Molecular dynamics of the anticodon domain of yeast tRNA(Phe): codon-anticodon interaction

We have studied the effect of codon-anticodon interaction on the structure and dynamics of transfer RNAs using molecular dynamics simulations over a nanosecond time scale. From our molecular dynamical investigations of the solvated anticodon domain of yeast tRNA(Phe) in the presence and absence of the codon trinucleotides UUC and UUU, we find that, although at a gross level the structures are quite similar for the free and the bound domains, there are small but distinct differences in certain parts of the molecule, notably near the Y37 base. Comparison of the dynamics in terms of interatomic or inter-residual distance fluctuation for the free and the bound domains showed regions of enhanced rigidity in the loop region in the presence of codons. Because fluorescence experiments suggested the existence of multiple conformers of the anticodon domain, which interconvert on a much larger time scale than our simulations, we probed the conformational space using five independent trajectories of 500 ps duration. A generalized ergodic measure analysis of the trajectories revealed that at least for this time scale, all the trajectories populated separate parts of the conformational space, indicating a need for even longer simulations or enhanced sampling of the conformational space to give an unequivocal answer to this question.     

Photoinduced conformational dynamics of a photoswitchable peptide: a nonequilibrium molecular dynamics simulation study

Employing nonequilibrium molecular dynamics simulations, a comprehensive computational study of the photoinduced conformational dynamics of a photoswitchable bicyclic azobenzene octapeptide is presented. The calculation of time-dependent probability distributions along various global and local reaction coordinates reveals that the conformational rearrangement of the peptide is rather complex and occurs on at least four timescales: 1) After photoexcitation, the azobenzene unit of the molecule undergoes nonadiabatic photoisomerization within 0.2 ps. 2) On the picosecond timescale, the cooling (13 ps) and the stretching (14 ps) of the photoexcited peptide is observed. 3) Most reaction coordinates exhibit a 50-100 ps component reflecting a fast conformational rearrangement. 4) The 500-1000 ps component observed in the simulation accounts for the slow diffusion-controlled conformational equilibration of the system. The simulation of the photoinduced molecular processes is in remarkable agreement with time-resolved optical and infrared experiments, although the calculated cooling as well as the initial conformational rearrangements of the peptide appear to be somewhat too slow. Based on an ab initio parameterized vibrational Hamiltonian, the time-dependent amide I frequency shift is calculated. Both intramolecular and solvent-induced contributions to the frequency shift were found to change by < or = 2 cm(-1), in reasonable agreement with experiment. The potential of transient infrared spectra to characterize the conformational dynamics of peptides is discussed in some detail.     

Molecular dynamics study of the conformations of glycosidic linkages in sialic acid modified ganglioside GM3 analogues

The objective of the present study is to model the analogues of monosialoganglioside (GM3) by making modifications in its sialic acid residue with different substitutions in aqueous environment and to determine their structural stability based upon computational molecular dynamics. Molecular mechanics and molecular dynamics investigation was carried out to study the conformational preferences of the analogues of GM3. Dynamic simulations were carried out on the analogues of GM3 varying in the substituents at C-1, C-4, C-5, C-8 and C-9 positions of their sialic acid or Neuraminic acid (NeuAc) residue. The analogues are soaked in a periodic box of TIP3P water as solvent and subjected to a 10 ns molecular dynamics (MD) simulation using AMBER ff03 and gaff force fields with 30 ps equilibration. The analogue of GM3 with 9-N-succNeuAc (analogue5, C9 substitution) was observed to have the lowest energy of ?6112.5 kcal/mol. Graphical analysis made on the MD trajectory reveals the direct and water mediated hydrogen bonds existing in these sialic acid analogues. The preferable conformations for glycosidic linkages of GM3 analogues found in different minimum energy regions in the conformational maps were identified. This study sheds light on the conformational preferences of GM3 analogues which may be essential for the design of GM3 analogues as inhibitors for different ganglioside specific pathogenic proteins such as bacterial toxins, influenza toxins and neuraminidases.     

Molecular dynamics simulation of oligosaccharides containing N-acetyl neuraminic acid

αD -N-acetyl neuraminic acid (Neu5Ac, sialic acid) is a commonly occurring carbohydrate residue in various cell surface glycolipids and glycoproteins. This residue is linked terminally or internally to Gal residues via an α(2 → 3) or α(2 → 6) linkage. In the cell surface receptor, sialyl-Lewis^X, a terminal α(2 → 3) linkage is present. Previous studies from our laboratory have shown that in solution Lewis^X adopts a relatively rigid structure. In order to model the Neu5Ac residue, vacuum molecular dynamics of this monosaccharide were compared with simulations that explicitly include solvent water. The dynamical average of the monosaccharide conformation obtained from the two simulations was similar. Vacuum calculations for the disaccharide Neu5Ac α(2 → 3) Gal β-O-methyl show that a number of low energy minima are accessible to this disaccharide. Molecular dynamics simulations starting from the low energy minima show conformational transitions with a time scale of 10–50 ps among several of the minima while large barriers between other minima prevent transitions on the time scale studied. Simulations of this disaccharide in the presence of solvent show fewer conformational transitions, illustrating a dampening effect of the solvent that has been observed in some other studies. Our results are most consistent with an equilibrium among multiple conformations for the Neu5Ac α(2 → 3) Gal β linkage. © 1994 John Wiley & Sons, Inc.     

Molecular dynamics simulations and the conformational mobility of blood group oligosaccharides

Molecular dynamics simulations were carried out without explicit consideration of solvent to explore the conformational mobility of blood group A and H oligosaccharides. The potential energy force field of Rasmussen and co-workers was used with the CHARMM program on a number of disaccharide and trisaccharide models composed of fucose, galactose, glucose, N-acetyl glucosamine, and N-acetyl galactosamine chosen to represent various fragments of blood group oligosaccharides. In agreement with results of earlier studies, stable chair conformations were found for each pyranoside from which no transitions were detected in simulations as long as 800 ps. Exocyclic dihedral angles, including that of C5-C6, generally show numerous transitions on a time scale of approximately 5-30 ps. The dihedral angles of some but not all glycosidic linkages of blood group oligosaccharides show transitions on the time scale of 30-50 ps, implying that the extent of internal motion in blood group oligosaccharides depends strongly on linkage stereochemistry. For certain blood group A and H oligosaccharides that show limited internal motion in these simulations, we argue that the calculations are consistent with our previous analysis of 1H nuclear Overhauser enhancement (NOE) data that imply single conformations over a wide range of temperature and solvent conditions. While the trajectories are consistent with 13C T1 values that have been interpreted as indicating rigid conformations, measurements of 13C-NOE and T1 as a function of magnetic field strength are proposed as an improved method for experimental detection of the internal motion that is suggested for certain oligosaccharides in these simulations. The results of these simulations differ substantially from those of peptides of a similar molecular weight in that the oligosaccharides show much less internal motion.     

A kinetic model for the internal motions of proteins: diffusion between multiple harmonic wells.

The dynamics of collective protein motions derived from Molecular Dynamics simulations have been studied for two small model proteins: initiation factor I and the B1 domain of Protein G. First, we compared the structural fluctuations, obtained by local harmonic approximations in different energy minima, with the ones revealed by large scale molecular dynamics (MD) simulations. It was found that a limited set of harmonic wells can be used to approximate the configurational fluctuations of these proteins, although any single harmonic approximation cannot properly describe their dynamics. Subsequently, the kinetics of the main (essential) collective protein motions were characterized. A dual-diffusion behavior was observed in which a fast type of diffusion switches to a much slower type in a typical time of about 1-3 ps. From these results, the large backbone conformational fluctuations of a protein may be considered as "hopping" between multiple harmonic wells on a basically flat free energy surface.     

Conformational space of alpha 1-4 glycosidic linkage: a molecular dynamics study

Molecular Dynamics simulations have been carried out for 100 ps on crystal structure of beta-cyclodextrin in vacuo and with explicit inclusion of solvent at constant pressure and constant temperature using the GROMOS MD algorithm, with a time step of 0.005 ps. The conformational space of the glycosidic linkage was studied by calculating two virtual dihedrals connecting the successive glucose units for the 2000 structures saved during the two simulations. Three preferred regions for alpha 1-4 glycosidic linkage were found in both the simulations. The use of these virtual dihedral angles in representing the glycosidic linkage is also brought out from these studies.     

Conformation of [8-arginine]vasopressin and V1 antagonists in dimethyl sulfoxide solution derived from two-dimensional NMR spectroscopy and molecular dynamics simulation

Structural and dynamic properties of [8-arginine]vasopressin and a class of highly potent vasopressin V1 antagonists which contain 3-mercapto-3,3-cyclopentamethylene propionic acid (Mca) in position 1 of the vasopressin sequence have been determined. On the basis of two-dimensional NMR experiments in dimethyl sulfoxide solution, interproton distances were derived according to which model conformations were built and refined using molecular dynamics simulations. The conformation of vasopressin and the V1 antagonists differ mainly in the region of the mutated residue. The antagonistic property was found to be related to an inversed chirality of the disulfide bridge. In all investigated molecules, characteristic beta-turn structure elements were found for the backbone conformation of the endocyclic residues Tyr2-Asn5. For this portion of the peptide sequence, various conformational equilibria were detected which matched different time scales. For [Arg8]vasopressin, averaged NMR parameters were obtained which could be explained by rapid interconversion between different beta-turn geometries, whereas multiple slowly exchanging conformations were observed for the V1 antagonists. V1 antagonists containing sarcosine in position 7 exhibited multiple spectral patterns for the exocyclic part attributed to cis/trans isomerization. The X-ray structure of deamino-oxytocin [Wood, S. P., Tickle, I. J., Treharne, A. M., Pitts, J. E., Mascarenhas, Y., Li, J. Y., Husain, J., Cooper, S., Blundell, T. L., Hruby, V. J., Buku, A., Fischman, A. J. & Wyssbrod, H. R. (1986) Science 232, 633-636] was found to represent one sample of the conformational space covered by the multiple conformations found for [Mca1, Arg8]vasopressin.     

Vasopressin and vasotocin facilitate reversal of a brightness discrimination

Male albino rats received vasopressin, vasotocin, pressinoic acid or placebo and were tested on an aversively motivated brightness discrimination task. Treatment with both vasopressin and vasotocin had no effect on acquisition but facilitated the reversal of the discrimination. Pressinoic acid had an inconsistent effect. The results are interpreted to show that the C terminal of the peptides vasopressin and vasotocin influence potency of these peptides. Furthermore, the results are interpreted as showing that both vasotocin and vasopressin influence selective attention during aversively motivated tasks.     

Locally accessible conformations of proteins: multiple molecular dynamics simulations of crambin.

Multiple molecular dynamics (MD) simulations of crambin with different initial atomic velocities are used to sample conformations in the vicinity of the native structure. Individual trajectories of length up to 5 ns sample only a fraction of the conformational distribution generated by ten independent 120 ps trajectories at 300 K. The backbone atom conformational space distribution is analyzed using principal components analysis (PCA). Four different major conformational regions are found. In general, a trajectory samples only one region and few transitions between the regions are observed. Consequently, the averages of structural and dynamic properties over the ten trajectories differ significantly from those obtained from individual trajectories. The nature of the conformational sampling has important consequences for the utilization of MD simulations for a wide range of problems, such as comparisons with X-ray or NMR data. The overall average structure is significantly closer to the X-ray structure than any of the individual trajectory average structures. The high frequency (less than 10 ps) atomic fluctuations from the ten trajectories tend to be similar, but the lower frequency (100 ps) motions are different. To improve conformational sampling in molecular dynamics simulations of proteins, as in nucleic acids, multiple trajectories with different initial conditions should be used rather than a single long trajectory.     

Aspects of model building applied to the C-terminal domain of the L12 protein from chloroplast ribosomes: a molecular dynamics study

A 170 picosecond molecular dynamics trajectory has been calculated starting from a model-built structure of chloroplast CTF. Local conformational changes occur during the equilibration period. Thereafter, a dynamically stable structure is attained. The conformational changes involve a turn connecting two structural subdomains which has an amino acid insertion and several substitutions with respect to the E. coli sequence. Potential energy minimisation alone fails to detect such a change. The overall folding and atomic positional fluctuations are very similar to those found in MD simulations of the E. coli molecule. The combined use of computer graphics based model building and MD calculations has lead to a thermally stable putative structure for the chloroplast CTF.     

Arginine vasopressin (AVP) replacement of helper cell requirement in IFN-gamma production. Evidence for a novel AVP receptor on mouse lymphocytes

Arginine vasopressin (AVP), a nine-amino acid neurohypophyseal hormone, is capable of replacing the helper cell requirement for IFN-gamma production by Lyt-2+ mouse splenic lymphocytes. We present data here showing that the AVP helper signal occurs via interaction with a novel R on splenic lymphocytes and involves primarily the N-terminal six-amino acid cyclic ring (pressinoic acid) with the C-terminal three-amino acid end of AVP playing a minor role. Pressinoic acid was capable of providing help at concentrations similar to those of AVP, whereas oxytocin and isoleucine pressinoic acid were 10- and 100-fold less effective, respectively. Isoleucine pressinoic acid has the same structure as pressinoic acid except for the substitution of isoleucine for phenylalanine in position 3 of the sequence. Consistent with the function data, R binding competitions with splenic lymphocyte membrane preparations showed that AVP and pressinoic acid competed similarly with [3H]AVP, whereas oxytocin and isoleucine pressinoic acid were much less effective competitors. Further characterization of the AVP lymphocyte R was performed using AVP analogues having well defined agonist and antagonist activities on either V1 (vasopressor) R or V2 (antidiuretic) R. The AVP helper signal was blocked by the V1 antagonist [d(CH2)1(5) Tyr(methyl)]AVP but not by another V1 antagonist, [d(CH2)1(5)D-Tyr(ethyl)2Val4]AVP. Both V1-R antagonists were able to block [3H]AVP binding to the V1-R on liver cells, whereas only the V1 antagonist that blocked AVP help was able to compete effectively for the spleen AVP-R. Neither a V2 agonist nor a V2 antagonist had any effect on AVP help in IFN-gamma production. These data strongly indicate the presence of a novel AVP-R on spleen lymphocytes, which is related to the classic V1-R on liver cell membranes.     

Conformational sampling by NMR solution structures calculated with the program DIANA evaluated by comparison with long-time molecular dynamics calculations in explicit water

The NMR solution structure of bovine pancreatic trypsin inhibitor (BPTI) obtained by distance geometry calculations with the program DIANA is compared with groups of conformers generated by molecular dynamics (MD) simulations in explicit water at ambient temperature and pressure. The MD simulations started from a single conformer and were free or restrained either by the experimental NOE distance restraints or by time-averaged restraints; the groups of conformers were collected either in 10 ps intervals during 200 ps periods of simulation, or in 50 ps intervals during a 1 ns period of simulation. Overall, these comparisons show that the standard protein structure determination protocol with the program DIANA provides a picture of the protein structure that is in agreement with MD simulations using “realistic” potential functions over a nanosecond timescale. For well-constrained molecular regions there is a trend in the free MD simulation of duration 1 ns that the sampling of the conformation space is slightly increased relative to the DIANA calculations. In contrast, for surface-exposed side-chains that are less extensively constrained by the NMR data, the DIANA conformers tend to sample larger regions of conformational space than conformers selected from any of the MD trajectories. Additional insights into the behavior of surface side-chains come from comparison of the MD runs of 200 ps or 1 ns duration. In this time range the sampling of conformation space by the protein surface depends strongly on the length of the simulation, which indicates that significant side-chain transitions occur on the nanosecond timescale and that much longer simulations will be needed to obtain statistically significant data on side-chain dynamics.     

Dynamics of the Peripheral Membrane Protein P2 from Human Myelin Measured by Neutron Scattering—A Comparison between Wild-Type Protein and a Hinge Mutant

Myelin protein P2 is a fatty acid-binding structural component of the myelin sheath in the peripheral nervous system, and its function is related to its membrane binding capacity. Here, the link between P2 protein dynamics and structure and function was studied using elastic incoherent neutron scattering (EINS). The P38G mutation, at the hinge between the β barrel and the α-helical lid, increased the lipid stacking capacity of human P2 in vitro, and the mutated protein was also functional in cultured cells. The P38G mutation did not change the overall structure of the protein. For a deeper insight into P2 structure-function relationships, information on protein dynamics in the 10 ps to 1 ns time scale was obtained using EINS. Values of mean square displacements mainly from protein H atoms were extracted for wild-type P2 and the P38G mutant and compared. Our results show that at physiological temperatures, the P38G mutant is more dynamic than the wild-type P2 protein, especially on a slow 1-ns time scale. Molecular dynamics simulations confirmed the enhanced dynamics of the mutant variant, especially within the portal region in the presence of bound fatty acid. The increased softness of the hinge mutant of human myelin P2 protein is likely related to an enhanced flexibility of the portal region of this fatty acid-binding protein, as well as to its interactions with the lipid bilayer surface requiring conformational adaptations.     

Molecular dynamics simulation of Lewis blood groups and related oligosaccharides.

Molecular dynamics simulations without explicit inclusion of solvent molecules have been performed to study the motions of Lewisa and Lewisb blood group oligosaccharides, and two blood group A tetrasaccharides having type I and type II core chains. The blood group H trisaccharide has also been studied and compared with the blood group A type II core chain. The potential energy surface developed by Rasmussen and co-workers was used with the molecular mechanics code CHARMM. The lowest energy minima of the component disaccharide fragments were obtained from conformational energy mapping. The lowest energy minima of these disaccharide fragments were used to build the tri- and tetrasaccharides that were further minimized before the actual heating/equilibration and dynamics simulations. The trajectories of the disaccharide fragments, e.g., Fuc alpha- (1----4)GlcNAc, Gal beta-(1----4)GlcNAc, etc., show transitions among various minima. However, the oligosaccharides were found to be dynamically stable and no transitions to other minimum energy conformations were observed in the time series of the glycosidic dihedral angles even during trajectories as long as 300 ps. The stable conformations of the glycosidic linkages in the oligosaccharides are not necessarily the same as the minimum energy conformation of the corresponding isolated disaccharides. The average fluctuations of the glycosidic angles in the oligosaccharides were well within the range of +/- 15 degrees. The results of these trajectory calculations were consistent with the relatively rigid single-conformation models derived for these oligosaccharides from 1H-nmr data.     

Stimulation of rat pituitary phospholipid methyltransferase by vasopressin but not oxytocin

Rat pituitary extracts contain two methyltransferases that catalyze stepwise methylation of phosphatidyl-ethanolamine to phosphatidylcholine using S-adenosylmethionine as the methyl donor. The activities of both of these enzymes were stimulated by 40 μM lysine or arginine vasopresin but not oxytocin, arginine vasotocin and Pro-Leu-Gly NH₂. The concentration of lysine-vasopressin required for the half-maximal stimulation of phospholipid methylation was 27 μM. A comparison of the chemical structure of different peptides with their ability to stimulate phospholipid methylation suggests that the stimulatory activity resides in the covalent ring structure (pressinoic acid) of the vasopressin molecule.     

Hydration structure and dynamics of B- and Z-DNA in the presence of counterions via molecular dynamics simulations

Following our previous attempts at understanding the structural and dynamical properties of water and counterions hydrating nucleic acids, we have performed molecular dynamics simulations for B- and Z-DNA. In these simulations, the nucleic acids were held rigid. In the case of B-DNA, one turn of B-DNA double helix was considered in the presence of 1500 water molecules and 20 counterions (K⁺). The simulations were performed for 4.0 ps after equilibrating the system. For Z-DNA, we considered one turn of the double helix in the presence of 1851 water molecules and 24 counterions (K⁺). The simulations were carried out for 3.5 ps after equilibration. The average temperature of these simulations was ～ 360 K for Z-DNA and ～ 345 K for B-DNA. In these simulations the hydrogen atoms were explicitly taken into account. For both simulations, a fifth-order predictor-corrector was used for solving the translational equations of motion. The rotational motion of the water molecules was represented in terms of quaternion algebra and the rotational equations of motion were solved with a second-order quaternion method using a sixth-order predictor-corrector method. A time step of 0.5 · 10^?15 s was used in these simulations. The structural and the dynamical properties of water solvating the counterions, and the phosphate groups of the DNA, were computed to understand the hydration structure. Diffusion coefficients and velocity correlation functions were calculated for both ions and the water molecules. The velocity correlation functions for the ions exhibit a caged behavior. The dipole correlation functions for the water molecules indicate that the water molecules close to the helix retain the memory of their initial orientations for longer periods of time than those away from the helix. During the time period of our simulation (3–4 ps) the ion probability distributions show a well-defined pattern and suggest limited mobility for the ions, being close to the helix.     

A "structural" water molecule in the family of fatty acid binding proteins

A single water molecule (w135), buried within the structure of rat intestinal fatty acid binding protein (I-FABP), is investigated by NMR, molecular dynamics simulations, and analysis of known crystal structures. An ordered water molecule was found in structurally analogous position in 24 crystal structures of nine different members of the family of fatty acid binding proteins. There is a remarkable conservation of the local structure near the w135 binding site among different proteins from this family. NMR cross-relaxation measurements imply that w135 is present in the I-FABP:ANS (1-sulfonato-8-(1')anilinonaphthalene) complex in solution with the residence time of >300 ps. Mean-square positional fluctuations of w135 oxygen observed in MD simulations (0.18 and 0.13 A2) are comparable in magnitude to fluctuations exhibited by the backbone atoms and result from highly constrained binding pocket as revealed by Voronoi volumes (averages of 27.0 +/- 1.8 A3 and 24.7 +/- 2.2 A3 for the two simulations). Escape of w135 from its binding pocket was observed only in one MD simulation. The escape process was initiated by interactions with external water molecules and was accompanied by large deformations in beta-strands D and E. Immediately before the release, w135 assumed three distinct states that differ in hydrogen bonding topology and persisted for about 15 ps each. Computer simulations suggest that escape of w135 from the I-FABP matrix is primarily determined by conformational fluctuations of the protein backbone and interactions with external water molecules.     

Structural flexibility of the nucleosome core particle at atomic resolution studied by molecular dynamics simulation

Comparative explicit solvent molecular dynamics (MD) simulations have been performed on a complete nucleosome core particle with and without N-terminal histone tails for more than 20 ns. Main purpose of the simulations was to study the dynamics of mobile elements such as histone N-terminal tails and how packing and DNA-bending influences the fine structure and dynamics of DNA. Except for the tails, histone and DNA molecules stayed on average close to the crystallographic start structure supporting the quality of the current force field approach. Despite the packing strain, no increase of transitions to noncanonical nucleic acid backbone conformations compared to regular B-DNA was observed. The pattern of kinks and bends along the DNA remained close to the experiment overall. In addition to the local dynamics, the simulations allowed the analysis of the superhelical mobility indicating a limited relative mobility of DNA segments separated by one superhelical turn (mean relative displacement of approximately +/-0.2 nm, mainly along the superhelical axis). An even higher rigidity was found for relative motions (distance fluctuations) of segments separated by half a superhelical turn (approximately +/-0.1 nm). The N-terminal tails underwent dramatic conformational rearrangements on the nanosecond time scale toward partially and transiently wrapped states around the DNA. Many of the histone tail changes corresponded to coupled association and folding events from fully solvent-exposed states toward complexes with the major and minor grooves of DNA. The simulations indicate that the rapid conformational changes of the tails can modulate the DNA accessibility within a few nanoseconds.     

Conformational flexibility and binding interactions of the G protein βγ heterodimer

Previous NMR experiments on unbound G protein βγ heterodimer suggested that particular residues in the binding interface are mobile on the nanosecond timescale. In this work we performed nanosecond‐timescale molecular dynamics simulations to investigate conformational changes and dynamics of Gβγ in the presence of several binding partners: a high‐affinity peptide (SIGK), phosducin, and the GDP‐bound α subunit. In these simulations, the high mobility of GβW99 was reduced by SIGK, and it appeared that a tyrosine might stabilize GβW99 by hydrophobic or aromatic stacking interactions in addition to hydrogen bonds. Simulations of the phosducin‐Gβγ complex showed that the mobility of GβW99 was restricted, consistent with inferences from NMR. However, large‐scale conformational changes of Gβγ due to binding, which were hypothesized in the NMR study, were not observed in the simulations, most likely due to their short (nanosecond) duration. A pocket consisting of hydrophobic amino acids on Gα appears to restrict GβW99 mobility in the crystal structure of the Gαβγ? heterotrimer. The simulation trajectories are consistent with this idea. However, local conformational changes of residues GβW63, GβW211, GβW297, GβW332, and GβW339 were detected during the MD simulations. As expected, the magnitude of atomic fluctuations observed in simulations was greater for α than for the βγ subunits, suggesting that α has greater flexibility. These observations support the notion that to maintain the high mobility of GβW99 observed by solution NMR requires that the Gβ–α interface must open up on time scale longer than can be observed in nanosecond scale simulations. Proteins 2011. © 2010 Wiley‐Liss, Inc.