 + -GLA is conserved for the complex. The limited energy-transfer confirms that in the presence of Ca 2 +, the interacting bis-ANS molecules only
approach a part of the tryptophan residues or even remain relatively distant from
them.
As native apo-GLA and Ca 2 + -GLA are close to their thermal destabilization at
the considered temperatures, the reduction of the perturbation in the presence of
Ca 2 + is not due to the stabilization of GLA as a whole. It is rather due to stiffening
of local structure by Ca H • Close inspection of the protein structure proves the
ability for such local effects. As described earlier, one of the hydrophobic clusters
of a-lactalbumin, Phe-53, Trp-60, Tyr-l03 and Trp-104 (Fig. 15.1), is situated in
the crevice that divides the protein in two structural halves and contains aromatic groups that belong to both halves. The Ca 2 + -binding loop of alactalbumins (with ligand residues Lys-79, Asp-82, Asp-84, Asp-87 and Asp-88)
contributes to the amino-terminal side of a 31O-helix (residues 76-82) and to the
carboxyl-terminus of the a-helix C (residues 86-99) (Acharya et al. 1989). Ca 2 +
binding closes the crevice and may prevent access ofbis-ANS to these hydrophobic residues.
This local stabilization may have as a consequence that the bis-ANS molecules
interacting with native Ca 2 + -GLA are only situated at the cluster distant from the
specific Ca 2 + -binding loop. In our further research we concentrate on the determination of the exact binding sites of the dye in the GLA-bis-ANS complexes in
the presence and absence of Ca 2 +.
223
nally compact native GLA. The pronounced decrease of the tryptophan fluorescence intensity obtained for apo-GLA in the presence ofbis-ANS at 4°C (Fig. 15.7
A) is accompanied by a fluorescence increase of bis-ANS near 500 nm (not
shown). It indicates that an important part of the tryptophan fluorescence energy
is transferred to the dye. The interaction ofbis-ANS with Ca 2 +-GLA at 37°C does
not result in a comparable energy-transfer (Fig. 15.7 B). Even the tryptophan
fluorescence of the bis-ANS-photolabeled-Ca 2 + -GLA complex is not reduced to
the same degree as for apo-GLA.
4.6
Conclusion
In 10 mM Tris-HCI, pH 7.5 and at 4°C in 2 mM EGTA as well as at 37°C in 2 mM
Ca 2 +, native GLA is close to its thermal transition. Nevertheless, there are a number of interesting differences in the interaction of the dye with the apo- and Ca 2 +form of the protein.
Native apo-GLA binds 2 bis-ANS molecules. The interactions with both probe
molecules can be described by independent equilibria. Therefore, it is likely that
both interacting bis-ANS molecules bind to a different hydrophobic cluster. The
efficient quenching of the tryptophan fluorescence also indicates that the binding
of bis-ANS to the apo-protein occurs at locations in the vicinity of these aromatic
groups. In addition, the far- UV CD spectrum of the complex becomes similar to
that of the protein in its molten globule state. In contrast,S bis-ANS molecules
are bound in a cooperative way to native Ca 2 +-GLA. The far-UV CD spectrum of
native Ca 2 + -GLA is conserved for the complex. The limited energy-transfer confirms that in the presence of Ca 2 +, the interacting bis-ANS molecules only
approach a part of the tryptophan residues or even remain relatively distant from
them.
As native apo-GLA and Ca 2 + -GLA are close to their thermal destabilization at
the considered temperatures, the reduction of the perturbation in the presence of
Ca 2 + is not due to the stabilization of GLA as a whole. It is rather due to stiffening
of local structure by Ca H • Close inspection of the protein structure proves the
ability for such local effects. As described earlier, one of the hydrophobic clusters
of a-lactalbumin, Phe-53, Trp-60, Tyr-l03 and Trp-104 (Fig. 15.1), is situated in
the crevice that divides the protein in two structural halves and contains aromatic groups that belong to both halves. The Ca 2 + -binding loop of alactalbumins (with ligand residues Lys-79, Asp-82, Asp-84, Asp-87 and Asp-88)
contributes to the amino-terminal side of a 31O-helix (residues 76-82) and to the
carboxyl-terminus of the a-helix C (residues 86-99) (Acharya et al. 1989). Ca 2 +
binding closes the crevice and may prevent access ofbis-ANS to these hydrophobic residues.
This local stabilization may have as a consequence that the bis-ANS molecules
interacting with native Ca 2 + -GLA are only situated at the cluster distant from the
specific Ca 2 + -binding loop. In our further research we concentrate on the determination of the exact binding sites of the dye in the GLA-bis-ANS complexes in
the presence and absence of Ca 2 +.