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Bor1p is a secondary transporter in yeast that is responsible for boron transport. Bor1p belongs to the SLC4 family which controls bicarbonate exchange and pH regulation in animals as well as borate uptake in plants. The SLC4 family is more distantly related to members of the Amino acid‐Polyamine‐organoCation (APC) superfamily, which includes well studied transporters such as LeuT, Mhp1, AdiC, vSGLT, UraA, SLC26Dg. Their mechanism generally involves relative movements of two domains: a core domain that binds substrate and a gate domain that in many cases mediates dimerization. To shed light on conformational changes governing transport by the SLC4 family, we grew helical membrane crystals of Bor1p from Saccharomyces mikatae and determined a structure at ∼6 Å resolution using cryo‐electron microscopy. To evaluate the conformation of Bor1p in these crystals, a homology model was built based on the related anion exchanger from red blood cells (AE1). This homology model was fitted to the cryo‐EM density map using the Molecular Dynamics (MD) Flexible Fitting method and then relaxed by all‐atom MD simulation in explicit solvent and membrane. Mapping of water accessibility indicates that the resulting structure represents an inward‐facing conformation. Comparisons of the resulting Bor1p model with the X‐ray structure of AE1 in an outward‐facing conformation, together with MD simulations of inward‐facing and outward‐facing Bor1p models, suggest rigid body movements of the core domain relative to the gate domain. These movements are consistent with the rocking‐bundle transport mechanism described for other members of the APC superfamily.

The cellular translocon, present in all three domains of life, is one of the most versatile and important biological nanopores. This complex molecular apparatus is directly responsible for the secretion of globular proteins across membranes as well as the insertion of integral membrane proteins into lipid bilayers. Recently determined structures of the archaean SecY translocon reveal an hour-glass-shaped pore, which accommodates the nascent peptide chain during translocation. While these structures provide important insights into ribosome binding to the translocon, threading of the nascent chain into the channel, and lateral gate opening for releasing the folded helical peptide into the membrane bilayer, the exact folding pathway of the peptide inside the protein-conducting channel during translocation and prior to the lateral release into the bilayer remains elusive. In the present study, we use molecular dynamics simulations to investigate atomic resolution peptide folding in hour-glass-shaped pore models that are based on the SecY translocon channel structure. The theoretical setup allows systematic variation of key determinants of folding, in particular the degree of confinement of the peptide and the hydration level of the pore. A 27-residue hydrophobic peptide was studied that is preferentially inserted into membranes by the translocon. Our results show that both pore diameter as well as channel hydration are important determinants for folding efficiency and helical stability of the peptide, therefore providing important insights into translocon gating and lateral peptide partitioning.

Secondary transporters utilize the free energy stored in the sodium gradient to move a solute against a concentration gradient. The transport process involves a sequence of conformational changes that exposes the substrate and ion binding sites alternatively to the extracellular and the intracellular compartment. At least three conformational states are required for this “alternating access model”: An outward facing open state, connecting ion and substrate binding site to the outside; an occluded state where ion and substrate are buried inside the protein; and an inward facing open state that allows egress of substrate into the cytosol. We recently published a study of these three crystallographically defined states in the prokaryotic nucleobase:sodium symporter Mhp1 [1], which has a similar fold to many other transporters such as LeuT, vSGLT or BetB. We showed that alternating access can be understood as a sequence of multiple gating events. Here we present long, micro-second molecular dynamics simulations that further explore the conformational space available to the gating elements. They reveal that the two “thin gates”, formed by the ends of helices TM5 and TM10 move on the 100 ns time scale. The state of the thin gates is coupled to the “thick gate” (formed by a 30 degree rotation of the “hash” domain relative to the “bundle”). The extracellular thin gate can only open if the thick gate is in the outward facing conformation, a synchronization necessary for the alternating access model. The protein also transitions spontaneously into a fourth “inward-facing occluded” state. Comparison to simulations of LeuT and vSGLT indicates that the gating elements in those transporters can function in similar manner to Mhp1. Our results suggest a structural model of the transport cycle in sodium-coupled symporters.[1] T. Shimamura et al., Science, 328 (2010) 470-473.

Na+/H+ antiporters are vital for maintaining homeostasis in bacterial cells, in particular for survival in high-salt environments. In humans, these transporters are important drug targets, because their dysfunction is linked to a variety of diseases, including cancer and cardiovascular pathophysiology. However, to date the molecular mechanism by which these proteins transport ions remains poorly understood. We recently solved the structure of the archaeal transporter Thermus Thermophilus NapA in an outward-facing conformation where the putative ion binding site is exposed to the cellular exterior [1]. Using disulfide links to trap the protein, we have now obtained an inward-facing structure of NapA, revealing a large conformational change consistent with the alternating access mechanism long proposed for these transporters. We performed molecular dynamics (MD) simulations demonstrating the disulfide-linked structure to be stable in the absence of the links. Simulations of both inward- and outward-facing conformations show that the dimerization domain anchors the protein in the membrane while the core domain translates the binding site 6-7 A in an elevator-like mechanism. Na+ binding is also dependent on protonation states of the conserved aspartates and lysine for both conformations. This binding is consistent with the hypothesis that the conserved lysine plays a direct role in binding protons in Na+/H+ antiporters that transport 2 H+ [2]. Taken together, structural and simulation data for two distinct conformations of the same transporter give an unambiguous view into the molecular mechanism underlying Na+/H+ antiport.[1] Lee et. al. Nature, 501:573-577, 09 2013. http://dx.doi.org/10.1038/nature12484.[2] Lee et. al. J Gen Physiol, 144:529-544, 2014. http://dx.doi.org/10.1085/jgp.201411219.

To fully understand the transport mechanism of Na(+)/H(+) exchangers, it is necessary to clearly establish the global rearrangements required to facilitate ion translocation. Currently, two different transport models have been proposed. Some reports have suggested that structural isomerization is achieved through large elevator-like rearrangements similar to those seen in the structurally unrelated sodium-coupled glutamate-transporter homolog GltPh. Others have proposed that only small domain movements are required for ion exchange, and a conventional rocking-bundle model has been proposed instead. Here, to resolve these differences, we report atomic-resolution structures of the same Na(+)/H(+) antiporter (NapA from Thermus thermophilus) in both outward- and inward-facing conformations. These data combined with cross-linking, molecular dynamics simulations and isothermal calorimetry suggest that Na(+)/H(+) antiporters provide alternating access to the ion-binding site by using elevator-like structural transitions.

Boron is an essential micronutrient for plants and animals. Transport of boron regulates uptake and protects against high levels of boron in plants and yeast. The yeast boron transporter Bor1p belongs to the SLC4 Anion Exchanger family of transporters that also includes the human chloride/bicarbonate exchanger. We have overexpressed and purified the Bor1p orthologue from Saccharomyces mikatae using Saccharomyces cerevisiae as a host. Helical crystals were produced by reconstituting the transporter together with cardiolipin and electron micrographs of frozen-hydrated tubes were recorded with a field emission gun at 200 kV using a direct electron detector. Crystals adopted two different helical symmetries from which two independent reconstructions were produced from 75 particles at a resolution of ∼7 A using a Fourier-Bessel approach. In order to interpret the dimeric densities revealed by the reconstructions, we built a homology model for Bor1p based on the crystal structure of the uracil transporter (UraA), which is the closest relative to Anion Exchangers within the APC superfamily of transporters. Initial homology models were produced from 10 alternative alignments of these distantly related proteins. The alignments were refined by mapping sequence conservation onto these 3D structures. The best model was then fitted to the cryo-EM map by adjusting the position of individual helices and the resulting structure was equilibrated in a lipid bilayer for 200 ns using molecular dynamics. The resulting structure suggests conformational changes relative to UraA in which helices at the dimer interface are tilted relative to the transporter domain, thus providing access to the substrate binding site from the extracellular side of the membrane. Comparison of our Bor1p structure with that from UraA are likely to reflect the structural changes that accompany the alternating access mechanism employed by this family of transporters.

Sodium/proton (Na+/H+) antiporters, located at the plasma membrane in every cell, are vital for cell homeostasis. In humans, their dysfunction has been linked to diseases, such as hypertension, heart failure and epilepsy, and they are well-established drug targets. The best understood model system for Na+/H+ antiport is NhaA from Escherichia coli, for which both electron microscopy and crystal structures are available. NhaA is made up of two distinct domains: a core domain and a dimerization domain. In the NhaA crystal structure a cavity is located between the two domains, providing access to the ion-binding site from the inward-facing surface of the protein. Like many Na+/H+ antiporters, the activity of NhaA is regulated by pH, only becoming active above pH 6.5, at which point a conformational change is thought to occur. The only reported NhaA crystal structure so far is of the low pH inactivated form. Here we describe the active-state structure of a Na+/H+ antiporter, NapA from Thermus thermophilus, at 3 Å resolution, solved from crystals grown at pH 7.8. In the NapA structure, the core and dimerization domains are in different positions to those seen in NhaA, and a negatively charged cavity has now opened to the outside. The extracellular cavity allows access to a strictly conserved aspartate residue thought to coordinate ion binding directly, a role supported here by molecular dynamics simulations. To alternate access to this ion-binding site, however, requires a surprisingly large rotation of the core domain, some 200against the dimerization interface. We conclude that despite their fast transport rates of up to 1,500 ions per second, Na+/H+ antiporters operate by a two-domain rocking bundle model, revealing themes relevant to secondary-active transporters in general.

Fusion of neurosecretory vesicles with the plasma membrane is mediated by SNARE proteins, which transfer a force to the membranes. However, the mechanism by which this force transfer induces fusion pore formation is still unknown. The neuronal vesicular SNARE protein synaptobrevin 2 (syb2) is anchored in the vesicle membrane by a single C terminal transmembrane (TM) helix. In coarse grain molecular dynamics simulations self-assembly of the membrane occurred with the syb2 TM helix inserted as expected from experimental data. The free energy profile for the position of the TM domain in the membrane was determined applying harmonic potentials to the peptide in its unbiased position, pulling it towards new biased equilibrium positions. The energy profile determined in this way predicts the energy landscapes for pulling syb2 towards the extravesicular side as expected for SNARE complex zippering. Applying a constant pulling force of 160 pN detaches the synaptobrevin C terminus from the vesicle's inner leaflet lipid head groups within ∼100ns and pulls the C terminus deeper into the membrane. This C terminal movement should occur on the physiological millisecond time scale at ∼120 pN force. It is facilitated and hindered by specific mutations in parallel with experimentally observed facilitation and inhibition of fusion. These results suggest a mechanism where fusion pore formation is induced by movement of the charged syb2 C terminus into the hydrophobic core of the membrane in response to the force generated by C terminal zippering of the SNARE complex. This displacement of the charged C terminus is expected to destabilize the membrane providing a plausible pathway to fusion pore formation. Supported by NIH grants R01GM085808, R21NS072577, EDICT Project grant 201924, an MRC fellowship and the Wellcome Trust.

Fusion of neurosecretory vesicles with the plasma membrane is mediated by SNARE proteins, which transfer a force to the membranes. However, the mechanism by which this force transfer induces fusion pore formation is still unknown. The neuronal vesicular SNARE protein synaptobrevin 2 (syb2) is anchored in the vesicle membrane by a single C-terminal trans- membrane (TM) helix. In coarse-grain molecular-dynamics simulations, self-assembly of the membrane occurred with the syb2 TM domain inserted, as expected from experimental data. The free-energy profile for the position of the syb2 membrane anchor in the membrane was determined using umbrella sampling. To predict the free-energy landscapes for a reaction pathway pulling syb2 toward the extravesicular side of the membrane, which is the direction of the force transfer from the SNARE complex, harmonic potentials were applied to the peptide in its unbiased position, pulling it toward new biased equilibrium positions. Appli- cation of piconewton forces to the extravesicular end of the TM helix in the simulation detached the synaptobrevin C-terminus from the vesicle's inner-leaflet lipid headgroups and pulled it deeper into the membrane. This C-terminal movement was facil- itated and hindered by specific mutations in parallel with experimentally observed facilitation and inhibition of fusion. Direct appli- cation of such forces to the intravesicular end of the TM domain resulted in tilting motion of the TM domain through the membrane with an activation energy of ~70 kJ/mol. The results suggest a mechanism whereby fusion pore formation is induced by movement of the charged syb2 C-terminus within the membrane in response to pulling and tilting forces generated by C-terminal zippering of the SNARE complex.

The structure of a homopentameric alpha7 nicotinic acetylcholine receptor is modelled by combining structural information from two sources: the X-ray structure of a water soluble acetylcholine binding protein from Lymnea stagnalis, and the electron microscopy derived structure of the transmembrane domain of the Torpedo nicotinic receptor. The alpha7 nicotinic receptor model is generated by simultaneously optimising: (i) chain connectivity, (ii) avoidance of stereochemically unfavourable contacts, and (iii) contact between the beta1-beta2 and M2-M3 loops that have been suggested to be involved in transmission of conformational change between the extracellular and transmembrane domains. A Gaussian network model was used to predict patterns of residue mobility in the alpha7 model. The results of these calculations suggested a flexibility gradient along the transmembrane domain, with the extracellular end of the domain more flexible that the intracellular end. Poisson-Boltzmann (PB) energy calculations and atomistic (molecular dynamics) simulations were used to estimate the free energy profile of a Na+ ion as a function of position along the axis of the pore-lining M2 helix bundle of the transmembrane domain. Both types of calculation suggested a significant energy barrier to exist in the centre of the (closed) pore, consistent with a "hydrophobic gating" model. Estimations of the PB energy profile as a function of ionic strength suggest a role of the extracellular domain in determining the cation selectivity of the alpha7 nicotinic receptor. These studies illustrate how molecular models of members of the nicotinic receptor superfamily of channels may be used to study structure-function relationships.

The transmembrane protein NhaA from Escherichia coli is a prototypical sodium/proton antiporter. It enables the bacterium to grow under high salt conditions while homologous proteins in eukaryotes are involved in pH and cell volume regulation. A number of acidic and basic residues have been shown to be essential for the transport of one sodium ion for two protons but the mechanistic details of their involvement have not been fully determined. Furthermore, the conformational changes involved in the transport mechanism were not known. We present an unpublished crystal structure of NhaA in the inward facing conformation and of the homolog NapA in the outward facing conformation [1]. Using modelling and computer simulations we show how NhaA can function according to the alternating access model, resulting in large relative domain motions that are incompatible with previous structural models for transport in NhaA. Our structure of NhaA contains a salt bridge between the two conserved residues Asp163 and Lys300. With the help of molecular dynamics simulations we critically examine competing models for the molecular mechanism of the stoichiometric transport of two protons for one sodium ion, including one in which Lys300 maintains an active role in proton transport.[1] Lee et al, Nature 501 (2013), 573.

Water-filled pores of nanometer dimensions play important roles in chemistry and biology, e.g., as channels through biological membranes. Biological nanopores are frequently gated, i.e., they switch between an open and a closed state. In several ion channel structures the gate is formed by a ring of hydrophobic side chains that do not physically occlude the pore. Here we investigate whether a hydrophobic pore can act as a gate via molecular dynamics simulations of the passage of water through atomistic models of nanopores embedded within a membrane mimetic. Both the geometry of a nanopore and the hydrophilicity vs hydrophobicity of its lining determine whether water enters the channel. For purely hydrophobic pores there is an abrupt transition from a closed state (no water in the pore cavity) to an open state (cavity water at approximately bulk density) once a critical pore radius is exceeded. This critical radius depends on the length of the pore and the radius of the mouth region. Furthermore, a closed hydrophobic nanopore can be opened by adding dipoles to its lining.

In living cells specialized water channels, the glycero-aquaporins, are responsible for rapid passive transport of water through the membrane. Crystal structures showed that these protein pores have diameters comparable to the size of a single water molecule (around 0.3 nm) and lengths around 2 to 3 nm. Despite being strongly confined, water moves rapidly through these and other narrow peptide pores such as gramicidin. Understanding how water behaves in confinement has not been easy, not the least because there is no experimental technique available to unambiguously measure water flux through a single water pore that would be comparable to single channel patch clamp recordings on ion channels. Thus, if we want to understand how a physiological function such as the water flux derives from the structure, namely the geometry and chemical character of the water pathway, we can do this only with models that relate macroscopic transport coefficients to microscopic properties of the pore. Although there is no satisfactory microscopic theory for water transport across all the pore dimensions relevant for biology (1,2) it is clear that the single most important parameter for water permeation is the radius of the pore. Biological, water-permeable pores have radii ranging from 0.14 nm (gramicidin A, K+-channel selectivity filter) through ∼0.8 nm (ligand gated ion channels in the open state) to around 10 nm (the open region of the nuclear pore complex) (3). Transport through pores that are wide compared to the typical interaction and correlation lengths in liquid water (≫ 1 nm or more than ∼4 water diameters) can be described well by continuum theories. There is also progress being made on narrow pores that can only accommodate a 1D chain of water molecules; here the important simplification is that water molecules cannot pass each other. However, as soon as we enter the “multi-pass” regime of R > 2rwater ≈ 0.28 nm no theories are available. The molecular dynamics simulations by Portella and de Groot (4) in this issue hint at what a new theory for the transition from the no-pass to the multi-pass regime could look like. In these simulations water can move freely through “toy model” pores of variable radius, length, and surface character. The pores are based on the architecture of the gramicidin A channel and embedded in a membrane mimetic. Computer simulations afford full control over all parameters of the simulation, including many at the atomic level that are not accessible to experimentalists in the real world. The authors use this to good effect; for instance, the degree of hydrophilicity of a pore is adjusted by simply editing the backbone dipole moment. The simulations measure the two experimentally accessible permeation coefficients that characterize water transport. The osmotic permeability coefficient pf expresses the observation that osmotic water flux Jw = pfΔcsolute is driven by a concentration difference of an impermeable solute between two compartments separated by a semipermeable membrane. For single-file pores, pf was shown to be proportional to the rate of translocation of the whole n-water chain by one hop. From a physiological point of view such a collective hop is the important observable: one water molecule enters the pore on one side, another one exits on the other and the net effect is the movement of one water molecule across the membrane. The second observable is the diffusive permeability coefficient pd. It is proportional to the number of translocation events of individual water molecules through the pore per unit time and thus primarily characterizes the pore-water interaction. It can be measured by following dilute isotope-tagged water molecules in solution that interact in the same way as the untagged ones. Because it takes n + 1 individual hops along the chain to achieve an individual translocation event there exists a relationship between the measurable permeabilities and the number of water molecules n in the single file chain, pfpd=n+1. (1) Equation 1 has been used successfully to determine the channel length of single-file pores such as gramicidin A from osmotic measurements (2); the length is simply calculated as n times the average water-water distance. However, the theoretical value of the pf/pd ratio for wider molecular pores than single file ones has not been established unequivocally and Alan Finkelstein's remark from 1987 is still true: “There is presently no satisfactory physical theory for diffusion and flow through pores with radii only a few times larger than that of the solvent molecule.” (2) One of the many intriguing findings of Portella and de Groot (4) is that a more general, although (at the moment) phenomenological, relation, pfpd=q(R)(〈n〉+1), (2) seems to hold for any water filled molecular pore (at least up to a radius of ∼0.6 nm). Here q(R) is the number of effective single file columns that fit into the pore of radius R; for a true single file pore q(R) = 1 holds. Equation 2 relaxes two important constraints that were required to derive Eq. 1: The “no-pass” and the “no-vacancy” condition. It seems that we can interpret flow through wider pores as consisting of effective 1D chains of water molecules. Second, by identifying the ratio of the permeabilities with the average occupancy of the single file it also becomes possible to apply permeability measurements in a meaningful way to hydrophobic pores that may only be partially occupied. Computer simulations indicated that sufficiently hydrophobic pores would be, depending on the degree of hydrophobicity, filled with water vapor, or fluctuate between liquid filled and vapor filled states. Although plausible on physical grounds—liquid water at ambient conditions is close to its liquid-vapor phase coexistence (5) and thus any influence such as a surface or a change in chemical potential may tilt the balance toward the vapor state—it is not clear that such liquid-vapor oscillations actually exist in nature. This work, however, suggests how to experimentally measure such behavior. By measuring pf/pd − 1 for a narrow pore one determines the average pore occupancy; choosing R so small that q = 1 simplifies the considerations. Because the liquid-vapor equilibrium is easily shifted in either direction by a change in pressure or temperature (6) one should only have to repeat the measurements under different conditions and look for a sudden change in 〈n(T, P) 〉. It should, however, also be kept in mind that simulations ultimately need to find justification in experiments (and experiments need a model, based on theory and simulation). Before Eq. 2 can be really used to interpret measurements it will require some additional experimental validation. This may be quite a formidable task that needs to be addressed by theoreticians and experimentalists together, similar to the ongoing attempts to explain the dependence of pf on the pore length from experiments (7) and simulations (8). Even so, the work of Portella and de Groot should already inspire the formulation of new theories and clarify the meaning of the experimental observables. For instance, together with the insight that water permeation can be described by a simple collective coordinate (9) it lets us qualitatively interpret pf and pd in a slightly different light: pd is related to the probability that a distinguishable water molecule completes a translocation whereas pf measures the translocation of indistinguishable particles. It seems that we may be able to learn more about the microscopic water structure in a pore from macroscopic measurements than thought previously.

The transmembrane protein NhaA from Escherichia coli is a prototypical sodium/proton antiporter. It enables the bacterium to grow under high salt conditions while homologous proteins in eukaryotes are involved in pH and cell volume regulation. A number of acidic and basic residues have been shown to be essential for the transport of one sodium ion for two protons but the mechanistic details of their involvement have not been fully determined. In particular, a highly conserved lysine residue (Lys300) near the center of the membrane had so far been only given a possible indirect role in the transport mechanism. We present a new atomic resolution structure of the inward facing conformation that shows a novel salt bridge between Lys300 and the conserved Asp163. Microsecond molecular dynamics simulations indicate that the salt bridge is sensitive to the presence of a sodium ion that spontaneously binds to the conserved aspartate residue 164. The simulations show how binding of sodium ion can be coupled to a structural change which might trigger a conformational change to an outward facing conformation. Taken together, the structural and simulation data generate a new hypothesis for how Lys300 could be directly involved in proton transport.

The outer membrane protein OprP from Pseudomonas aeruginosa forms an anion-selective pore, especially selective for phosphate ions. The protein is homo-trimeric, with each pore lined by three positively charged loops (L3, L5, and T7) folded into its lumen. OprP plays a key role in high-affinity phosphate uptake under the condition of phosphate starvation. To better understand the mechanism of phosphate-selective permeation, we employed three simulation techniques: (i) equilibrium molecular dynamics simulations (MD); (ii) steered MD (SMD); (iii) umbrella sampling to calculate a potential of mean force (PMF) for phosphate and chloride ions. The PMFs reveal a deep energy well midway along the OprP channel. Two adjacent phosphate-binding sites (W1 and W2), each with a well depth of ∼8kT, are identified close to the L3 loop in the most constricted region of the pore. The transfer of phosphate between sites W1 and W2 is correlated with changes in conformation of the sidechain of K121, which serves as a ‘charged brush’ to facilitate phosphate passage between the two subsites. The PMF for chloride has also been computed and can be compared with that of phosphate. Our simulations suggest that OprP does not conform to the conventional picture of a channel with a relatively flat energy landscape for permeant ions, but rather resembles a membrane-inserted binding protein with a high specificity that allows access to a centrally located binding site from both the extracellular and the periplasmic spaces.

Many proteins function as dynamic molecular machines that cycle between well-defined states. A mechanistic and atomic-scale understanding starts with crystal, NMR or electron microscopy structures in these states. Typically, none or only very limited structural information is available for the intermediates along the transition. Computational methods can simulate transitions between states but due to the absence of intermediate structures it is hard to verify that the simulated transition path is correct. One exception is the enzyme adenylate kinase. It is well studied and a large number of crystal structures are available. Vonrhein et al [1] suggested early on that some of these structures would be transition intermediates due to stabilization by crystal contacts and created a `movie' from nine structures. We took this idea one step further and compare 45 experimental structures to hundreds of transitions of E. coli AdK simulated with the dynamic importance sampling method (DIMS). We find that DIMS trajectories, which only require a crystal structure for the starting and the end point of the transitions, contain all intermediate crystal structures (RMSD for matches

Proteins exist in aqueous solution. Hydration can be viewed as a description of how the protein disturbs the structure and dynamics of water. Water molecules in the vicinity of proteins are generally seen as either external or internal water molecules. Internal water molecules occupy cavities, exchange on a time-scale of 0.1--10 microseconds with bulk water, are almost as conserved as amino acids, and are therefore likely to be important for function. External water molecules tend to be found in protein crevices and are typically not conserved, even between crystal structures of the same protein. We introduce a method to analyze the behavior of water molecules in molecular dynamics (MD) simulations in terms of graphs. The graph encodes a simple hopping model: Nodes in the graph correspond to hydration sites, typically defined from the density in computer simulations or observed water sites in crystal structures. Directed edges correspond to transitions (“hops”) between sites, with transition rates computed from MD simulations. We apply this analysis to the water-filled cavity of intestinal fatty acid binding protein (I-FABP) in its apo and holo (palmitate-bound) state. This demonstrates how ligand binding influences the well-defined set of hydration sites in and around the protein's cavity. The ligand displaces a number of hydration sites but does not affect others close by. The parameters extracted from the network model allow us to model the movement of water molecules with a Markov Chain Monte Carlo model. The graphical construct reproduces the average site occupancy found in the MD simulations and the fluctuations of the occupancy.This approach suggests new types of sampling and analysis that can be applied to extend the range of molecular dynamics models and the role of water in ligand binding.

Accurate propagation of genetic material is essential for life. Yet, DNA is constantly being bombarded by various external factors resulting in millions of lesions per day. Lesions include modified bases, backbone nicks, and single stranded gaps. The presence of such lesions could lead to differences in the conformational dynamics of DNA, suggesting a potential mechanism for DNA repair enzymes to locate damages. To investigate how the presence of lesions affects the conformational dynamics of DNA, Forster resonance energy transfer (FRET) was used. The DNA samples contained internally modified Cy3/Cy5 dyes. Each DNA sample was designed to have a different lesion such as a nick or abasic site. Higher FRET efficiencies were expected if the DNA was more dynamic. However, only small differences in the FRET efficiency were found. Therefore, time-resolved anisotropy was used in combination with FRET, where faster acceptor emission depolarization is indicative of more dynamic DNA. To gain an understanding of the atomistic details, molecular dynamics (MD) simulations were performed and MD trajectories analyzed in terms of global and local structural quantities such as bending angle and helicoidal parameters. Both experiments and simulations showed that nicked DNA behaves similarly to intact DNA. However, the introduction of a single stranded gap of nucleotides increases the dynamic behavior of the DNA. While a nick alone did not change the conformational dynamics of the DNA, the introduction of a mismatch base or abasic site increased conformational dynamics by weakening the hydrogen bonding and stacking interactions around the nick. The combination of experimental and computational results suggests that the disruption of local structural stability leads to global conformational changes.