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Molecular dynamics simulations have been performed on a complex in which clusters of boron in the form of molecules of the nanodiamond ortho-carborane (C2B10H12) have been inserted into the four large nonpolar cavities of a nanotube of the right-handed coiled-coil (RHCC)tetrabrachion. The techniques of multi-configurational thermodynamic integration, steered molecular dynamics and umbrella sampling have been combined to investigate the energetics of storage of ortho-carborane in the cavities and to map out the free energy landscape of the RHCC-tetrabrachion-ortho-carboranecomplex along the central channel and along directions transverse to the central channel. The purpose of the study was to explore potential pathways for the diffusion of ortho-carborane between the cavities and the solvent and to assess the stability of the complex as a possible drug delivery system for boron neutron capture therapy (BNCT). The investigation reveals a complex free energy landscape with a multitude of peaks and valleys, all of which can be related to specific architectural elements of the RHCC-nanotube, and the activation barriers for ortho-carborane capture and release support the requirements for rapid cargo uptake coupled with tight binding to the cavities.

Non-equilibrium molecular dynamics simulations of vibrational energy flow induced by the imposition of a thermal gradient have been performed on the μ2-dimeric enzyme glutamate-1-semialdehyde aminomutase (GSAM), the key enzyme in the biosynthesis of chlorophyll, in order to identify energy transport pathways and to elucidate their role as potential allosteric communication networks for coordinating functional dynamics, specifically the negative cooperativity observed in the motion of the two active site gating loops. Fully atomistic MD simulations of thermal diffusion were executed with a GROMACS simulation package on a fully solvated GSAM enzyme by heating various active site target ligands (initially, catalytic intermediates and cofactors) to 300K while holding the remainder of the protein and the solvent bath at 10K and monitoring the temperature T(t) of all the enzyme residues as a function of time over a 1ns observation window. Energy is observed to be deposited in a relatively small number of discrete chains of residues most of which contribute to specific structural or biochemical functionality. Thermal linkages between all thermally active chains were established by isolating a specific pair of chains and performing a thermal diffusion simulation on the pair, one held at 300K and the other at 10K, with the rest of the protein frozen in its initial atomic configuration and thus thermally unresponsive. Proceeding in this way, it was possible to map out multiple pathways of vibrational energy flow leading from one of the active sites through a network of contiguous residues, many of which were evolutionarily conserved and linked by hydrogen bonds, into the other active site and ultimately to the other gating loop.

Tetrabrachion forms the key component of the S-layer of Staphylothermus marinus. Molecular dynamics simulations have been used to study the energetics of occupancy of cavity 3 of the right-handed coiled-coil stalk of tetrabrachion by both water molecules and cyclooctasulfur S8 crowns, as well as to determine possible pathways and free energy barriers for the diffusion of both water and cyclooctasulfur through the peptide walls of RHCC tetrabrachion between cavity 3 and bulk solvent. Calculations of the transfer free energy from solvent to cavity show that clusters of six, seven and eight water molecules are marginally stable in cavity 3, but that occupancy of the cavity by a cyclooctasulfur ring is favoured significantly over water clusters of all sizes. Thermal activation simulations at T = 400K revealed that water molecules diffusing through the wall pass through a sequence of metastable configurations where they are temporarily immobilized by forming networks of hydrogen bonds with specific wall residues. Calculations of the free energy of these metastable configurations using multi-configurational thermodynamic integration yielded a free energy profile with a principal free energy maximum ∆G~50 kJ/mol and a slight activation asymmetry in favour of the direction from cavity to solvent. Potential exit pathways for cyclooctasulfur were investigated with the methods of steered molecular dynamics and umbrella sampling. The cyclooctasulfur was steered through a gap in the tetrabrachion wall along a linear path from cavity 3 into the solvent and the resulting trajectory was subdivided into 22 sampling windows. The free energy profile created for the trajectory by umbrella sampling showed a sharp principal maximum as a function of the reaction coordinate with asymmetric free energy barriers ∆Gexit~220 kJ/mol and ∆Gentrance~100 kJ/mol for cavity exit and entrance, respectively. Keywords: Nonpolar cavities, Archaea S-layer proteins, Right-handed coiled coil, Hydration, Cyclooctasulfur, Molecular dynamics simulations, Free energy, Double-decoupling, Multi-configurational thermodynamic integration (MCTI), Steered molecular dynamics, Umbrella sampling, Weighted histogram analysis method (WHAM), Bootstrapping