An equilibrium and a kinetic stopped-flow fluorescence study of the binding of various metal ions to goat alpha-lactalbumin.

Various metal ions bind to the protein alpha-lactalbumin prepared from goat milk. The stability of the protein after metal binding is compared with that of the apo-protein by monitoring the fluorescence of the tryptophan residues under equilibrium conditions. The kinetics of the metal binding is studied by stopped-flow fluorescence spectroscopy.

Selectivity of tryptophan residues in mediating photolysis of disulfide bridges in goat alpha-lactalbumin.

Goat alpha-lactalbumin (GLA) contains four tryptophan (Trp) residues and four disulfide bonds. Illumination with near-UV light results in the cleavage of disulfide bridges and in the formation of free thiols. To obtain information about the reaction products, the illuminated protein was carbamidomethylated and digested with trypsin and the peptides were analyzed by mass spectrometry.

Influence of Trp mutation on native, intermediate, and transition states of goat alpha-lactalbumin: an equilibrium and kinetic study.

Equilibrium circular dichroism and kinetic stopped-flow fluorescence studies on the stability and the folding kinetics of a set of Trp to Phe mutants of goat alpha-lactalbumin (GLA) were used to characterize the native, intermediate, and transition states of these constructs. GLA contains four tryptophan residues, three of which, Trp26, Trp104, and Trp118, are located in the alpha-domain, while the fourth, Trp60, is located in the beta-domain. Trp26, Trp60, and Trp104 are part of a hydrophobic cluster, whereas Trp118 is situated in a more flexible region near the C-terminal end of the protein.

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 amino acid Asn136 in HIV-1 reverse transcriptase (RT) maintains efficient association of both RT subunits and enables the rational design of novel RT inhibitors.

The highly conserved Asn136 is in close proximity to the nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI)-specific lipophilic pocket of human immunodeficiency virus type 1 (HIV-1) RT. Site-directed mutagenesis has revealed that the catalytic activity of HIV-1 RT mutated at position Asn136 is heavily compromised. Only 0.