Limited proteolysis has been used to probe the partially folded state of bovine alpha-lactalbumin (BLA) at acid pH (A-state) or dissolved in aqueous trifluoroethanol (TFE-state). The sites of proteolytic fission have been determined by isolation of the various BLA fragments and comparison of their N-terminal amino acid sequence and amino acid composition after acid hydrolysis, as well as their molecular mass determined by mass spectrometry, with the known sequence of BLA. Incubation of BLA with pepsin at 20-22 degrees C and pH 2.0 in the presence of 0.1 M NaCl results in very rapid cleavage of the 123-residue chain at peptide bond Ala40-Ile41 and subsequently at Leu52-Phe53, leading to a nicked species of BLA constituted by the two fragments 1-40 and 53-123 cross-linked by the four disulfide bridges of the protein. Much slower proteolytic cleavage occurs at Tyr103-Trp104. The highly helical conformational state acquired by BLA when dissolved in aqueous buffer (pH 7.0) containing 50% (v/v) TFE was probed by the TFE-resistant thermolysin. Proteolytic cleavage occurs at the peptide bond Ala40-Ile41 and much more slowly at Phe80-Leu81. Moreover, the peptide bond Gln2-Leu3 at the N-terminus of the chain is partially cleaved by thermolysin. Conversely, native BLA in a pH 7.0 buffer is rather resistant to proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Transient protein–protein interactions are functionally relevant as a control mechanism in a variety of biological processes. Analysis of the 3D structure of protein–protein complexes indicates that water molecules trapped at the interface are very common; however, their role in the stability and specificity of protein homodimer interactions has been not addressed yet. To provide new insights into the energetic bases that govern the formation of highly hydrated interfaces, the dissociation process of bovine βlg variant A at a neutral pH was characterized here thermodynamically by conducting dilution experiments with an isothermal titration calorimeter. Association was enthalpically driven throughout the temperature range spanned. ΔH and ΔCp were significantly more negative than estimates based on surface area changes, suggesting the occurrence of effects additional to the dehydration of the contact surfaces between subunits. Near‐UV CD spectra proved to be independent of protein concentration, indicating a rigid body‐like association. Furthermore, the process proved not to be coupled to significant changes in the protonation state of ionizable groups or counterion exchange. In contrast, both osmotic stress experiments and a computational analysis of the dimer's 3D structure indicated that a large number of water molecules are incorporated into the interface upon association. Numerical estimates considering the contributions of interface area desolvation and water immobilization accounted satisfactorily for the experimental ΔCp. Thus, our study highlights the importance of explicitly considering the effects of water sequestering to perform a proper quantitative analysis of the formation of homodimers with highly hydrated interfaces. Proteins 2008. © 2007 Wiley‐Liss, Inc.

An understanding of the interplay between structure and energetics is crucial for the optimization of modern protein engineering techniques. In this context, the study of natural isoforms is a subject of major interest, as it provides the scenario for analyzing mutations that have endured during biological evolution. In this study, we performed a comparative analysis of the ligand-recognition and homodimerization energetics of bovine β-lactoglobulin variants A (βlgA) and B (βlgB). These variants differ by only two amino-acid substitutions: 64th (Asp(A) → Gly(B)), which is fully exposed to the solvent, and 118th (Val(A) → Ala(B)), immersed in the hydrophobic core of the protein. Calorimetric measurements revealed significant enthalpic and entropic differences between the isoforms in both binding processes. A structural comparison suggests that a variation in the conformation of the loop C-D, induced by mutation Asp/Gly, could be responsible for the differences in ligand-binding energetics. While recognition of lauric acid was entropically driven, recognition of sodium dodecyl sulfate was both entropically and enthalpically driven, confirming the key role of the ligand polar moiety. Because of a more favorable enthalpy, the dimerization equilibrium constant of βlgB was larger than that of βlgA at room temperature, while the two dimers became similarly stable at 35 °C. The isoforms exchanged the same number of structural water molecules and protons and shared similar stereochemistry at the dimer interface. MD simulations revealed that the subunits of both variants become more flexible upon dimer formation. It is hypothesized that a larger increase of βlgA mobility could account for the dimerization energetic differences observed.

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