Coordinate-Dependent Drift-Diffusion Reveals the Kinetic Intermediate Traps of Top7-Based Proteins

The computer-designed Top7 served as a scaffold to produce immunoreactive proteins by grafting of the 2F5 HIV-1 antibody epitope (Top7-2F5) followed by biotinylation (Top7- 2F5-biotin). The resulting nonimmunoglobulin affinity proteins were effective in inducing and detecting the HIV-1 antibody. However, the grafted Top7-2F5 design led to protein aggregation, as opposed to the soluble biotinylated Top7-2F5-biotin. The structure-based model predicted that the thermodynamic cooper-ativity of Top7 increases after grafting and biotin-labeling, reducing their intermediate state populations. In this work, the folding kinetic traps that might contribute to the aggregation propensity are investigated by the diffusion theory. Since the engineered proteins have similar sequence and structural homology, they served as protein models to study the kinetic intermediate traps that were uncovered by characterizing the position-dependent drift-velocity (v(Q)) and the diffusion (D(Q)) coefficients. These coordinate-dependent coefficients were taken into account to obtain the folding and transition path times over the free energy transition states containing the intermediate kinetic traps. This analysis may be useful to predict the aggregated kinetic traps of scaffold-epitope proteins that might compose novel diagnostic and therapeutic platforms.

Automated summary

The computer-designed Top7 scaffold was used to produce immunoreactive proteins by grafting the 2F5 HIV-1 antibody epitope, which effectively induced and detected HIV-1 antibodies. However, protein aggregation was observed in the grafted Top7-2F5 design, while the soluble biotinylated Top7-2F5-biotin did not have this issue. The study predicted an increase in thermodynamic cooperativity of Top7 after grafting and biotin-labeling, reducing intermediate state populations. Investigating folding kinetic traps and coordinate-dependent coefficients may be useful in predicting aggregated kinetic traps of scaffold-epitope proteins for novel diagnostic and therapeutic platforms.