Accelerated molecular dynamics (aMD) simulations greatly improve the efficiency of conventional molecular dynamics (cMD) for sampling biomolecular conformations, but they require proper reweighting for free energy calculation. In this work, we systematically compare the accuracy of different reweighting algorithms including the exponential average, Maclaurin series, and cumulant expansion on three model systems: alanine dipeptide, chignolin, and Trp-cage. Exponential average reweighting can recover the original free energy profiles easily only when the distribution of the boost potential is narrow (e.g., the range ≤20kBT) as found in dihedral-boost aMD simulation of alanine dipeptide. In dual-boost aMD simulations of the studied systems, exponential average generally leads to high energetic fluctuations, largely due to the fact that the Boltzmann reweighting factors are dominated by a very few high boost potential frames. In comparison, reweighting based on Maclaurin series expansion (equivalent to cumulant expansion on the first order) greatly suppresses the energetic noise but often gives incorrect energy minimum positions and significant errors at the energy barriers (∼2–3kBT). Finally, reweighting using cumulant expansion to the second order is able to recover the most accurate free energy profiles within statistical errors of ∼kBT, particularly when the distribution of the boost potential exhibits low anharmonicity (i.e., near-Gaussian distribution), and should be of wide applicability. A toolkit of Python scripts for aMD reweighting “PyReweighting” is distributed free of charge at .

We present a QM/MM ab initio molecular dynamics study of the peptide hydrolysis reaction catalyzed by HIV-1 protease. The QM/MM calculations are based on previous extensive classical MD simulations on the protein in complex with a model substrate Rothlisberger, U. Protein Sci. 2002, 11, 2393-2402. Gradient-corrected BLYP density functional theory (DFT) describes the reactive part of the active site, and the AMBER force field describes the rest of the protein, the solvent, and the counterions. An unbiased enhanced sampling of the QM/MM free-energy surface is performed to identify a plausible reaction coordinate for the second step of the reaction. The enzymatic reaction is characterized by two reaction freeenergy barriers of ∼18 and ∼21 kcal mol -1 separated by a metastable gem-diol intermediate. In both steps, a proton transfer that involves the substrate and the two catalytic Asp molecules is observed. The orientation and the flexibility of the reactants, governed by the surrounding protein frame, are the key factors in determining the activation barrier. The calculated value for the barrier of the second step is slightly larger than the value expected from experimental data (∼16 kcal mol -1 ). An extensive comparison with calculations on gas-phase model systems at the Hartree-Fock, DFT-BP, DFT-BLYP, DFT-B3LYP, MP2, CCD, and QM/MM DFT-BLYP levels of theory suggests that the DFT-BLYP functional has the tendency to underestimate the energy of the gem-diol intermediate by ∼5-7 kcal mol -1 .The aspartyl protease from human immunodeficiency virus type 1 (HIV-1 PR) targets the AIDS epidemic. The enzyme is essential for viral metabolism 1 because it cleaves the long polypeptide chain that is expressed in infected host cells in specific positions to generate the active proteins that are required for viral maturation.HIV-1 PR is a homodimer with the active site located at the interface between the two subunits. The cleavage site is an Asp dyad (Asp25 and Asp25′, Figure 1), located inside a large activesite pocket that allows the enzyme to recognize and cleave sequences of six amino acids selectively. 2 Several aspects of the enzymatic reaction mechanism have been the focus of a variety of computational techniques, including molecular mechanics, 3-5 tight-binding, 6 semiempirical, 7,8 and ab initio 9-15 methods. This theoretical work has been complemented by kinetic, thermodynamic, and structural data. 8,[16][17][18][19][20][21][22][23][24][25][26][27][28][29] The picture emerging from these studies can be summarized as follows. The free form of the enzyme (E) is stabilized by a low-barrier H bond (LBHB) 30 locking the Asp dyad in an almost coplanar conformation. In a first physical step, E binds to substrate SUB to form the enzyme-substrate complex ESUB, which might (ESUB(a), Figure 1) or might not (ESUB(b), Figure 1) maintain the LBHB. 2 H and 15 N kinetic isotope effect measurements 17,18,28 have established that in HIV-1 PR (i) a hydrated intermediate is reversibly formed and (ii) protonation of the peptide bond nit...

Defining the molecular details and consequences of the association of water-soluble proteins with membranes is fundamental to understanding protein-lipid interactions and membrane functioning. Phospholipase A 2 (PLA 2 ) enzymes, which catalyze the hydrolysis of phospholipid substrates that compose the membrane bilayers, provide the ideal system for studying protein-lipid interactions. Our study focuses on understanding the catalytic cycle of two different human PLA 2 s: the cytosolic Group IVA cPLA 2 and calcium-independent Group VIA iPLA 2 . Computer-aided techniques guided by deuterium exchange mass spectrometry data, were used to create structural complexes of each enzyme with a single phospholipid substrate molecule, whereas the substrate extraction process was studied using steered molecular dynamics simulations. Molecular dynamic simulations of the enzyme-substrate-membrane systems revealed important information about the mechanisms by which these enzymes associate with the membrane and then extract and bind their phospholipid substrate. Our data support the hypothesis that the membrane acts as an allosteric ligand that binds at the allosteric site of the enzyme's interfacial surface, shifting its conformation from a closed (inactive) state in water to an open (active) state at the membrane interface.GIVA cPLA 2 | GVIA iPLA 2 | PAPC | MD simulations | DXMS