Biophysical characterization of protein folding and misfolding.

The HPr proteins were characterized as folding by a two-state folding mechanism. Here, we present a comparison of the equilibrium and kinetic folding for the HPr protein from Bacillus subtilis, E coli and a key variant from these proteins. For the wild-type protein we find that GHX is greater than GUDC, suggesting that the HPr does not fold by a simple two-state mechanism. This discrepancy is revealed by testing the two-state nature of the folding reaction of HPr with mutation. We show that removing a single charge side chain (Asp 69) converts the HPr protein back to a simple two-state mechanism. Ribonuclease Sa and two charge-reversal variants can be converted into amyloidin vitro by the addition of 2,2,2-triflouroethanol (TFE). We report here amyloid fibril formation for these proteins as a function of pH. The pH at maximal fibril formation correlates with the pH dependence of protein solubility, but not with stability, for these variants. Additionally, we show that the pH at maximal fibril formation for a number of ivwell-characterized proteins is near the pI, where the protein is expected to be the least soluble. This suggests that protein solubility is an important determinant of fibril formation

Role of cation in enhancing the conversion of the Alzheimer\u27s peptide into amyloid fibrils using protic ionic liquids

We report on the impact of changes in the protic ionic liquid (pIL) cation on the fibrilisation kinetics and the conversion of the A 16-22 from monomers to amyloid fibrils. When we compare the use of primary, secondary, and tertiary amines we find that the primary amine results in the greatest conversion into amyloid fibrils. We show that the pIL is directly interacting with the peptide and this likely drives the difference in conversion and kinetics observed

Prediction of Protein Solubility from Calculation of Transfer Free Energy

Solubility plays a major role in protein purification, and has serious implications in many diseases. We studied the effects of pH and mutations on protein solubility by calculating the transfer free energy from the condensed phase to the solution phase. The condensed phase was modeled as an implicit solvent, with a dielectric constant lower than that of water. To account for the effects of pH, the protonation states of titratable side chains were sampled by running constant-pH molecular dynamics simulations. Conformations were then selected for calculations of the electrostatic solvation energy: once for the condensed phase, and once for the solution phase. The average transfer free energy from the condensed phase to the solution phase was found to predict reasonably well the variations in solubility of ribonuclease Sa and insulin with pH. This treatment of electrostatic contributions combined with a similar approach for nonelectrostatic contributions led to a quantitative rationalization of the effects of point mutations on the solubility of ribonuclease Sa. This study provides valuable insights into the physical basis of protein solubility