Effects of crowding by mono-, di-, and tetrasaccharides on cytochrome c-cytochrome c peroxidase binding: comparing experiment to theory.
In cells, protein-protein interactions occur in an environment that is crowded with other molecules, but in vitro studies are almost exclusively performed in dilute solution. To gain information about the effects of crowding on protein complex formation, we used isothermal titration calorimetry to measure the stoichiometry, the free energy change, and the enthalpy change for the binding of yeast iso-1-ferricytochrome c to yeast ferricytochrome c peroxidase in dilute solution and in solutions crowded with the sugars glucose, sucrose, and stachyose. The stoichiometry is 1:1 under all conditions. The sugars stabilize the complex, but by only 0.1-0.5 kcal.mol(-)(1), and the increased stability is not correlated with the change in enthalpy of complex formation. We then compared the measured stability changes to values obtained from several analyses that are currently used to predict crowding-induced changes in biomolecular equilibria. None of the analyses are completely successful by themselves, and the results suggest that a complete analysis must account for both excluded-volume and chemical interactions.
Morar
A S
AS
Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-3290, USA.
Wang
X
X
Pielak
G J
GJ
D002151:Calorimetry; D003574:Cytochrome c Group / Q000737:chemistry / Q000378:metabolism*; D003578:Cytochrome-c Peroxidase / Q000737:chemistry / Q000378:metabolism*; D004735:Energy Transfer; D004795:Enzyme Stability; D005947:Glucose / Q000737:chemistry*; D008956:Models, Chemical; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism; D009844:Oligosaccharides / Q000737:chemistry*; D009997:Osmotic Pressure; D010316:Particle Size; D011485:Protein Binding; D012996:Solutions; D013395:Sucrose / Q000737:chemistry*; D013816:Thermodynamics
D003574:Cytochrome c Group; D009097:Multienzyme Complexes; D009844:Oligosaccharides; D012996:Solutions; C005695:stachyose; D013395:Sucrose; D003578:Cytochrome-c Peroxidase; D005947:Glucose