Macromolecular crowders and osmolytes modulate the structural and catalytic properties of alkaline molten globular 5-aminolevulinate synthase

Abstract

The molten globule state is a dynamic ensemble of conformational subsets, where proteins lack well-defined tertiary structure, but retain native-like content of secondary structure and a relatively compact fold. Using various spectroscopic techniques, we characterized the effects of macromolecular crowders and osmolytes on the structural and catalytic properties of the alkaline molten globule state of murine erythroid 5-aminolevulinate synthase (mALAS2^alk defined at pH 9.5/37 °C). The tertiary structure rigidity of mALAS2^alk, as discerned from near-UV circular dichroic (CD) measurements, increased in the presence of the osmolytes N-trimethylamine oxide (TMAO) and glycerol. In contrast, the macromolecular crowders Dextran 200 and Ficoll 400, even at concentrations of 20% (w/v), were far less effective in rigidifying the tertiary structure. At this concentration of Dextran 200, the far-UV ellipticity of mALAS2^alk intensified, implicating stabilization of secondary structural elements in the crowded environment. Furthermore, in the presence of Dextran, the solubility of mALAS2^alk strongly depended on the molar concentration of the AMPSO buffer, suggesting changes in the surface hydration of the enzyme. Through ligand-induced enhancement of the tertiary structure rigidity, the solubility of mALAS2^alk increased under crowded conditions that otherwise favored precipitation of the holoenzyme. The k_cat value of mALAS2^alk (pH 9.5/37 °C) increased significantly upon the addition of cosolvents, although the rates remained lower than the k_cat determined under physiological conditions (pH 7.5/37 °C). Our data suggest that the molecular properties of at least some molten globular proteins might be modulated within the highly crowded and osmolyte enriched intracellular environment.