Effects of small molecule crowding on protein structure and equilibrium thermodynamics of protein -protein interaction

Intra- (protein folding and stability) and inter- (signal transduction, cell-cell recognition and bioenergetics) molecular interactions are fundamental for growth and development. When studying these interactions control factors relevant for the cellular function must be taken into consideration. Inside the cell small- and macromolecules can occupy up to 50% of the total volume and reach concentrations of 450 g/L. Crowding, the result of high cellular solute concentrations, can have large effects on macromolecular equilibria.;My work is an equilibrium thermodynamics study on the effects of molecular crowding on intra- and interprotein interactions. I use sugars as crowding reagents. A description of crowding, crowders, and the most widely-applied analyses currently used to predict crowding-induced effects is given in CHAPTER I.;CHAPTER II is an overview of the biophysical methods that I use to examine protein structure and protein complex stability.;In CHAPTER III I show that sugars stabilize cytochrome c but do not alter its size and structure. NMR data show that melezitose-induced chemical shift changes in native cytochrome c are small, 1H and 15N redox shifts in dilute and melezitose solution are highly correlated, and the hydrodynamic radii of native cytochrome c in dilute and glucose solutions agree within the experimental errors.;In CHAPTERs IV and V I used isothermal titration calorimetry to determine the thermodynamic parameters for the physiologically-relevant cytochrome c/cytochrome c peroxidase complex formation in the presence of mono- (D(+)-glucose), di- (sucrose), tri- (melezitose, raffinose) and tetrasaccharides (stachyose). Three sugars (glucose, sucrose, stachyose) gave calorimetric results similar to those in dilute solution. However, trisaccharides stabilize the second cytochrome c binding site on cytochrome c peroxidase. I applied the 2:1 binding model to trisaccharide data, which allows for intersite interaction. Mutagenesis studies show that the second site is near Asp148. The steady-state kinetics of peroxidase is not affected by the presence of sugars.;Chapter VI hypothesizes that crowding stabilizes a collapsed state of natively-unfolded proteins. Sugars not only decrease the hydrodynamic radius of P protein and acid-denatured cytochrome c, they also induce native-like secondary structure. The radius of alpha-synuclein in dilute solution is less than that predicted for a random coil, and adding glucose causes further collapse. However, alpha-synuclein lacks organized structure in both dilute solution and glucose. I propose that the different behavior resides in the extent of the hydrophobic core.