ABSTRACT
The effects of solution conditions on protein collapse were studied by measuring the hydrodynamic radii of two unfolded proteins, alpha-synuclein and acid-denatured ferricytochrome c, in dilute solution and in 1 M glucose. The radius of alpha-synuclein in dilute solution is less than that predicted for a highly denatured state, and adding 1 M glucose causes further collapse. Circular dichroic data show that alpha-synuclein lacks organized structure in both dilute solution and 1 M glucose. On the other hand, the radius of acid-denatured cytochrome c in dilute solution is consistent with that of a highly denatured state, and 1 M glucose induces collapse to the size and structure of native cytochrome c. Taken together, these data show that alpha-synuclein, a natively unfolded protein, is collapsed even in dilute solution, but lacks structure.
Subject(s)
Cytochrome c Group/chemistry , Nerve Tissue Proteins/chemistry , Solvents/chemistry , Circular Dichroism , Glucose , Hydrogen-Ion Concentration , Magnetic Resonance Spectroscopy/methods , Protein Denaturation , Protein Structure, Secondary , Solutions , Synucleins , Temperature , alpha-SynucleinABSTRACT
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.
Subject(s)
Cytochrome c Group/metabolism , Cytochrome-c Peroxidase/metabolism , Glucose/chemistry , Oligosaccharides/chemistry , Sucrose/chemistry , Calorimetry , Cytochrome c Group/chemistry , Cytochrome-c Peroxidase/chemistry , Energy Transfer , Enzyme Stability , Models, Chemical , Multienzyme Complexes/chemistry , Multienzyme Complexes/metabolism , Osmotic Pressure , Particle Size , Protein Binding , Solutions , ThermodynamicsABSTRACT
We present a simple and inexpensive method for producing 15N-labeled Saccharomyces cerevisiae iso-1-cytochrome c in Escherichia coli. The labeled protein gives excellent NMR spectra.
Subject(s)
Cytochrome c Group/biosynthesis , Escherichia coli/enzymology , Magnetic Resonance Spectroscopy , Nitrogen Isotopes , Saccharomyces cerevisiae/enzymologyABSTRACT
Proteins denature at low pH because of intramolecular electrostatic repulsions. The addition of salt partially overcomes this repulsion for some proteins, yielding a collapsed conformation called the A-state. A-states have characteristics expected for the molten globule, a notional kinetic protein folding intermediate. Here we show that the addition of neutral sugars to solutions of acid-denatured equine ferricytochrome c induces formation of the A-state in the absence of added salt. We characterized the structure and stability of the sugar-induced A-state with circular dichroism spectropolarimetry (CD) and NMR-monitored hydrogen-deuterium exchange experiments. We also examined the stability of the sugar-induced A-state as a function of sugar size and concentration. The results are interpreted using several models and we conclude that the stabilizing effect is consistent with increased steric repulsion between the protein and the sugar solutions.