Chemical Shift Tensors of Cimetidine Form A Modeled with Density Functional Theory Calculations: Implications for NMR Crystallography.

The principal components of the 13C chemical shift tensors for the ten crystallographically distinct carbon atoms of the active pharmaceutical ingredient cimetidine Form A have been measured using the FIREMAT technique. Density functional theory (DFT) calculations of 13C and 15N magnetic shielding tensors are used to assign the 13C and 15N peaks. DFT calculations were performed on cimetidine and a training set of organic crystals using both plane-wave and cluster-based approaches. The former set of calculations allowed several structural refinement strategies to be employed, including calculations utilizing a dispersion-corrected force field that was parametrized using 13C and 15N magnetic shielding tensors. The latter set of calculations featured the use of resource-intensive hybrid-DFT methods for the calculation of magnetic shielding tensors. Calculations on structures refined using the new force-field correction result in improved values of 15N magnetic shielding tensors (as gauged by agreement with experimental chemical shift tensors), although little improvement is seen in the prediction of 13C shielding tensors. Calculations of 13C and 15N magnetic shielding tensors using hybrid functionals show better agreement with experimental values in comparison to those using GGA functionals, independent of the method of structural refinement; the shielding of carbon atoms bonded to nitrogen are especially improved using hybrid DFT methods.

A combined computational and experimental approach reveals the structure of a C/EBPß-Spi1 interaction required for IL1B gene transcription.

We previously reported that transcription of the human IL1B gene, encoding the proinflammatory cytokine interleukin 1ß, depends on long-distance chromatin looping that is stabilized by a mutual interaction between the DNA-binding domains (DBDs) of two transcription factors: Spi1 proto-oncogene at the promoter and CCAAT enhancer-binding protein (C/EBPß) at a far-upstream enhancer. We have also reported that the C-terminal tail sequence beyond the C/EBPß leucine zipper is critical for its association with Spi1 via an exposed residue (Arg-232) located within a pocket at one end of the Spi1 DNA-recognition helix. Here, combining in vitro interaction studies with computational docking and molecular dynamics of existing X-ray structures for the Spi1 and C/EBPß DBDs, along with the C/EBPß C-terminal tail sequence, we found that the tail sequence is intimately associated with Arg-232 of Spi1. The Arg-232 pocket was computationally screened for small-molecule binding aimed at IL1B transcription inhibition, yielding l-arginine, a known anti-inflammatory amino acid, revealing a potential for disrupting the C/EBPß-Spi1 interaction. As evaluated by ChIP, cultured lipopolysaccharide (LPS)-activated THP-1 cells incubated with l-arginine had significantly decreased IL1B transcription and reduced C/EBPß's association with Spi1 on the IL1B promoter. No significant change was observed in direct binding of either Spi1 or C/EBPß to cognate DNA and in transcription of the C/EBPß-dependent IL6 gene in the same cells. These results support the notion that disordered sequences extending from a leucine zipper can mediate protein-protein interactions and can serve as druggable targets for regulating gene promoter activity.

Dopamine Transporter Dynamics of N-Substituted Benztropine Analogs with Atypical Behavioral Effects.

Atypical dopamine transporter (DAT) inhibitors, despite high DAT affinity, do not produce the psychomotor stimulant and abuse profile of standard DAT inhibitors such as cocaine. Proposed contributing features for those differences include off-target actions, slow onsets of action, and ligand bias regarding DAT conformation. Several 3α-(4',4''-difluoro-diphenylmethoxy)tropanes were examined, including those with the following substitutions: N-(indole-3''-ethyl)- (GA1-69), N-(R)-2''-amino-3''-methyl-n-butyl- (GA2-50), N-2''aminoethyl- (GA2-99), and N-(cyclopropylmethyl)- (JHW013). These compounds were previously reported to have rapid onset of behavioral effects and were presently evaluated pharmacologically alone or in combination with cocaine. DAT conformational mode was assessed by substituted-cysteine accessibility and molecular dynamics (MD) simulations. As determined by substituted-cysteine alkylation, all BZT analogs except GA2-99 showed bias for a cytoplasmic-facing DAT conformation, whereas cocaine stabilized the extracellular-facing conformation. MD simulations suggested that several analog-DAT complexes formed stable R85-D476 "outer gate" bonds that close the DAT to extracellular space. GA2-99 diverged from this pattern, yet had effects similar to those of other atypical DAT inhibitors. Apparent DAT association rates of the BZT analogs in vivo were slower than that for cocaine. None of the compounds was self-administered or stimulated locomotion, and each blocked those effects of cocaine. The present findings provide more detail on ligand-induced DAT conformations and indicate that aspects of DAT conformation other than "open" versus "closed" may facilitate predictions of the actions of DAT inhibitors and may promote rational design of potential treatments for psychomotor-stimulant abuse.

2-Substituted 3ß-Aryltropane Cocaine Analogs Produce Atypical Effects without Inducing Inward-Facing Dopamine Transporter Conformations.

Previous structure-activity relationship studies indicate that a series of cocaine analogs, 3ß-aryltropanes with 2ß-diarylmethoxy substituents, selectively bind to the dopamine transporter (DAT) with nanomolar affinities that are 10-fold greater than the affinities of their corresponding 2α-enantiomers. The present study compared these compounds to cocaine with respect to locomotor effects in mice, and assessed their ability to substitute for cocaine (10 mg/kg, i.p.) in rats trained to discriminate cocaine from saline. Despite nanomolar DAT affinity, only the 2ß-Ph2COCH2-3ß-4-Cl-Ph analog fully substituted for cocaine-like discriminative effects. Whereas all of the 2ß compounds increased locomotion, only the 2ß-(4-ClPh)PhCOCH2-3ß-4-Cl-Ph analog had cocaine-like efficacy. None of the 2α-substituted compounds produced either of these cocaine-like effects. To explore the molecular mechanisms of these drugs, their effects on DAT conformation were probed using a cysteine-accessibility assay. Previous reports indicate that cocaine binds with substantially higher affinity to the DAT in its outward (extracellular)- compared with inward-facing conformation, whereas atypical DAT inhibitors, such as benztropine, have greater similarity in affinity to these conformations, and this is postulated to explain their divergent behavioral effects. All of the 2ß- and 2α-substituted compounds tested altered cysteine accessibility of DAT in a manner similar to cocaine. Furthermore, molecular dynamics of in silico inhibitor-DAT complexes suggested that the 2-substituted compounds reach equilibrium in the binding pocket in a cocaine-like fashion. These behavioral, biochemical, and computational results show that aryltropane analogs can bind to the DAT and stabilize outward-facing DAT conformations like cocaine, yet produce effects that differ from those of cocaine.

Biological Testing of Organophosphorus-Inactivated Acetylcholinesterase Oxime Reactivators Identified via Virtual Screening.

There is a pressing need for new therapeutics to reactivate covalently inactivated acetylcholinesterase (AChE) due to exposure to organophosphorus (OP) compounds. Current reactivation therapeutics (RTs) are not broad-spectrum and suffer from other liabilities, specifically the inability to cross the blood-brain-barrier. Additionally, the chemical diversity of available therapeutics is small, limiting opportunities for structure-activity relationship (SAR) studies to aid in the design of more effective compounds. In order to find new starting points for the development of oxime-containing therapeutic reactivators and to increase our base of knowledge, we have employed a combination of computational and experimental procedures to identify additional compounds with the real or potential ability to reactivate AChE while augmenting and complementing current knowledge. Computational methods were used to identify previously uninvestigated oxime-containing molecules. Experimentally, six compounds were found with reactivation capabilities comparable to, or exceeding, those of 2-pralidoxime (2-PAM) against a panel of AChE inactivated by paraoxon, diisopropylfluorophosphate (DFP), fenamiphos, and methamidophos. One compound showed enhanced reactivation ability against DFP and fenamiphos, the least tractable of these OPs to be reactivated.

Structural dynamics of the monoamine transporter homolog LeuT from accelerated conformational sampling and channel analysis.

The bacterial leucine transporter LeuT retains significant secondary structure similarities to the human monoamine transporters (MAT) such as the dopamine and serotonin reuptake proteins. The primary method of computational study of the MATs has been through the use of the crystallized LeuT structure. Different conformations of LeuT can give insight into mechanistic details of the MAT family. A conformational sampling performed through accelerated molecular dynamics simulations testing different combinations of the leucine substrate and bound sodium ions revealed seven distinct conformational clusters. Further analysis has been performed to target salt-bridge residues R30-D404, Y108-F253, and R5-D369 and transmembrane domains on both the seven isolated structures and the total trajectories. In addition, solvent accessibility of LeuT and its substrate binding pockets has been analyzed using a program for calculating channel radii. Occupation of the Na2 site stabilizes the outward conformation and should bind to the open outward conformation before the leucine and Na1 sodium while two possible pathways were found to be available for intracellular transport.

Exploring the physicochemical properties of oxime-reactivation therapeutics for cyclosarin, sarin, tabun, and VX inactivated acetylcholinesterase.

The inactivation of acetylcholinesterase (AChE) by organophosphorus agent (OP) compounds is a serious problem regardless of how the individual was exposed. The reactivation of OP-inactivated AChE is dependent on the OP conjugate, and commonly a specific oxime is better at reactivating a specific OP conjugate than several diverse OP conjugates. The presented research explores the physicochemical properties needed for the reactivation of OP-inactivated AChE. Four different OPs, cyclosarin, sarin, tabun, and VX, were analyzed using the same set of oxime reactivators. A trial descriptor pool of semiempirical, traditional, and molecular interaction field descriptors was used to construct an ensemble of QSAR models for each OP-conjugate pair. Based on the molecular information and the cross-validation ability, individual QSAR models were selected to be part of an OP-conjugate consensus model. The OP-conjugate specific models provide important insight into the physicochemical properties required to reactivate the OP conjugates of interest. The reactivation of AChE inactivated with either cyclosarin or tabun requires the oxime therapeutic to possess an overall polar-positive surface area. Oxime therapeutics for the reactivation of sarin-inactivated AChE are conformationally dependent while oxime reverse therapeutics for VX require a compact region with a highly hydrophilic region and two positively charged pyridine rings.

ß-Amyloid and neprilysin computational studies identify critical residues implicated in binding specificity.

The zinc metalloprotease neprilysin (NEP) promiscuously degrades small bioactive peptides. NEP is among a select group of metalloenzymes that degrade the amyloid beta-peptide (Aß) in vivo and in situ. Since accumulation of neurotoxic Aß aggregates in the brain appears to be a causative agent in the pathophysiology of Alzheimer's disease (AD), increased clearance of Aß resulting from overexpression of NEP exhibits therapeutic potential for AD. However, higher NEP peptidase activity may be harmful without an increased specificity for Aß over other competing substrates. Crystal structures of NEP-inhibitor complexes and their characterization have highlighted potential amino acid interactions involved in substrate binding and are used as templates to guide our methodology in docking Aß in NEP. Results from protein-ligand docking calculations predict S2' subsite residues Arg 102 and Arg 110 of NEP participate in specific interactions with Aß. These interactions provide insight into developing NEP specificity for Aß.

Global transitions of proteins explored by a multiscale hybrid methodology: application to adenylate kinase.

Efficient and accurate mapping of transition pathways is a challenging problem in allosteric proteins. We propose here a to our knowledge new methodology called collective molecular dynamics (coMD). coMD takes advantage of the collective modes of motions encoded by the fold, simultaneously evaluating the interactions and energetics via a full-atomic MD simulation protocol. The basic approach is to deform the structure collectively along the modes predicted by the anisotropic network model, upon selecting them via a Monte Carlo/Metropolis algorithm from among the complete pool of all accessible modes. Application to adenylate kinase, an allosteric enzyme composed of three domains, CORE, LID, and NMP, shows that both open-to-closed and closed-to-open transitions are readily sampled by coMD, with large-scale motions of the LID dominating. An energy-barrier crossing occurs during the NMP movements. The energy barrier originates from a switch between the salt bridges K136-D118 at the LID-CORE interface and K57-E170 and D33-R156 at the CORE-NMP and LID-NMP interfaces, respectively. Despite its simplicity and computing efficiency, coMD yields ensembles of transition pathways in close accord with detailed full atomic simulations, lending support to its utility as a multiscale hybrid method for efficiently exploring the allosteric transitions of multidomain or multimeric proteins.

Conformational free-energy landscapes for a peptide in saline environments.

The conformations that proteins adopt in solution are a function of both their primary structure and surrounding aqueous environment. Recent experimental and computational work on small peptides, e.g., polyK, polyE, and polyR, have highlighted an interesting and unusual behavior in the presence of aqueous ions such as ClO4⁻, Na⁺, and K⁺. Notwithstanding the aforementioned studies, as of this writing, the nature of the driving force induced by the presence of ions and its role on the conformational stability of peptides remains only partially understood. Molecular-dynamics simulations have been performed on the heptapeptide AEAAAEA in NaCl and KCl solutions at concentrations of 0.5, 1.0, and 2.0 M. Metadynamics in conjunction with a three-dimensional model reaction coordinate was used to sample the conformational space of the peptide. All simulations were run for 2 µs. Free-energy landscapes were computed over the model reaction coordinate for the peptide in each saline assay as well as in the absence of ions. Circular dichroism spectra were also calculated from each trajectory. In the presence of Na⁺ and K⁺ ions, no increase in helicity is observed with respect to the conformation in pure water.

LeuT conformational sampling utilizing accelerated molecular dynamics and principal component analysis.

Monoamine transporters (MATs) function by coupling ion gradients to the transport of dopamine, norepinephrine, or serotonin. Despite their importance in regulating neurotransmission, the exact conformational mechanism by which MATs function remains elusive. To this end, we have performed seven 250 ns accelerated molecular dynamics simulations of the leucine transporter, a model for neurotransmitter MATs. By varying the presence of binding-pocket leucine substrate and sodium ions, we have sampled plausible conformational states representative of the substrate transport cycle. The resulting trajectories were analyzed using principal component analysis of transmembrane helices 1b and 6a. This analysis revealed seven unique structures: two of the obtained conformations are similar to the currently published crystallographic structures, one conformation is similar to a proposed open inward structure, and four conformations represent novel structures of potential importance to the transport cycle. Further analysis reveals that the presence of binding-pocket sodium ions is necessary to stabilize the locked-occluded and open-inward conformations.

Experimentally restrained molecular dynamics simulations for characterizing the open states of cytochrome P450cam.

Residual dipolar couplings (RDCs) were used as restraints in fully solvated molecular dynamics simulations of reduced substrate- and carbonmonoxy-bound cytochrome P450(cam) (CYP101A1), a 414-residue soluble monomeric heme-containing camphor monooxygenase from the soil bacterium Pseudomonas putida. The (1)D(NH) residual dipolar couplings used as restraints were measured in two independent alignment media. A soft annealing protocol was used to heat the starting structures while incorporating the RDC restraints. After production dynamics, structures with the lowest total violation energies for RDC restraints were extracted to identify ensembles of conformers accessible to the enzyme in solution. The simulations result in substrate orientations different from that seen in crystallographic structures and a more open and accessible enzyme active site and largely support previously reported differences between the open and closed states of CYP101A1.

Discovery of novel selective serotonin reuptake inhibitors through development of a protein-based pharmacophore.

The serotonin transporter (SERT), a member of the neurotransmitter sodium symporter (NSS) family, is responsible for the reuptake of serotonin from the synaptic cleft to maintain neurotransmitter homeostasis. SERT is established as an important target in the treatment of anxiety and depression. Because a high-resolution crystal structure is not available, a computational model of SERT was built based upon the X-ray coordinates of the leucine transporter LeuT, a bacterial NSS homologue. The model was used to develop the first SERT structure-based pharmacophore. Virtual screening (VS) of a small molecule structural library using the generated SERT computational model yielded candidate ligands of diverse scaffolds. Pharmacological analysis of the VS hits identified two SERT-selective compounds, potential lead compounds for further SERT-related medication development.

Design, synthesis, and testing of an 6-O-linked series of benzimidazole based inhibitors of CDK5/p25.

Alzheimer's disease (AD) is a progressive neurodegenerative disease resulting in cognitive and behavioral impairment. The two classic pathological hallmarks of AD include extraneuronal deposition of amyloid ß (Aß) and intraneuronal formation of neurofibrillary tangles (NFTs). NFTs contain hyperphosphorylated tau. Tau is the major microtubule-associated protein in neurons and stabilizes microtubules (MTs). Cyclin dependent kinase 5 (CDK5), when activated by the regulatory binding protein p25, phosphorylates tau at a number of proline-directed serine/threonine residues, resulting in formation of phosphorylated tau as paired helical filaments (PHFs) then in subsequent deposition of PHFs as NFTs. Beginning with the structure of Roscovitine, a moderately selective CDK5 inhibitor, we sought to conduct structural modifications to increase inhibitory potency of CDK5 and increase selectivity over a similar enzyme, cyclin dependent kinase 2 (CDK2). The design, synthesis, and testing of a series of 1-isopropyl-4-aminobenzyl-6-ether-linked benzimidazoles is presented.

Sodium perchlorate effects on the helical stability of a mainly alanine peptide.

Sodium perchlorate salt (NaClO(4)) is commonly used as an internal intensity standard in ultraviolet resonance Raman (UVRR) spectroscopy experiments. It is well known that NaClO(4) can have profound effects on peptide stability. The impact of NaClO(4) on protein stability in UVRR experiments has not yet been fully investigated. It is well known from experiment that protein stability is strongly affected by the solution composition (water, salts, osmolytes, etc.). Therefore, it is of the utmost importance to understand the physical basis on which the presence of salts and osmolytes in the solution impact protein structure and stability. The aim of this study is to investigate the effects of NaClO(4), on the helical stability of an alanine peptide in water. Based upon replica-exchange molecular dynamics data, it was found that NaClO(4) solution strongly stabilizes the helical state and that the number of pure helical conformations found at room temperature is greater than in pure water. A thorough investigation of the anion effects on the first and second solvation shells of the peptide, along with the Kirkwood-Buff theory for solutions, allows us to explain the physical mechanisms involved in the observed specific ion effects. A direct mechanism was found in which ClO(4)(-) ions are strongly attracted to the folded backbone.

Molecular dynamics of leucine and dopamine transporter proteins in a model cell membrane lipid bilayer.

The dopamine transporter (DAT) operates via facilitated diffusion, harnessing an inward Na(+) gradient to drive dopamine from the extracellular synaptic cleft to the neuron interior. The DAT is relevant to central nervous system disorders such as Parkinson disease and attention-deficit hyperactivity disorder and is the primary site of action for the abused psychostimulants cocaine and amphetamines. Crystallization of a DAT homolog, the bacterial leucine transporter LeuT, provided the first reliable 3-D DAT template. Here, the LeuT crystal structure and the DAT molecular model have been combined with their respective substrates, leucine and dopamine, in lipid bilayer molecular dynamics simulations toward tracking substrate movement along the protein's substrate/ion permeation pathway. Specifically, movement of residue pairs that comprise the "external gate" was followed as a function of substrate presence. The transmembrane (TM) 1 arginine-TM 10 aspartate strut formed less readily in DAT compared with LeuT, with or without substrate present. For LeuT but not DAT, the addition of substrate enhanced the chances of forming the TM 1-10 bridge. Also, movement of the fourth extracellular loop EL-4 in the presence of substrate was more pronounced for DAT, the EL-4 unwinding to a degree. The overall similarity between the LeuT and DAT molecular dynamics simulations indicated that LeuT was a legitimate model to guide DAT structure-function predictions. There were, nevertheless, differences significant enough to allow for DAT-unique insights, which may include how cocaine, methylphenidate (Ritalin, NIDA Drug Supply, Rockville, MD), and other DAT blockers are not recognized as substrates even though they can access the primary substrate binding pocket. Proteins 2010. (c) 2009 Wiley-Liss, Inc.

Salt dependence of an alpha-helical peptide folding energy landscapes.

We used CD, UV resonance Raman spectroscopy, and molecular dynamics simulation to examine the impact of salts on the conformational equilibria and the Ramachandran Psi angle (un)folding Gibbs free energy landscape coordinate of a mainly polyalanine alpha-helical peptide, AP of sequence AAAAA(AAARA)(3)A. NaClO(4) stabilizes alpha-helical-like conformations more than does NaCl, which stabilizes more than Na(2)SO(4) at identical ionic strengths. This alpha-helix stabilization ordering is the reverse of the Hofmeister series of anions in their ability to disorder water hydrogen bonding. Much of the NaClO(4) alpha-helix stabilization results from ClO(4)(-) association with the AP terminal -NH(3)(+) groups and Arg side chains. ClO(4)(-) stabilizes 3(10)-helix conformations but destabilizes turn conformations. The decreased Cl(-) and SO(4)(2-) AP alpha-helix stabilization probably results from a decreased association with the Arg and terminal -NH(3)(+) groups. Cl(-) is expected to have a smaller binding affinity and thus stabilizes alpha-helical conformations intermediately between NaClO(4) and Na(2)SO(4). Electrostatic screening stabilizes pi-bulge conformations.

Dopamine transporter comparative molecular modeling and binding site prediction using the LeuT(Aa) leucine transporter as a template.

Pharmacological and behavioral studies indicate that binding of cocaine and the amphetamines by the dopamine transporter (DAT) protein is principally responsible for initiating the euphoria and addiction associated with these drugs. The lack of an X-ray crystal structure for the DAT or any other member of the neurotransmitter:sodium symporter (NSS) family has hindered understanding of psychostimulant recognition at the atomic level; structural information has been obtained largely from mutagenesis and biophysical studies. The recent publication of a crystal structure for the bacterial leucine transporter LeuT(Aa), a distantly related NSS family homolog, provides for the first time a template for three-dimensional comparative modeling of NSS proteins. A novel computational modeling approach using the capabilities of the Molecular Operating Environment program MOE 2005.06 in conjunction with other comparative modeling servers generated the LeuT(Aa)-directed DAT model. Probable dopamine and amphetamine binding sites were identified within the DAT model using multiple docking approaches. Binding sites for the substrate ligands (dopamine and amphetamine) overlapped substantially with the analogous region of the LeuT(Aa) crystal structure for the substrate leucine. The docking predictions implicated DAT side chains known to be critical for high affinity ligand binding and suggest novel mutagenesis targets in elucidating discrete substrate and inhibitor binding sites. The DAT model may guide DAT ligand QSAR studies, and rational design of novel DAT-binding therapeutics.

Interaction of the phospholipid head group with representative quartz and aluminosilicate structures: an ab initio study.

Silica dust particles in the form of quartz (but not kaolin) have been hypothesized to promote pulmonary diseases such as silicosis. The hypothesis is that quartz and kaolin have a comparable membranolytic potential on a specific surface area basis, and they have a comparable cytotoxic potential for lavaged pulmonary macrophages. Suppression of the cytotoxic activity occurs when these dust particles are treated with dipalmitoylphosphatidylcholine (DPPC), a common phospholipid component of the lung pulmonary surfactant. However, the enzyme phospholipase A2 is known to digest the phospholipid component more readily in the presence of quartz than kaolin. Since surface silanol (Si-OH) and aluminol (Al-OH) groups may interact differently with the phospholipid, an understanding of the selective removal of phospholipid by PLA2 may explain in vivo differences in cytotoxicity between quartz and kaolin. To develop some insight into this phenomenon, the interaction between a phospholipid and silica particles was examined by performing ab initio DFT calculations on clusters constructed with small (one or two silica tetrahedral units) representative parts of the silicate surface and the phospholipid head group. The clusters consisted of a phospholipid head group or functional groups from the head group complexed with Si(OSiH 3) 3OH, Al(OSiH 3) 3OH (-) or Al(OSiH 3) 3OH 2. Fully optimized geometries of the complexes were used to determine binding energies, -OH vibrational frequency shifts, and NMR chemical shieldings. Results indicate that interaction of the protonated aluminol group (Al-OH 2 (+)) with the phosphate portion of the head group is strongest, while interaction of the -OH 2 (+) group with the trimethyl-choline moiety of the head group is weakest. The presence of the choline moiety increased the magnitude of the -OH vibrational frequency shifts, and the shifts were significantly larger in complexes with protonated aluminol groups relative to silanol complexes. Analysis of ChelpG atomic charges shows that a net transfer of charge occurs from the silica unit to the head group within the complexes.

Structure of aqueous sodium perchlorate solutions.

Salt solutions have been the object of study of many scientists through history, but one of the most important findings came along when the Hofmeister series were discovered. Their importance arises from the fact that they influence the relative solubility of proteins, and solubility is directly related to one of today's holy grails: protein folding. In this work we characterize one of the more-destabilizing salts in the series, sodium perchlorate, by studying it as an aqueous solution at various concentrations ranging from 0.08 to 1.60 mol/L. Molecular dynamics simulations at room temperature permitted a detailed study of the organization of solvent and cosolvent, in terms of its radial distribution functions, along with the study of the structure of hydrogen bonds in the ions' solvation shells. We found that the distribution functions have some variations in their shape as concentration changes, but the position of their peaks is mostly unaffected. Regarding water, the most salient fact is the noticeable (although small) change in the second hydration shell and even beyond, especially for g(O(w)***O(w)), showing that the locality of salt effects should not be restricted to considerations of only the first solvation shell. The perturbation of the second shell also appears in the study of the HB network, where the difference between the number of HBs around a water molecule and around the Na(+) cation gets much smaller as one goes from the first to the second solvation shell, yet the difference is not negligible. Nevertheless, the effect of the ions past their first hydration shell is not enough to make a noticeable change in the global HB network. The Kirkwood-Buff theory of liquids was applied to our system, in order to calculate the activity derivative of the cosolvent. This coefficient, along with a previously calculated preferential binding, allowed us to establish that if a folded AP peptide is immersed in the studied solution, becoming the solute, then increasing the salt concentration will make the helix more stable.