I. Partitioning of soft water-soluble polymers into beta-barrel channels in their functional states The cell is a crowded place. As an example, volume concentration of macromolecules in the cytoplasm of Escherichia coli is as high as 30-40%, leading to significant deviations of macromolecular reaction rates and equilibria from those in diluted samples. The current consensus is that the functional consequences of molecular crowding stem from two phenomena: hard-core repulsions, otherwise referred to as entropic effects, and soft chemical interactions. This year we have demonstrated that the entropic effects are generally more subtle than just the hard-core repulsions, because they necessarily include the phenomenon of forced macromolecule partitioning into protein cavities. With consequences for the selective partitioning of large molecules in cells, studies of polymer partitioning into nanopores are also instructive for refining single-molecule sensing as well as for polymer-assisted transport and packaging. To address these questions, we analyzed forced size-dependent partitioning of binary mixtures of differently sized polyethylene glycols (PEGs) in nanosize cavities. We probed three structurally different channels: Voltage-Dependent Anion Channel from outer mitochondrial membrane (VDAC), bacterial porin OmpC (outer membrane protein C), and bacterial channel-forming toxin alpha-Hemolysin. Our interpretation was based on the idea that relatively less-penetrating polymers push the more easily penetrating ones into nanosize channels in excess of their bath concentration. All three channels exhibited forced partitioning that could be understood conceptually as well as quantitatively within a polymers-pushing-polymers model, allowing good estimates for the size-dependent pore penetration energy differences. As a result of our work, beyond proof of concept, forced partitioning in cells can now be recognized and used as a new tool for molecule-selective transport and active osmotically regulated packaging. In particular, comparison of the theory with experiments had proved to be excellent for VDAC. Polymer partitioning data for the other two channels were consistent with theory if additional assumptions regarding the energy penalty of pore penetration are included. However, because of the several implicit assumptions, including those that idealized channel geometry, these deviations from the theoretical predictions were reasonably expected. The obtained results demonstrate that the general concept of polymers-pushing-polymers is helpful in understanding and quantification of concrete examples of size-dependent forced partitioning of polymers into protein nanopores. II. Progress in theoretical and experimental studies of the voltage-dependent anion channel of the outer mitochondrial membrane Present understanding of molecular mechanisms of functioning of beta-barrel channels, such as VDAC, general and specific bacterial porins, and different toxin channels, is far behind the progress in the field of conventional highly-selective ion channels. Specifically, mitochondrial channels compose a separate class characterized by unique properties that distinguish them from other channels, including evolutionary related bacterial porins. Mitochondrial channels are directly involved in regulation of the normal function of mitochondria and in metabolic changes in response to environmental challenges and different kinds of stress, such as apoptosis. For example, VDAC, the channel of the outer mitochondrial membrane, serves as a major pathway for the metabolites exchange between the cytosol and mitochondria and thus controls a significant portion of the outer membrane permeability. Small ions and water-soluble mitochondrial metabolites, such as ATP, ADP, all cross the mitochondrial outer membrane through one pathway, the VDAC. Therefore, determination of the molecular basis of VDAC's ion selectivity, metabolite permeability, and regulation is crucial to understanding its function and physiological role. Our recent progress in VDAC studies demonstrates that the computational techniques not only complement experimental electrophysiological data but also lead to an a priori identification of the binding sites for ATP or for the cytosolic regulators of VDAC, thus connecting existing structures to the long-standing proposition of VDAC's crucial role in providing and regulating metabolite transport in and out of mitochondria. Using a very promising new computational technology based on the combination of 2-dimensional umbrella sampling simulations with the distributed replica-exchange protocol we showed that ATP in the VDAC pore exhibits surprisingly broad distribution of states. For the first time our analysis accounted for the flexibility of ATP molecule revealing 10 most probable structural clusters along the pore. Notably, one of the pathways can be associated with the highest possible ATP flux rates. It appears that in a majority of cases the rate-limiting step is a dissociation of ATP from the-helix of the VDAC N-terminus. Importantly, all basic residues in the VDAC's N-terminus, which participate in formation of an ATP binding site, were also found to bind to the acidic residues in the C-terminal tail of alpha-tubulin. This conclusion is supported by our previous experiments and calculations which show that ATP is excluded from the tubulin-blocked state of VDAC. Thus, the availability of the recently solved structures, our lab latest experimental data, and powerful state-of-the-art computational techniques combined with modern analytical approaches lead to the breakthrough in our understanding of the relationship between VDAC function and structure. III. Physical theory of channel-facilitated transport Physical theory of channel-facilitated transport gives us a set of rigorous tools for quantitative analysis of experimental findings. This year we had extended our studies of entropic effects in transport problems. Whenever transport of particles takes place in the presence of three-dimensional non-uniform confining geometries, the description of the particle dynamics in terms of the one-dimensional Smoluchowski equation effectively introduces entropy potentials which account for the variations in confinement geometry along particles path. Such structures with varying confinement are ubiquitous in both technology and biology. Ion channels - protein structures embedded in cell and organelle membranes - offer a class of probably the smallest structures where the main concepts of the confined diffusion are still applicable. One of our studies deals with the range of validity of the reduction of axial diffusion in two-dimensional channels to the effective one-dimensional description in terms of the modified Fick-Jacobs equation. We demonstrated that such a reduction is applicable when the channel width variation rate does not exceed unity. In another study we analyzed diffusion of point particles in linearly corrugated two-dimensional channels. Such geometry allowed us to obtain an approximate analytical expression that gives the particle effective diffusivity as a function of the geometric parameters of the channel. To establish its accuracy and the range of applicability, the expression was tested against Brownian dynamics simulation results. The tests showed that the expression works very well for long channel periods, but fails when the period is not long enough compared to the minimum width of the channel. To fix this deficiency, we proposed a simple empirical correction to the analytical expression the structure wavelength correction. The resulting expression for the effective diffusivity proved to be in excellent agreement with the simulation results for all values of the channel period.