Fluorescence contributions of the individual Trp residues in goat alpha-lactalbumin.

Goat alpha-lactalbumin (GLA) contains four tryptophan (Trp) residues. In order to obtain information on the fluorescence contribution of the individual Trp residues in native GLA, we recorded the fluorescence spectra of four GLA mutants, W26F, W60F, W104F, and W118F, in each of which a single Trp residue was replaced with phenylalanine (Phe). Comparison of the fluorescence spectra of the four mutants with that of wild-type GLA indicated that, in native GLA, three Trp residues (Trp60, Trp104, and Trp118) are strongly quenched and account for the partial indirect quenching of Trp26. As a consequence, the fluorescence of wild-type GLA and of the mutants W60F, W104F, and W118F mainly results from Trp26. An inspection of the crystal structure indicated that, in addition to the disulfide bonds that are in direct contact with the indole groups of Trp60 and Trp118, backbone peptide bonds that are in direct contact with the indole groups of Trp60, Trp104, and Trp118, contribute to the direct quenching effects. Interestingly, the lack of direct quenching of Trp26 explains why the cleavage of disulfide bonds by UV light is mediated more by the highly fluorescent Trp26 than by the less fluorescent Trp104 and Trp118.

Cyclic peptide models of the Ca2+-binding loop of alpha-lactalbumin.

A series of cyclic peptides with different linkers were designed and synthesized to model the elbow-type Ca2+-binding loop of alpha-lactalbumin (LA). All amino acids of the Ca2+-binding loop are strikingly well conserved among LAs of different species with the sequence Lys79-Phe-Leu-Asp82-Asp-Asp-Leu-Thr- Asp87-Asp88, where three carboxylates of Asp82, Asp87, and Asp88 and the amide carbonyl oxygen atoms of Lys79 and Asp84 participate in Ca2+ binding. Alanine-containing models were also prepared for monitoring the role of the binding (82, 87-88) and nonbinding Asp residues (83-84) in coordinating the cation. The structural features of synthetic peptides and their Ca2+-binding properties were investigated in solution by circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy. In water, the CD curves show a strong negative band below 200 nm as a sign of the presence of unfolded conformers. In TFE, all cyclic peptides were found to have a CD spectrum, reflecting the presence of folded (turn) conformers. The effect of Ca2+ was dependent on the structure and concentration of the model and the Ca2+ to peptide ratio (r(cat)). A surprising time dependence of the FTIR spectra of Ca2+ complexes of the Ala-containing peptides was observed. The shape of the broad amide I band showed no more change after approximately 60 min. Contrary to this, the deprotonation of the side chain COOH group(s) and formation of the final coordination sphere of Ca2+ took more time. Infrared spectra showed that in the Ca2+ complex of model comprising the binding Asp residues of LA, the cation is coordinated to the COO- groups of all three Asps, while in the complex of model comprising nonbinding Asp residues of LA, the two neighboring Asp side chains form a bridged Ca2+-binding system.

Tryptophan to phenylalanine substitutions allow differentiation of short- and long-range conformational changes during denaturation of goat alpha-lactalbumin.

To test the occurrence of local particularities during the unfolding of Ca2+-loaded goat alpha-lactalbumin (GLA) we replaced Trp60 and -118, either one or both, by Phe. In contrast with alternative studies, our recombinant alpha-lactalbumins are expressed in Pichia pastoris and do not contain the extra N-terminal methionine. The substitution of Trp60 leads to a reduction of the global stability. The effect of the Trp118Phe substitution on the conformation and stability of the mutant, however, is negligible. Comparison of the fluorescence spectra of these mutants makes clear that Trp60 and -118 are strongly quenched in the native state. They both contribute to the quenching of Trp26 and -104 emission. By the interplay of these quenching effects, the fluorescence intensity changes upon thermal unfolding of the mutants behave very differently. This is the reason for a discrepancy of the apparent transition temperatures derived from the shift of the emission maxima (Tm,Fl lambda) and those derived from DSC (Tm,DSC). However, the transition temperatures derived from fluorescence intensity (Tm,Fl int) and from DSC (Tm,DSC), respectively, are quite similar, and thus, no local rearrangements are observed upon heat-induced unfolding. At room temperature, the occurrence of specific local rearrangements upon GdnHCl-induced denaturation of the different mutants is deduced from the apparent free energies of their transition state obtained from stopped-flow fluorescence measurements. By phi-value analysis it appears that, while the surroundings of Trp118 are exposed in the kinetic transition state, the surroundings of Trp60 remain native.

Photoexcitation of tryptophan groups induces reduction of two disulfide bonds in goat alpha-lactalbumin.

Illumination of goat alpha-lactalbumin (GLA) with 280 or 295 nm light results in tryptophan-mediated photolysis of disulfide bonds within the protein. The photolysis is not dependent on the absence or presence of Ca(2+) and is observed as well on illumination of native and of partially unfolded GLA. However, photolysis of native GLA results in a partial unfolding of the protein. The latter phenomenon is most clearly observed on fluorescence measurements at low temperatures (near 3 degrees C). The photolysis induces some dimerization and oligomerization, but most GLA molecules remain monomeric. To obtain more information about the reaction products, the illuminated protein is treated with iodoacetamide to label the free thiol groups, it is fragmented with trypsin, and the fragments are analyzed by mass spectrometry. Via this approach, we observe that the cleavage of disulfide bonds is restricted to Cys6-Cys120 and Cys73-Cys91 bonds. The photolytic cleavage of either of these disulfide bonds results in the formation of a single free thiol, a phenomenon restricted to Cys120 and Cys91, respectively. We also found indications that a thioether linkage is formed between Cys73 and Trp60. The alkylsulfenylation of Trp60 presumably results from a combination of primary thiyl and tryptyl radicals.

Structural basis for difference in heat capacity increments for Ca(2+) binding to two alpha-lactalbumins.

Thermodynamic parameters for the unfolding of as well as for the binding of Ca(2+) to goat alpha-lactalbumin (GLA) and bovine alpha-lactalbumin (BLA) are deduced from isothermal titration calorimetry in a buffer containing 10 mM Tris-HCl, pH 7.5 near 25 degrees C. Among the different parameters available, the heat capacity increments (Delta C(p)) offer the most direct information for the associated conformational changes of the protein variants. The Delta C(p) values for the transition from the native to the molten globule state are rather similar for both proteins, indicating that the extent of the corresponding conformational change is nearly identical. However, the respective Delta C(p) values for the binding of Ca(2+) are clearly different. The data suggest that a distinct protein region is more sensitive to a Ca(2+)-dependent conformational change in BLA than is the case in GLA. By analysis of the tertiary structure we observed an extensive accumulation of negatively charged amino acids near the Ca(2+)-binding site of BLA. In GLA, the cluster of negative charges is reduced by the substitution of Glu-11 by Lys. The observed difference in Delta C(p) values for the binding of Ca(2+) is presumably in part related to this difference in charge distribution.