Abstract
Trp‐cage miniprotein was used to investigate the role of a salt‐bridge (Asp^9^–Arg^16^) in protein formation, by mutating residues at both sides, we mapped its contribution to overall stability and its role in folding mechanism. We found that both of the above side‐chains are also part of a dense interaction network composed of electrostatic, H‐bonding, hydrophobic, etc. components. To elucidate the fold stabilizing effects, we compared and contrasted electronic circular dichroism and NMR data of miniproteins equipped with a salt‐bridge with those of the salt‐bridge deleted mutants. Data were acquired both in neutral and in acidic aqueous solutions to decipher the pH dependency of both fully and partially charged partners. Our results indicate that the folding of Trp‐cage miniproteins is more complex than a simple two‐state process as we detected an intermediate state that differs significantly from the native fold. The intermediate formation is related to the salt‐bridge stabilization; in the miniprotein variants equipped with salt‐bridge the population of the intermediate state at acidic pH is significantly higher than it is for the salt‐bridge deleted mutants. In this molecular framework Arg^16^ stabilizes more than Asp^9^ does, because of its higher degree of 3D‐fold cooperation. In conclusion, the Xxx$^{9} \leftrightarrow$Yyy^16^ salt‐bridge is not an isolated entity of this fold; rather it is an integrated part of a complex interaction network. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.