Abstract
The intracellular concentration of protein may be as high as 400 mg per ml; thus it seems inevitable that within the cell, numerous protein-protein contacts are constantly occurring. A basic biochemical principle states that the equilibrium of an association reaction can be shifted by ligand binding. This indicates that if within the cell many protein-protein interactions are indeed taking place, some fundamental characteristics of proteins would necessarily differ from those observed in traditional biochemical systems. Accordingly, we measured the effect of eight different proteins on the formation of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM) from guanidinium chloride unfolded monomers. The eight proteins at concentrations of micrograms per ml induced an important increase on active dimer formation. Studies on the mechanism of this phenomenon showed that the proteins stabilize the dimeric structure of TbTIM, and that this is the driving force that promotes the formation of active dimers. Similar data were obtained with TIM from three other species. The heat changes that occur when TbTIM is mixed with lysozyme were determined by isothermal titration calorimetry; the results provided direct evidence of the weak interaction between apparently unrelated proteins. The data, therefore, are strongly suggestive that the numerous protein-protein interactions that occur in the intracellular space are an additional control factor in the formation and stability of proteins.
Highlights
The mechanisms that lead to the formation of the threedimensional functionally active structure of proteins are being extensively investigated
We determined the effect of eight different proteins on the reactivation of homodimeric triosephosphate isomerase (TIM) from guanidinium chloride (GdnHCl) unfolded monomers, and on the stability of the dimer
The results showed that in the absence of bovine serum albumin (BSA), the specific activity of TbTIM decreased as its concentration was progressively diminished (Fig. 3A); this reflects the dissociation of the dimer as the concentration of protein is gradually diminished
Summary
The mechanisms that lead to the formation of the threedimensional functionally active structure of proteins are being extensively investigated. An additional problem that has been in the mind of many investigators is that the test tube conditions in which the enzymes are traditionally studied differ dramatically from those that operate in the intracellular milieu In this regard, a relevant point is that within the cell the concentration of protein may be as high 400 mg per ml [4,5,6,7,8], a situation that has been termed crowding. In an instructive article, Schellman [9] made a quantitative evaluation of Le Chatelier’s principle that indicates that for example, the binding of ligands to a dimer will shift the equilibrium of the association reaction toward the dimer; ligand binding to one of the intermediate species in dimer formation will affect adversely the formation of the dimer On these grounds, we explored if a model protein has the capacity to interact with other apparently unrelated proteins. We determined the effect of eight different proteins on the reactivation of homodimeric triosephosphate isomerase (TIM) from guanidinium chloride (GdnHCl) unfolded monomers, and on the stability of the dimer
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