Effect of ligand binding on a protein with a complex folding landscape

Abstract

Ligand binding to a protein can stabilize it significantly against unfolding. The variation of the folding free energy, ΔΔG, due to ligand binding can be derived from a simple reaction scheme involving exclusive binding to the native state. One obtains the following expression: , where K is the ligand dissociation constant and L is its concentration, R is the universal gas constant and T is the temperature. This expression has been shown to correctly describe experimental results on multiple proteins. In the current work we studied the effect of ligand binding on the stability of the multi-domain protein adenylate kinase from E. coli (AKE). Unfolding experiments were conducted using single-molecule FRET spectroscopy, which allowed us to directly obtain the fraction of unfolded protein in a model-free way from FRET efficiency histograms. Surprisingly, it was found that the effect of two inhibitors (ApA and AMPPNP) and a substrate (AMP) on the stability of AKE was much smaller than expected based on K values obtained independently using microscale thermophoresis. To shed light on this issue, we measured the K for ApA over a range of chemical denaturant concentrations where the protein is still folded. It was found that K increases dramatically over this range, likely due to the population of folding intermediates, whose binding to the ligand is much weaker than that of the native state. We propose that binding to folding intermediates may dominate the effect of ligands on the stability of multi-domain proteins, and could therefore have a strong impact on protein homeostasis in vivo.

Direct observation of ultrafast large-scale dynamics of an enzyme under turnover conditions

“…3 B ) that were similar to those observed with ATP, though shifted to lower concentrations, suggesting that it is the binding of the substrates that switches the enzyme from one FEP to the other, irrespective of the chemical step. We also studied domain closure dynamics with AMP-PNP, a nonhydrolysable analog of ATP with a similar K d ( 23 ), and found that even at a high concentration and in the presence of AMP, the equilibrium did not fully shift to the closed conformation as in the presence of ATP and AMP ( Fig. 3 C ).…”

Thermophoresis for characterizing biomolecular interaction

“…In this substantial approach, MST and SPR were used for the interaction study. On the other hand, the small molecules inhibitory effect was investigated extensively via MST [59][60][61][62][63][64]. Where, Shang et al [60] successfully investigated small molecule as inhibitor G-protein-coupled Rho guanine nucleotide exchange factors.…”

Ancient Thioredoxins Evolved to Modern Day Stability-Function Requirement by Altering Native State Ensemble

“…In parallel to the advancements in phylogenetics, recent biophysical studies of proteins have shown that all positions are dynamically linked to each other within a network of interactions, where the strength of each link varies across the protein. This network of interaction leads to intrinsic fluctuations -encoded in the structure and sequence -that govern protein function [4,[16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32]. The obsolete view of the single native structure has been long replaced by "an ensemble of substates" that accurately represent the native state [25].…”