Nuclear pore complexes (NPCs) gate the only conduits for nucleocytoplasmic transport in eukaryotes. Their gate is formed by nucleoporins containing large intrinsically disordered domains with multiple phenylalanine-glycine repeats (FG domains). In combination, these are hypothesized to form a structurally and chemically homogeneous network of random coils at the NPC center, which sorts macromolecules by size and hydrophobicity. Instead, we found that FG domains are structurally and chemically heterogeneous. They adopt distinct categories of intrinsically disordered structures in non-random distributions. Some adopt globular, collapsed coil configurations and are characterized by a low charge content. Others are highly charged and adopt more dynamic, extended coil conformations. Interestingly, several FG nucleoporins feature both types of structures in a bimodal distribution along their polypeptide chain. This distribution functionally correlates with the attractive or repulsive character of their interactions with collapsed coil FG domains displaying cohesion toward one another and extended coil FG domains displaying repulsion. Topologically, these bipartite FG domains may resemble sticky molten globules connected to the tip of relaxed or extended coils. Within the NPC, the crowding of FG nucleoporins and the segregation of their disordered structures based on their topology, dimensions, and cohesive character could force the FG domains to form a tubular gate structure or transporter at the NPC center featuring two

The blood-brain barrier (BBB) is formed by specialized tight junctions between endothelial cells that line brain capillaries to create a highly selective barrier between the brain and the rest of the body. A major problem to overcome in drug design is the ability of the compound in question to cross the BBB. Neuroactive drugs are required to cross the BBB to function. Conversely, drugs that target other parts of the body ideally should not cross the BBB to avoid possible psychotropic side effects. Thus, the task of predicting the BBB permeability of new compounds is of great importance. Two gold-standard experimental measures of BBB permeability are logBB (the concentration of drug in the brain divided by concentration in the blood) and logPS (permeability surface-area product). Both methods are time-consuming and expensive, and although logPS is considered the more informative measure, it is lower throughput and more resource intensive. With continual increases in computer power and improvements in molecular simulations, in silico methods may provide viable alternatives. Computational predictions of these two parameters for a sample of 12 small molecule compounds were performed. The potential of mean force for each compound through a 1,2-dioleoyl-sn-glycero-3-phosphocholine bilayer is determined by molecular dynamics simulations. This system setup is often used as a simple BBB mimetic. Additionally, one-dimensional position-dependent diffusion coefficients are calculated from the molecular dynamics trajectories. The diffusion coefficient is combined with the free energy landscape to calculate the effective permeability (Peff) for each sample compound. The relative values of these permeabilities are compared to experimentally determined logBB and logPS values. Our computational predictions correlate remarkably well with both logBB (R(2) = 0.94) and logPS (R(2) = 0.90). Thus, we have demonstrated that this approach may have the potential to provide reliable, quantitatively predictive BBB permeability, using a relatively quick, inexpensive method.

We used atomic force microscopy to measure the binding forces between Mucin1 (MUC1) peptide and a single-chain variable fragment (scFv) antibody selected from a scFv library screened against MUC1. This binding interaction is central to the design of molecules used for targeted delivery of radioimmunotherapeutic agents for prostate and breast cancer treatment. Our experiments separated the specific binding interaction from nonspecific interactions by tethering the antibody and MUC1 molecules to the atomic force microscope tip and sample surface with flexible polymer spacers. Rupture force magnitude and elastic characteristics of the spacers allowed identification of the rupture events corresponding to different numbers of interacting proteins. We used dynamic force spectroscopy to estimate the intermolecular potential widths and equivalent thermodynamic off rates for monovalent, bivalent, and trivalent interactions. Measured interaction potential parameters agree with the results of molecular docking simulation. Our results demonstrate that an increase of the interaction valency leads to a precipitous decline in the dissociation rate. Binding forces measured for monovalent and multivalent interactions match the predictions of a Markovian model for the strength of multiple uncorrelated bonds in a parallel configuration. Our approach is promising for comparison of the specific effects of molecular modifications as well as for determination of the best configuration of antibody-based multivalent targeting agents.atomic force microscopy ͉ multivalency ͉ radioimmunmotherapy ͉ binding affinity I nteractions between biological molecules drive a vast variety of cellular processes and span a wide range of strength and complexity. Multivalent interactions where several binding units combine to produce superior binding strength play an important role in adaptive immune response (1) and intercellular adhesion (2), as well as in the mechanism of action of many pharmaceuticals (3). Clinical researchers have used multivalency as an affinity-enhancing approach (4, 5) in a variety of immunotherapies and imaging techniques to target specific tissues (6, 7).Linking several molecules into a large multivalent binding construct also creates bulky agents that exhibit reduced tissue penetration and have a higher probability of accumulation in liver (8). Therefore, a better understanding of the multivalent binding is necessary for the creation of optimized agents that balance binding efficiency and molecular size. Quantitative characterization of multivalent interactions is also important for understanding the basic biophysics of complex molecular systems.The last decade saw an explosion of interaction force measurement techniques that allowed researchers to measure and apply molecular level stresses (9-11). Atomic force microscopy (AFM) probes ligand-receptor interactions by simply pulling off the ligand from the receptor using external force (12). Kinetic approaches to the binding force measurements, such as dynamic force spectroscopy ...

A panel of five zinc-chelated aza-macrocycle ligands and their ability to catalyze the hydration of carbon dioxide to bicarbonate, H(2)O + CO(2) → H(+) + HCO(3)(–), was investigated using quantum-mechanical methods and stopped-flow experiments. The key intermediates in the reaction coordinate were optimized using the M06-2X density functional with aug-cc-pVTZ basis set. Activation energies for the first step in the catalytic cycle, nucleophilic CO(2) addition, were calculated from gas-phase optimized transition-state geometries. The computationally derived trend in activation energies was found to not correspond with the experimentally observed rates. However, activation energies for the second, bicarbonate release step, which were estimated using calculated bond dissociation energies, provided good agreement with the observed trend in rate constants. Thus, the joint theoretical and experimental results provide evidence that bicarbonate release, not CO(2) addition, may be the rate-limiting step in CO(2) hydration by zinc complexes of aza-macrocyclic ligands. pH-independent rate constants were found to increase with decreasing Lewis acidity of the ligand-Zn complex, and the trend in rate constants was correlated with molecular properties of the ligands. It is suggested that tuning catalytic efficiency through the first coordination shell of Zn(2+) ligands is predominantly a balance between increasing charge-donating character of the ligand and maintaining the catalytically relevant pK(a) below the operating pH.

The monomeric protein catechol O-methyltransferase (COMT) of rat liver is one of a number of methyl, from AdoMet, transferring enzymes that share a common catalytic domain. As a representative enzyme, COMT has been chosen for molecular dynamic (MD) simulation studies in order to ascertain if there are correlated motions in the ES complex that would be useful in decreasing the energy of activation for the S N 2 methyl transfer reaction. Such correlative motions have been found and are discussed. The MD trajectory can also provide insight into the observed preference of meta-O-methylation over para-O-methylation for ionic substrates such as dopamine and norepinephrine. The catechol ring has a tilt of approximately 30°compared to that of the X-ray structure. This directs any substituent at the 5-position of the catecholate ring, which is para to the O-methylation site, into a hydrophobic pocket formed by Trp38 and Tyr200. This pocket accommodates hydrophobic substituents such that para-O-methylation is favored. Polar substituents are repelled by this hydrophobic pocket making meta-O-methylation favorable.

Hybrid quantum mechanics͞molecular mechanics calculations using Austin Model 1 system-specific parameters were performed to study the S N2 displacement reaction of chloride from 1,2-dichloroethane (DCE) by nucleophilic attack of the carboxylate of acetate in the gas phase and by Asp-124 in the active site of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10. The activation barrier for nucleophilic attack of acetate on DCE depends greatly on the reactants having a geometry resembling that in the enzyme or an optimized gas-phase structure. It was found in the gas-phase calculations that the activation barrier is 9 kcal͞mol lower when dihedral constraints are used to restrict the carboxylate nucleophile geometry to that in the enzyme relative to the geometries for the reactants without dihedral constraints. The calculated quantum mechanics͞molecular mechanics activation barriers for the enzymatic reaction are 16.2 and 19.4 kcal͞mol when the geometry of the reactants is in a near attack conformer from molecular dynamics and in a conformer similar to the crystal structure (DCE is gauche), respectively. This haloalkane dehalogenase lowers the activation barrier for dehalogenation of DCE by 2-4 kcal͞mol relative to the single point energies of the enzyme's quantum mechanics atoms in the gas phase. S N2 displacements of this sort in water are infinitely slower than in the gas phase. The modest lowering of the activation barrier by the enzyme relative to the reaction in the gas phase is consistent with mutation experiments.H aloalkane dehalogenases catalyze the hydrolytic cleavage of carbon-halogen bonds in aliphatic and aromatic halogenated compounds. The haloalkane dehalogenase from the nitrogen-fixing hydrogen bacterium Xanthobacter autotrophicus GJ10 (DhlA) prefers 1,2-dichloroethane (DCE) as substrate and converts it to 2-chloroethanol and chloride (1). A catalytic triad consisting of Asp-124, His-289, and Asp-260 is the central residue in the dehalogenation reaction (Scheme 1). On binding DCE in the predominantly hydrophobic active site, it undergoes S N 2 displacement of chloride by nucleophilic attack of Asp-124-COO Ϫ to form an ester intermediate at the rate of 50 Ϯ 10 s Ϫ1(1, 2). The ester intermediate is subsequently hydrolyzed by an activated water molecule. The dyad of His-289 and Asp-260 is thought to be responsible for activating the water molecule (3). This enzyme functions most efficiently at pH 8.2, likely because the imidazole NE2 of His-289 needs to be unprotonated for the hydrolysis reaction to proceed. Dehalogenases are of great interest because they are able to react with halogenated molecules under mild conditions (4). Many halogenated molecules are pollutants, and bioremediation is a highly desirable method for removing these harmful molecules from the environment. Dehalogenases have not existed in nature for a long time andhave not yet evolved into optimal enzymes. For example, the catalytic efficiency for DhlA is 4,550 M Ϫ1 ⅐s Ϫ1 for DCE (5), as compared with 5.6 ϫ 10 7 M Ϫ1 ⅐s Ϫ1for...

The αvβ3 integrin, expressed on the surface of various normal and cancer cells, is involved in numerous physiological processes such as angiogenesis, apoptosis, and bone resorption. Because this integrin plays a key role in angiogenesis and metastasis of human tumors, αvβ3 integrin ligands are of great interest to advances in targeted-therapy and cancer imaging. In this report, one-bead-one-compound (OBOC) combinatorial libraries containing the RGD motif were designed and screened against K562 myeloid leukemia cells that had been transfected with human αvβ3 integrin gene. Cyclic peptide LXW7 was identified as a leading ligand with a build-in handle that binds specifically to αvβ3 and showed comparable binding affinity (IC50 = 0.68±0.08 μM) to some of the well-known RGD “head-to-tail” cyclic pentapeptide ligands reported in the literature. The biotinylated form of LXW7 ligand showed similar binding strength as LXW7 against αvβ3 integrin, whereas biotinylated RGD cyclopentapeptide ligands revealed a 2 to 8 fold weaker binding affinity than their free forms. LXW7 was able to bind to both U-87MG glioblastoma and A375M melanoma cell lines, both of which express high levels of αvβ3 integrin. In vivo and ex vivo optical imaging studies with biotinylated-ligand/streptavidin-Cy5.5 complex in nude mice bearing U-87MG or A375M xenografts revealed preferential uptake of biotinylated LXW7 in tumor. When compared with biotinylated RGD cyclopentapeptide ligands, biotinylated LXW7 showed higher tumor uptake but lower liver uptake.

Fluorine NMR chemical shifts from proteins containing fluorinated amino acids are usually dispersed over a wide range when the protein is in its native conformation. The shift dispersion essentially disappears when the protein is unfolded. Origins of the large protein structure-induced shielding effects are not clear, although they have been ascribed to electric field effects, short-range electron-electron interactions ("van der Waals" effects), and various local magnetic anisotropies. The present work explores the relative contributions of electric fields and short-range electronic interactions to fluorine shielding of 6-fluorotryptophan residues contained in the enzyme dihydrofolate reductase E. coli (DHFR), in binary complexes of this enzyme with NADPH and with methotrexate, and in a ternary complex with NADPH and methotrexate. Comparison of computed shielding effects to experimental data suggest an important role for short-range electronic interactions in determining fluorine shielding changes in these proteins and a lesser, but nonnegligible, contribution of electric fields and other anisotropies to observed shielding effects. However, the methods employed for calculation of fluorine shielding effects do not have great predictive power for this enzyme, contrary to what has been possible in other systems. The failure to obtain a clean diagnosis of shielding in this system may be a consequence of high conformational mobility.