Effects of macromolecular crowding on the structural stability of human α-lactalbumin.

The folding of protein, an important process for protein to fulfill normal functions, takes place in crowded physiological environments. α-Lactalbumin, as a model system for protein-folding studies, has been used extensively because it can form stable molten globule states under a range of conditions. Here we report that the crowding agents Ficoll 70, dextran 70, and polyethylene glycol (PEG) 2000 have different effects on the structural stability of human α-lactalbumin (HLA) represented by the transition to a molten globule state: dextran 70 dramatically enhances the thermal stability of Ca(2+)-depleted HLA (apo-HLA) and Ficoll 70 enhances the thermal stability of apo-HLA to some extent, while PEG 2000 significantly decreases the thermal stability of apo-HLA. Ficoll 70 and dextran 70 have no obvious effects on trypsin degradation of apo-HLA but PEG 2000 accelerates apo-HLA degradation by trypsin and destabilizes the native conformation of apo-HLA. Furthermore, no interaction is observed between apo-HLA and Ficoll 70 or dextran 70, but a weak, non-specific interaction between the apo form of the protein and PEG 2000 is detected, and such a weak, non-specific interaction could overcome the excluded-volume effect of PEG 2000. Our data are consistent with the results of protein stability studies in cells and suggest that stabilizing excluded-volume effects of crowding agents can be ameliorated by non-specific interactions between proteins and crowders.

Both the C-terminal polylysine region and the farnesylation of K-RasB are important for its specific interaction with calmodulin.

BACKGROUND: Ras protein, as one of intracellular signal switches, plays various roles in several cell activities such as differentiation and proliferation. There is considerable evidence showing that calmodulin (CaM) binds to K-RasB and dissociates K-RasB from membrane and that the inactivation of CaM is able to induce K-RasB activation. However, the mechanism for the interaction of CaM with K-RasB is not well understood. METHODOLOGY/PRINCIPAL FINDINGS: Here, by applying fluorescence spectroscopy and isothermal titration calorimetry, we have obtained thermodynamic parameters for the interaction between these two proteins and identified the important elements of K-RasB for its interaction with Ca(2+)/CaM. One K-RasB molecule interacts with one CaM molecule in a GTP dependent manner with moderate, micromolar affinity at physiological pH and physiologic ionic strength. Mutation in the polybasic domain of K-Ras decreases the binding affinity. By using a chimera in which the C-terminal polylysine region of K-RasB has been replaced with that of H-Ras and vice versa, we find that at physiological pH, H-Ras-(KKKKKK) and Ca(2+)/CaM formed a 1:1 complex with an equilibrium association constant around 10(5) M(-1), whereas no binding reaction of K-RasB-(DESGPC) with Ca(2+)/CaM is detected. Furthermore, the interaction of K-RasB with Ca(2+)/CaM is found to be enhanced by the farnesylation of K-RasB. CONCLUSIONS/SIGNIFICANCE: We demonstrate that the polylysine region of K-RasB not only contributes importantly to the interaction of K-RasB with Ca(2+)/CaM, but also defines its isoform specific interaction with Ca(2+)/CaM. The farnesylation of K-RasB is also important for its specific interaction with Ca(2+)/CaM. Information obtained here can enhance our understanding of how CaM interacts with K-RasB in physiological environments.

Binding of calcium ions to Ras promotes Ras guanine nucleotide exchange under emulated physiological conditions.

Both Ras protein and calcium play significant roles in various cellular processes via complex signaling transduction networks. However, it is not well understood whether and how Ca(2+) can directly regulate Ras function. Here we demonstrate by isothermal titration calorimetry that Ca(2+) directly binds to the H-Ras.GDP.Mg(2+) complex with moderate affinity at the first binding site followed by two weak binding events. The results from limited proteinase degradation show that Ca(2+) protects the fragments of H-Ras from being further degraded by trypsin and by proteinase K. HPLC studies together with fluorescence spectroscopic measurements indicate that binding of Ca(2+) to the H-Ras.GDP.Mg(2+) complex remarkably promotes guanine nucleotide exchange on H-Ras under emulated physiological Ca(2+) concentration conditions. Addition of high concentrations of either of two macromolecular crowding agents, Ficoll 70 and dextran 70, dramatically enhances H-Ras guanine nucleotide exchange extent in the presence of Ca(2+) at emulated physiological concentrations, and the nucleotide exchange extent increases significantly with the concentrations of crowding agents. Together, these results indicate that binding of calcium ions to H-Ras remarkably promotes H-Ras guanine nucleotide exchange under emulated physiological conditions. We thus propose that Ca(2+) may activate Ras signaling pathway by interaction with Ras, providing clues to understand the role of calcium in regulating Ras function in physiological environments.