Protonation/deprotonation effects on the stability of the Trp-cage miniprotein

Abstract

The Trp-cage miniprotein is a 20 amino acid peptide that exhibits many of the properties of globular proteins. In this protein, the hydrophobic core is formed by a buried Trp side chain. The folded state is stabilized by an ion pair between aspartic acid and an arginine side chain. The effect of protonating the aspartic acid on the Trp-cage miniprotein folding/unfolding equilibrium is studied by explicit solvent molecular dynamics simulations of the protein in the charged and protonated Asp9 states. Unbiased Replica Exchange Molecular Dynamics (REMD) simulations, spanning a wide temperature range, are carried out to the microsecond time scale, using the AMBER99SB forcefield in explicit TIP3P water. The protein structural ensembles are studied in terms of various order parameters that differentiate the folded and unfolded states. We observe that in the folded state the root mean square distance (rmsd) from the backbone of the NMR structure shows two highly populated basins close to the native state with peaks at 0.06 nm and 0.16 nm, which are consistent with previous simulations using the same forcefield. The fraction of folded replicas shows a drastic decrease because of the absence of the salt bridge. However, significant populations of conformations with the arginine side chain exposed to the solvent, but within the folded basin, are found. This shows the possibility to reach the folded state without formation of the ion pair. We also characterize changes in the unfolded state. The equilibrium populations of the folded and unfolded states are used to characterize the thermodynamics of the system. We find that the change in free energy difference due to the protonation of the Asp amino acid is 3 kJ mol(-1) at 297 K, favoring the charged state, and resulting in ΔpK(1) = 0.5 units for Asp9. We also study the differences in the unfolded state ensembles for the two charge states and find significant changes at low temperature, where the protonated Asp side chain makes multiple hydrogen bonds to the protein backbone.

Modulation of folding energy landscape by charge–charge interactions: Linking experiments with computational modeling

“…Traditionally, the modulation of electrostatic interactions in proteins was done by changing the pH or to a lesser degree changing the ionic strength of the solution (8,9). Such approaches are complicated by the difficulties of predicting the titration properties of individual amino acid residues in the context of ensembles of protein conformations that are sampled during the folding reaction (10). A more attractive approach is to modulate electrostatic interactions via substitutions that perturb the thermodynamic and kinetic properties of proteins using simple and computationally tractable model systems.…”

Linking computation and experiments to study the role of charge–charge interactions in protein folding and stability

“…Finally, substitutions at surface positions that were predicted not to contribute to the protein stability due to the charge-charge interaction were found to have stability similar to the wild type ubiquitin. The effects of single site substitutions on protein stability were subsequently tested on over 15 different proteins that varied in size, secondary structure content and overall topology [56][57][58][59][60][61][62][63][64][65][66][67]. In all cases it was found that computational modeling can qualitatively predict the effects of substitutions at surface charge positions on the proteins stability.…”

Side-chain effects on the structures of protonated amino acid dimers: A gas-phase infrared spectroscopy study

“…For biological molecules, protonation is a basic process that can have a profound impact on structure by strongly affecting non-covalent intra-and intermolecular interactions. [1][2][3][4][5][6][7] For peptides and proteins, the amine moiety is the most commonly preferred protonation site and protonated amines can engage in various interactions with other functional groups to play key roles in diverse processes such as protein/peptide folding, 2,4 proton transfer dynamics 1, 3 or peptide backbone dissociation. [8][9][10] Recently, these interactions have also been recognized to be of importance in the formation of metastable amino acid clusters [11][12][13][14] with the possible relevance to aggregation related diseases such as phenylketonuria.…”