Molecular dynamics is a powerful tool that has been long used for the simulation of biomolecules. It complements experiments, by providing detailed information about individual atomic motions. But there is an essential and often overlooked assumption that, left unchecked, could invalidate any results from it: is the simulated trajectory long enough, so that the system has reached thermodynamic equilibrium, and the measured properties are converged? Previous studies showed mixed results in relation to this assumption. This has profound implications, as the resulting simulated trajectories may not be reliable in predicting equilibrium properties. Yet, this is precisely what most molecular dynamics studies do. So the question arises: are these studies even valid?Here, we present a thorough analysis of up to a hundred microseconds long trajectories, of several system with varying size, to probe the convergence of different structural, dynamical and cumulative properties, and elaborate on the relevance of the concept of equilibrium, and its physical and biological meaning. The results show that properties with the most biological interest tend to converge in multi-microsecond trajectories, although other properties–like transition rates to low probability conformations–may require more time.

Tumor extracellular matrix (ECM) is a high-capacity target for the precision delivery of affinity ligand-guided drugs and imaging agents. Recently, we developed a PL1 peptide (sequence: PPRRGLIKLKTS) for systemic targeting of malignant ECM. Here, we map the dynamics of PL1 binding to its receptors Fibronectin Extra Domain B (FN-EDB) and Tenascin C C-isoform (TNC-C) by computational modeling and cell-free binding studies on mutated receptor proteins, and study cellular binding and internalization of PL1 nanoparticles in cultured cells. Molecular dynamics simulation and docking analysis suggested that the engagement of PL1 peptide with both receptors is primarily driven by electrostatic interactions. Substituting acidic amino acid residues with neutral amino acids at predicted PL1 binding sites in FN-EDB (D52N-D49N-D12N) and TNC-C (D39N-D45N) resulted in the loss of binding of PL1 nanoparticles. Remarkably, PL1-functionalized nanoparticles (NPs) were not only deposited on the target ECM but bound the cells and initiated a robust cellular uptake via a pathway resembling macropinocytosis. Our studies establish the mode of engagement of the PL1 peptide with its receptors and suggest applications for intracellular delivery of nanoscale payloads. The outcomes of this work can be used for the development of PL1-derived peptides with improved stability, affinity, and specificity for precision targeting of the tumor ECM and malignant cells.

Hypothetical scanning molecular dynamics (HSMD) is a relatively new method for calculating the absolute free energy and entropy. HSMD is extended here for the first time for calculating the absolute free energy of binding, ΔA⁰, as applied to the avidin−biotin complex. With HSMD the ligand is built (more accurately reconstructed) from nothing in solvent and in the protein, in contrast to the commonly used methods where the ligand is annihilated (by thermodynamic integration) in these environments. Therefore, the end-point problem encountered with the latter methods does not exist with HSMD and the need for restraints is avoided. Also, the entropy of the ligand and water in both environments is obtained directly as a byproduct of the simulation. The binding mechanism of biotin to avidin involves a mobile loop that is expected to be in an open conformation in unbound avidin, which is changed to a closed one upon binding, that is, the loop moves to cover biotin in the active site. The contribution of the loop’s conformational change to the total free energy of binding is calculated here for the first time. Our result, ΔA⁰ = −24.9 ± 7 covers the experimental value −20.7 kcal/mol within the error bars.