Crystal engineering of lattice metrics of perhalometallate salts and MOFs

The synthesis of the salt 3 and metallo-organic framework (MOF) [{(4,4^′-bipy)CoBr₂}_n] 4 by a range of solid state (mechanochemical and thermochemical) and solution methods is reported; they are isostructural with their respective chloride analogues 1 and 2. 3 and 4 can be interconverted by means of HBr elimination and absorption. Single phases of controlled composition and general formula [4,4^′-H₂bipy][CoBr_4-xCl_x] 5_x may be prepared from 2 and 4 by solid—gas reactions involving HBr or HCl respectively. Crystalline single phase samples of 5_x and [{(4,4^′-bipy)CoBr_2-xCl_x}_n] 6_x were prepared by solid-state mechanochemical routes, allowing fine control over the composition and unit cell volume of the product. Collectively these methods enable continuous variation of the unit cell dimensions of the salts [4,4^′-H₂bipy][CoBr_4-xCl_x] (5_x) and the MOFs [{(4,4^′-bipy)CoBr_2-xCl_x}_n] (6_x) by varying the bromide to chloride ratio and establish a means of controlling MOF composition and the lattice metrics, and so the physical and chemical properties that derive from it.     

Conformational polymorphism in a heteromolecular single crystal leads to concerted movement akin to collective rack-and-pinion gears at the molecular level

We describe a heteromolecular single crystal that exhibits three reversible and concerted reorganizations upon heating and cooling. The products of the reorganizations are conformational polymorphs. The reorganizations are postulated to proceed through three motions: (i) alkyl translations, (ii) olefin rotations, and (iii) rotational tilts. The motions are akin to rack-and-pinion gears at the molecular level. The rack-like movement is based on expansions and compressions of alkyl chains that are coupled with pinion-like 180° rotations of olefins. To accommodate the movements, phenol and thiophene components undergo rotational tilts about intermolecular hydrogen bonds. The movements are collective, being propagated in close-packed repeating units. This discovery marks a step to understanding how organic solids can support the development of crystalline molecular machines and devices through correlated and collective movements.     

Thermal modulation of birefringence observed in a crystalline molecular gyrotop

Recently, functional organic materials have been put into practical use. The application of molecular motions has the potential to create new molecule-based materials. For this reason, considerable attention has been focused on the chemistry and properties of molecular machines in which mechanical motions of parts of the molecules are observed. In particular, phenylene rotation in the crystalline state has been investigated using framed molecular gyrotops having a phenylene rotor encased in three long alkyl spokes. In this study, we show thermal modulation of birefringence in a crystal due to the states of dynamic equilibrium of a novel molecular gyrotop. A macrocage molecule having a bridged phenylene rotor was synthesized as a novel molecular gyrotop. Rapid rotation of the phenylene rotor of the molecular gyrotop was confirmed by solid-state (2)H NMR spectroscopy that showed changes in the optical properties of a single crystal, i.e., the thermal modulation of birefringence. These results are the first application of the dynamic states in a crystal causing an optical change. These phenomena were also confirmed by control experiments using a molecular gyrotop with a nonrotating xylene rotor. We anticipate our finding to be a starting point for the creation of a new field of material chemistry that will make use of the dynamic states of molecules.     

An isolated water droplet in the aqueous solution of a supramolecular tetrahedral cage

Water under nanoconfinement at ambient conditions has exhibited low-dimensional ice formation and liquid–solid phase transitions, but with structural and dynamical signatures that map onto known regions of water’s phase diagram. Using terahertz (THz) absorption spectroscopy and ab initio molecular dynamics, we have investigated the ambient water confined in a supramolecular tetrahedral assembly, and determined that a dynamically distinct network of 9 ± 1 water molecules is present within the nanocavity of the host. The low-frequency absorption spectrum and theoretical analysis of the water in the Ga₄L₆¹²⁻ host demonstrate that the structure and dynamics of the encapsulated droplet is distinct from any known phase of water. A further inference is that the release of the highly unusual encapsulated water droplet creates a strong thermodynamic driver for the high-affinity binding of guests in aqueous solution for the Ga₄L₆¹²⁻ supramolecular construct.

Supramolecular capsules create internal cavities that are thought to act like enzyme active sites (1). As aqueous enzymes provide inspiration for the design of supramolecular catalysts, one of the goals of supramolecular chemistry is the creation of synthetic “receptors” that have both a high affinity and a high selectivity for the binding of guests in water (2, 3). The Ga₄L₆¹²⁻ tetrahedral assembly formulated by Raymond and coworkers represents an excellent example of a water-soluble supramolecular cage that has provided host interactions that promotes guest encapsulation. Using steric interactions and electrostatic charge to chemically position the substrate while shielding the reaction from solvent, this host has been shown to provide enhanced reaction rates that approach the performance of natural biocatalysts (4–10). Moreover, aqueous solvation of the substrate, host, and encapsulated solvent also play an important role in the whole catalytic cycle. In particular, the driving forces that release water from the nanocage host to favor the direct binding with the substrate is thought to be a critical factor in successful catalysis, but is challenging to probe directly (7, 8, 11–14).In both natural and artificial nanometer-sized environments, confined water displays uniquely modified structure and dynamics with respect to the bulk liquid (15–18). Recently, these modified properties were also found to have significant implications for the mechanism and energetics of reactions taking place in confined water with respect to those observed in bulk aqueous solution (19–21). In a pioneering study on supramolecular assemblies, Cram and collaborators (22) concluded that the interior of those cages is a “new and unique phase of matter” for the incarcerated guests. In more recent studies, it was postulated that, similar to graphitic and zeolite nanopores (23, 24), confined water within supramolecular host cavities is organized in stable small clusters [(H₂O)_n, with n = 8 to 19] that are different from gas phase water clusters (25). In these studies, the hydrogen-bonded water clusters were reported to be mostly ice- or clathrate-like by X-ray and neutron diffraction in the solid state at both ambient and cryogenic temperatures (26–32). However, to the best of our knowledge, such investigations have not characterized the Ga₄L₆¹²⁻ supramolecular tetrahedral assembly in the liquid state near room temperature and pressure, where the [Ga₄L₆]¹²⁻ capsule can perform catalytic reactions (6, 8, 9).Here, we use terahertz (THz) absorption spectroscopy and ab initio molecular dynamics (AIMD) to characterize low-frequency vibrations and structural organization of water in the nanoconfined environment. THz is ideally suited to probe the intermolecular collective dynamics of the water hydrogen bond (HB) network with extremely high sensitivity, as illustrated for different phases of water (33–38), and for aqueous solutions of salts, osmolytes, alcohols, and amino acids (36, 39–42). The THz spectra of the water inside the nanocage has been quantitatively reproduced with AIMD, allowing us to confidently characterize the water network in the cage in order to provide a more complete dynamical, structural, and thermodynamic picture. We have determined that the spectroscopic signature of the confined water in the nanocage is a dynamically arrested state whose structure bears none of the features of water at any alternate thermodynamic state point such as pressurized liquid or ice. Our experimental and theoretical study provides insight into the role played by encapsulated water in supramolecular catalysis, creating a low entropy and low enthalpy water droplet readily displaced by a catalytic substrate.     

Crystal structure of Staphylococcus aureus transglycosylase in complex with a lipid II analog and elucidation of peptidoglycan synthesis mechanism

Bacterial transpeptidase and transglycosylase on the surface are essential for cell wall synthesis, and many antibiotics have been developed to target the transpeptidase; however, the problem of antibiotic resistance has arisen and caused a major threat in bacterial infection. The transglycosylase has been considered to be another excellent target, but no antibiotics have been developed to target this enzyme. Here, we determined the crystal structure of the Staphylococcus aureus membrane-bound transglycosylase, monofunctional glycosyltransferase, in complex with a lipid II analog to 2.3 Å resolution. Our results showed that the lipid II-contacting residues are not only conserved in WT and drug-resistant bacteria but also significant in enzymatic activity. Mechanistically, we proposed that K140 and R148 in the donor site, instead of the previously proposed E156, are used to stabilize the pyrophosphate-leaving group of lipid II, and E100 in the acceptor site acts as general base for the 4-OH of GlcNAc to facilitate the transglycosylation reaction. This mechanism, further supported by mutagenesis study and the structure of monofunctional glycosyltransferase in complex with moenomycin in the donor site, provides a direction for antibacterial drugs design.     

Use of experimental crystallographic phases to examine the hydration of polar and nonpolar cavities in T4 lysozyme

There is conflicting evidence as to whether cavities in proteins that are nonpolar and large enough to accommodate solvent are empty or are occupied by disordered water molecules. Here, we use multiple-wavelength x-ray data collected from crystals of the selenomethionine-substituted L99A/M102L mutant of T4 lysozyme to obtain a high-resolution electron density map free of bias that is unavoidably associated with conventional model-based structure determination and refinement. The mutant, L99A/M102L, has four cavities, two being polar in character and the other two nonpolar. Cavity 1 (polar, volume 45.2 ų) was expected to contain two well ordered water molecules, and this is confirmed in the experimental electron density map. Likewise, cavity 2 (polar, 16.9 ų) is confirmed to contain a single water molecule. Cavity 3 (nonpolar, 21.4 ų) was seen to be empty in conventional x-ray refinement, and this is confirmed in the experimental map. Unexpectedly, however, cavity 4 (nonpolar, volume 133.5 ų) was seen to contain diffuse electron density equivalent to ≈1.5 water molecules. Although cavity 4 is largely nonpolar, it does have some polar character, and this apparently contributes to the presence of solvent. The cavity is large enough to accommodate four to five water molecules, and it appears that a hydrogen-bonded chain of three or more solvent molecules could occupy the cavity at a given time. The results are consistent with theoretical predictions that cavities in proteins that are strictly nonpolar will not contain solvent until the volume is large enough to permit mutually satisfying water–water hydrogen bonds.     

The Influence of Solvent on the Crystal Packing of Ethacridinium Phthalate Solvates

The synthesis, structural characterization and influence of solvents on the crystal packing of solvated complexes of ethacridine with phthalic acid: 6,9-diamino-2-ethoxyacridinium phthalate methanol solvate (1), 6,9-diamino-2-ethoxyacridinium phthalate ethanol solvate (2), 6,9-diamino-2-ethoxyacridinium phthalate isobutanol solvate (3), and 6,9-diamino-2-ethoxyacridinium phthalate tert-butanol solvate monohydrate (4) are described in this article. Single-crystal XRD measurements revealed that the compounds 1–4 crystallized in the triclinic P-1 space group, and the 6,9-diamino-2-ethoxyacridinium cations, phthalic acid anions and solvent molecules interact via strong N–H···O, O–H···O, C–H···O hydrogen bonds, and C–H···π and π–π interactions to form different types of basic structural motifs, such as: heterotetramer bis[···cation···anion···] in compound 1 and 2, heterohexamer bis[···cation···alcohol···anion···] in compound 3, and heterohexamer bis[···cation···water···anion···] in compound 4. Presence of solvents molecule(s) in the crystals causes different supramolecular synthons to be obtained and thus has an influence on the crystal packing of the compounds analyzed.     

Site-specific vibrational spectral signatures of water molecules in the magic H3O+(H2O)20 and Cs+(H2O)20 clusters

Theoretical models of proton hydration with tens of water molecules indicate that the excess proton is embedded on the surface of clathrate-like cage structures with one or two water molecules in the interior. The evidence for these structures has been indirect, however, because the experimental spectra in the critical H-bonding region of the OH stretching vibrations have been too diffuse to provide band patterns that distinguish between candidate structures predicted theoretically. Here we exploit the slow cooling afforded by cryogenic ion trapping, along with isotopic substitution, to quench water clusters attached to the H₃O⁺ and Cs⁺ ions into structures that yield well-resolved vibrational bands over the entire 215- to 3,800-cm⁻¹ range. The magic H₃O⁺(H₂O)₂₀ cluster yields particularly clear spectral signatures that can, with the aid of ab initio predictions, be traced to specific classes of network sites in the predicted pentagonal dodecahedron H-bonded cage with the hydronium ion residing on the surface.Over the last decade, the cooperative mechanics underlying the microhydration of simple ions has undergone a renaissance due to rapid advances in experimental and theoretical methods. On the experimental side, it is routine to capture and study size-selected species cooled to cryogenic temperatures (1–5), and theoretical techniques are now capable of handling tens of atoms with all-electron, “supermolecule” approaches, where complex hydration networks are treated in the ansatz of polyatomic molecular physics (6). A dramatic example of the new insights afforded by this combined approach is the recent elucidation of the spectral signature associated with the hydronium ion when it is accommodated on the surface of a pentagonal dodecahedral cage formed by the “magic” H₃O⁺(H₂O)₂₀ cluster (7). The theoretical structure proposed earlier (8) (denoted I) is illustrated in Fig. 1, and the recently reported D₂ predissociation spectrum of the cryogenically cooled, D₂ tagged H₃O⁺(H₂O)₂₀ cluster is compared with that observed for the H₃O⁺(H₂O)₃ Eigen cation (9, 10) in Fig. 1 A and B, respectively. The key bands derived from the OH stretching motions of the surface-embedded hydronium (denoted νH3O+a) were assigned (7) to the broad features in the experimental spectrum about 500 cm⁻¹ below the corresponding bands in the free Eigen cation. The intramolecular HOH bending and OH stretching modes of the surface water molecules are not strongly shifted by introduction of the ion, however, where the latter appear as a broad envelope spanning the 3,000- to 3,700-cm⁻¹ range typical of liquid water. An interesting aspect of the D₂ tagged spectrum obtained with cryogenic cooling (as opposed to that reported on the same ion generated under the more rapid quenching conditions at play in a supersonic jet ion source) (8, 11) is that distinct features begin to emerge above the continuous background absorption in the OH stretching region above 3,400 cm⁻¹.Open in a separate windowFig. 1.Vibrational predissociation spectra of (A) H₃O⁺(H₂O)₂₀ and (B) the H₃O⁺(H₂O)₃ Eigen cation. Bands attributed (7) to the antisymmetric H₃O⁺ stretches, νH3O+a, and H₃O⁺ umbrella bending mode, νH3O+umb of the embedded hydronium ion are indicated by the red lines. The antisymmetric stretches of remote ADD type waters (circled in turquoise in A) were assigned (6, 7) to band A, whereas the free OH stretches from the AAD waters are circled in orange. The symmetric (s) and antisymmetric (as) OH stretches of the water molecules solvating the Eigen cation (circled in blue) are expected (7) to red shift into the broad continuum near position B in H₃O⁺(H₂O)₂₀. The H₃O⁺(H₂O)₂₀ structure, denoted I, was the lowest energy structure identified in a search using the B3LYP method (6, 8).This observation of sharp OH stretching structure in a moderately large water cluster is significant in the context of the ongoing, often controversial discussion regarding the origin of the diffuse OH stretching spectrum displayed by neat liquid water (12–14), as well as by dilute acids (15, 16). Indeed, over the last decade, intensive experimental (17, 18) and theoretical (19–21) efforts have addressed the underlying mechanics of this broadening, resulting in significant differences of opinion regarding the role of excitonic band structure, excited state dynamics, and Fermi resonances (22–25). Cluster behavior is highly relevant in this discussion as they allow one to isolate microscopic assemblies with well-defined H-bonding configurations and thus directly address the spectral signatures associated with the various network morphologies. In this paper, we contrast the behavior of the n = 20 hydrates of the H₃O⁺ and Cs⁺ ions, both of which are preferentially generated in the M⁺(H₂O)_n cluster ion distributions (i.e., are magic numbers) and are thought to be based on a pentagonal dodecahedron motif (12 interconnected H-bonded pentagons). An important difference between these ions is that H₃O⁺ is predicted to reside on the surface of the cage (6, 8, 26), whereas Cs⁺ is sequestered in its interior (27, 28). We follow the evolution of the bands in the perdeuterated isotopologues, Cs⁺(D₂O)₂₀ and D₃O⁺(D₂O)₂₀, and find strong support for the earlier assignment of the spectral signature of the embedded H₃O⁺ ion. Of most importance here, however, is the observation that the perdeuterated isotopologues yield significantly better resolved fine structure in the lower energy OD stretching manifold than was evident in the H₃O⁺(H₂O)₂₀ spectrum. Comparison of this pattern with harmonic predictions yields compelling assignments of the strongest transitions to four classes of water molecules, which, in turn, allows us to refine the H₃O⁺(H₂O)₂₀ structure I suggested earlier. Special aspects of the n = 20 structures are further investigated in a study of the low frequency (<1,000 cm⁻¹) librational motions over the size range n = 17–27, where significantly sharper structure is observed in the n = 20 spectrum relative to those displayed by nearby cluster sizes. The origin of these bands is considered in the context of harmonic spectra predicted for the geometries identified through assignment of the OH/OD stretching band pattern. Features associated with frustrated rotations of the trapped H₃O⁺ moiety, as well as of water molecules in four-coordinated network sites, are thus identified for the first time.     

A joint x-ray and neutron study on amicyanin reveals the role of protein dynamics in electron transfer

The joint x-ray/neutron diffraction model of the Type I copper protein, amicyanin from Paracoccus denitrificans was determined at 1.8 Å resolution. The protein was crystallized using reagents prepared in D₂O. About 86% of the amide hydrogen atoms are either partially or fully exchanged, which correlates well with the atomic depth of the amide nitrogen atom and the secondary structure type, but with notable exceptions. Each of the four residues that provide copper ligands is partially deuterated. The model reveals the dynamic nature of the protein, especially around the copper-binding site. A detailed analysis of the presence of deuterated water molecules near the exchange sites indicates that amide hydrogen exchange is primarily due to the flexibility of the protein. Analysis of the electron transfer path through the protein shows that residues in that region are highly dynamic, as judged by hydrogen/deuterium exchange. This could increase the rate of electron transfer by transiently shortening through-space jumps in pathways or by increasing the atomic packing density. Analysis of C-H⋯X bonding reveals previously undefined roles of these relatively weak H bonds, which, when present in sufficient number can collectively influence the structure, redox, and electron transfer properties of amicyanin.     

Structure and topology of monomeric phospholamban in lipid membranes determined by a hybrid solution and solid-state NMR approach

Phospholamban (PLN) is an essential regulator of cardiac muscle contractility. The homopentameric assembly of PLN is the reservoir for active monomers that, upon deoligomerization form 1:1 complexes with the sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA), thus modulating the rate of calcium uptake. In lipid bilayers and micelles, monomeric PLN exists in equilibrium between a bent (or resting) T state and a more dynamic (or active) R state. Here, we report the high-resolution structure and topology of the T state of a monomeric PLN mutant in lipid bilayers, using a hybrid of solution and solid-state NMR restraints together with molecular dynamics simulations in explicit lipid environments. Unlike the previous structural ensemble determined in micelles, this approach gives a complete picture of the PLN monomer structure in a lipid bilayer. This hybrid ensemble exemplifies the tilt, rotation, and depth of membrane insertion, revealing the interaction with the lipids for all protein domains. The N-terminal amphipathic helical domain Ia (residues 1–16) rests on the surface of the lipid membrane with the hydrophobic face of domain Ia embedded in the membrane bilayer interior. The helix comprised of domain Ib (residues 23–30) and transmembrane domain II (residues 31–52) traverses the bilayer with a tilt angle of ≈24°. The specific interactions between PLN and lipid membranes may represent an additional regulatory element of its inhibitory function. We propose this hybrid method for the simultaneous determination of structure and topology for membrane proteins with compact folds or proteins whose spatial arrangement is dictated by their specific interactions with lipid bilayers.     

Role of protein kinase a in human hepatocyte DNA synthesis

The cellular mechanisms associated with the replicative response of hepatocytes to growth factor stimulation is incompletely understood. Murine hepatocyte DNA synthesis is altered by cyclic AMP, suggesting that protein kinase A is involved in the cellular mechanisms associated with liver growth. The purpose of this study was to evaluate the role of protein kinase A in human hepatocyte DNA synthesis. Human hepatocytes were isolated and maintained in primary culture on rat tail collagen. DNA synthesis was evaluated by determining [³H]thymidine incorporation. Human hepatocytes between 24 and 96 hr following harvest increased DNA synthesis in response to epidermal growth factor but not in response to glucagon, a stimulant of adenyl cyclase, or dibutyryl cyclic AMP. Mitogen-stimulated DNA synthesis was decreased by dibutyryl cyclic AMP. Cyclic AMP isomers that block or stimulate the effect of cyclic AMP on protein kinase A did not significantly alter resting or mitogen-stimulated human hepatocyte DNA synthesis. The results suggest that increased protein kinase A activity does not produce human hepatocyte replicative DNA synthesis.Supported in part by USPHS DK27695.     

Study of the high-potential iron sulfur protein in Halorhodospira halophila confirms that it is distinct from cytochrome c as electron carrier

The role of high-potential iron sulfur protein (HiPIP) in donating electrons to the photosynthetic reaction center in the halophilic gamma-proteobacterium Halorhodospira halophila was studied by EPR and time-resolved optical spectroscopy. A tight complex between HiPIP and the reaction center was observed. The EPR spectrum of HiPIP in this complex was drastically different from that of the purified protein and provides an analytical tool for the detection and characterization of the complexed form in samples ranging from whole cells to partially purified protein. The bound HiPIP was identified as iso-HiPIP II. Its Em value at pH 7 in the form bound to the reaction center was approximately 100 mV higher (+140 +/- 20 mV) than that of the purified protein. EPR on oriented samples showed HiPIP II to be bound in a well defined geometry, indicating the presence of specific protein-protein interactions at the docking site. At moderately reducing conditions, the bound HiPIP II donates electrons to the cytochrome subunit bound to the reaction center with a half-time of < or =11 micros. This donation reaction was analyzed by using Marcus's outer-sphere electron-transfer theory and compared with those observed in other HiPIP-containing purple bacteria. The results indicate substantial differences between the HiPIP- and the cytochrome c2-mediated re-reduction of the reaction center.     

The crystal structure of the second Z-DNA binding domain of human DAI (ZBP1) in complex with Z-DNA reveals an unusual binding mode to Z-DNA

Mammalian DAI (DNA-dependent activator of IFN-regulatory factors), an activator of the innate immune response, senses cytosolic DNA by using 2 N-terminal Z-DNA binding domains (ZBDs) and a third putative DNA binding domain located next to the second ZBD. Compared with other previously known ZBDs, the second ZBD of human DAI (hZβ_DAI) shows significant variation in the sequence of the residues that are essential for DNA binding. In this article, the crystal structure of the hZβ_DAI/Z-DNA complex reveals that hZβ_DAI has a similar fold to that of other ZBDs, but adopts an unusual binding mode for recognition of Z-DNA. A residue in the first β-strand rather than residues in the β-loop contributes to DNA binding, and part of the (α3) recognition helix adopts a 3₁₀ helix conformation. The role of each residue that makes contact with DNA was confirmed by mutational analysis. The 2 ZBDs of DAI can together bind to DNA and both are necessary for full B-to-Z conversion. It is possible that binding 2 DAIs to 1 dsDNA brings about dimerization of DAI that might facilitate DNA-mediated innate immune activation.     

Evidence that the synthesis of glucosylphosphodolichol in yeast involves a 35-kDa membrane protein.

In an effort to identify the polypeptide chain of glucosylphosphodolichol synthase (EC 2.4.1.117), yeast microsomal membranes were allowed to react with 5-azido[beta-32P]UDPGlc, a photoactive analogue of UDPGlc, which is a substrate for this enzyme. Upon photolysis the 32P-labeled probe was shown to link covalently to a 35-kDa protein present in microsomal membranes prepared from several wild-type yeast strains. Binding was either reduced or absent in the microsomal membranes from two yeast mutants (alg5 and dpg1) that are known to be defective in the synthesis of glucosylphosphodolichol. The microsomes isolated from a heterozygous diploid strain alg5::dpg1 generated from these two mutants exhibited partial restoration of both the ability to photolabel the 35-kDa protein and the ability to catalyze the synthesis of glucosylphosphodolichol. Microsomal membranes from a mutant strain that synthesized glucosylphosphodolichol but lacked the ability to transfer the glucosyl residue to the growing lipid-linked oligosaccharide (alg6) exhibited labeling with 5-azido[beta-32P]UDPGlc comparable to that found in microsomes from the wild-type strain. In all cases photoinsertion of the probe into the 35-kDa protein correlated with the level of synthase assayed in the microsomal membranes. These results strongly support the conclusion that the 35-kDa protein labeled in these experiments is a component of glucosylphosphodolichol synthase.     

Significance of the DNA synthesis in hypertrophic cardiomyopathies

DNA content and proliferating cell nuclear antigen (PCNA) expression were investigated in normal hearts, in hypertrophic from hemodynamic overload hearts and in hypertrophic cardiomyopathy (HCM). The aim of this study was mainly to determine whether the hyperdiploid myocardial cells in all cases are in dynamic or static phase.The percentage of PCNA positive cells only in the HCM group was significantly higher (mean value=25.4%) than the percentage of hyperdiploid cells (mean value=9.3%). Therefore, the DNA replication occurs through a different process from that of normal cell cycle which lead to an increase in ploidy and eventually mitosis. These data should be interpreted not only as the result of a periodic amitotic DNA renewal and not even as the result of an increased apoptosis, but especially as a repair process of the DNA molecules affected by a various types of damages in HCMs.     

RNA synthesis in cells infected with herpes simplex virus. II. Evidence that a class of viral mRNA is derived from a high molecular weight precursor synthesized in the nucleus

Viral RNA extracted from the cytoplasmic polyribosomes of human epidermoid carcinoma no. 2 cells infected with herpes simplex virus (mRNA) had a sedimentation coefficient between 10S and 20S while that from nuclei of infected cells varied in size from 10S to >80S. Estimates of the maximum molecular weight of viral RNA from its sedimentation coefficients suggest that at least 10 per cent of the viral genome is transcribed as a single molecule. The ratio of RNA of different sizes found in the nuclei of cells pulse labeled for 12 minutes was approximately the same as those found in cells labeled for longer intervals implying that either some classes of viral mRNA were made as small molecules or that the large viral RNA molecules were cleaved soon after synthesis. Cytoplasmic mRNA competed to a level of at least 80 per cent in viral DNA-RNA hybridization tests with >50S RNA extracted from nuclei of infected cells. This is consistent with the hypothesis that viral mRNA is produced by cleavage of a large precursor RNA molecule.     

Identification of a high molecular weight polypeptide that may be part of the circadian clockwork in Acetabularia

In the chloroplast fraction of the unicellular and uninucleate green alga Acetabularia, we have detected a M_r ≈230,000 protein (p230) whose synthesis exhibits a pronounced endogenous diurnal rhythm. As judged by scanning densitometry of fluorographs of NaDodSO₄/polyacrylamide gels, the synthesis of other proteins in the same fraction was independent of the time in the cycle. The incorporation of [³⁵S]methionine into p230 was completely inhibited by cycloheximide, whereas chloramphenicol had no effect. This strongly suggests that p230 is translated on 80S ribosomes. Eighthour periods of exposure to cycloheximide produced a shift in the phase of the oscillation of p230 synthesis. The results are consistent with the hypothesis that p230 is essential for expression of circadian rhythms in Acetabularia.     

Quantum delocalization of protons in the hydrogen-bond network of an enzyme active site

Enzymes use protein architectures to create highly specialized structural motifs that can greatly enhance the rates of complex chemical transformations. Here, we use experiments, combined with ab initio simulations that exactly include nuclear quantum effects, to show that a triad of strongly hydrogen-bonded tyrosine residues within the active site of the enzyme ketosteroid isomerase (KSI) facilitates quantum proton delocalization. This delocalization dramatically stabilizes the deprotonation of an active-site tyrosine residue, resulting in a very large isotope effect on its acidity. When an intermediate analog is docked, it is incorporated into the hydrogen-bond network, giving rise to extended quantum proton delocalization in the active site. These results shed light on the role of nuclear quantum effects in the hydrogen-bond network that stabilizes the reactive intermediate of KSI, and the behavior of protons in biological systems containing strong hydrogen bonds.Although many biological processes can be well-described with classical mechanics, there has been much interest and debate as to the role of quantum effects in biological systems ranging from photosynthetic energy transfer, to photoinduced isomerization in the vision cycle and avian magnetoreception (1). For example, nuclear quantum effects, such as tunneling and zero-point energy (ZPE), have been observed to lead to kinetic isotope effects of greater than 100 in biological proton and proton-coupled electron transfer processes (2, 3). However, the role of nuclear quantum effects in determining the ground-state thermodynamic properties of biological systems, which manifest as equilibrium isotope effects, has gained significantly less attention (4).Ketosteroid isomerase (KSI) possesses one of the highest enzyme unimolecular rate constants and thus, is considered a paradigm of proton transfer catalysis in enzymology (5–11). The remarkable rate of KSI is intimately connected to the formation of a hydrogen-bond network in its active site (Fig. 1A), which acts to stabilize a charged dienolate intermediate, lowering its free energy by ∼11 kcal/mol (1 kcal = 4.18 kJ) relative to solution (Fig. S1) (6). This extended hydrogen-bond network in the active site links the substrate to Asp103 and Tyr16, with the latter further hydrogen-bonded to Tyr57 and Tyr32, which is shown in Fig. 1A.Open in a separate windowFig. 1.KSI⋅intermediate and KSI^D40N ? inhibitor complex. Schematic depiction of (A) the KSI⋅intermediate complex during the catalytic cycle (Fig. S1) and (B) a complex between KSI^D40N and phenol, an inhibitor that acts as an intermediate analog. Both the intermediate and the inhibitor are stabilized by a hydrogen-bond network in the active site of KSI. (C) Image of KSI^D40N with the tyrosine triad enlarged and the atoms O16, H16, O32, H32, and O57 labeled (shown with Tyr57 deprotonated) (16).The mutant KSI^D40N preserves the structure of the wild-type (WT) enzyme while mimicking the protonation state of residue 40 in the intermediate complex (Fig. 1B), therefore permitting experimental investigation of an intermediate-like state of the enzyme (6, 12–14). Experiments have identified that, in the absence of an inhibitor, one of the residues in the active site of KSI^D40N is deprotonated (12). Although one might expect the carboxylic acid of Asp103 to be deprotonated, the combination of recent ¹³C NMR and ultraviolet visible spectroscopy (UV-Vis) experiments has shown that the ionization resides primarily on the hydroxyl group of Tyr57, which possesses an anomalously low pK_a of 6.3 ± 0.1 (12). Such a large tyrosine acidity is often associated with specific stabilizing electrostatic interactions (such as a metal ion or cationic residue in close proximity), which is not the case here, suggesting that an additional stabilization mechanism is at play (15).One possible explanation is suggested by the close proximity of the oxygen (O) atoms on the side chains of the adjacent residues Tyr16 (O16) and Tyr32 (O32) to the deprotonated O on Tyr57 (O57) (Fig. 1C) (16). In several high-resolution crystal structures, these distances are found to be around 2.6 Å (14, 16, 17), which is much shorter than those observed in hydrogen-bonded liquids such as water, where O–O distances are typically around 2.85 Å. Such short heavy-atom distances are only slightly larger than those typically associated with low-barrier hydrogen bonds (18–20), where extensive proton sharing is expected to occur between the atoms. In addition, at these short distances, the proton’s position uncertainty (de Broglie wavelength) becomes comparable with the O–O distance, indicating that nuclear quantum effects could play an important role in stabilizing the deprotonated residue (Fig. 1C). In this work, we show how nuclear quantum effects determine the properties of protons in the active-site hydrogen-bond network of KSI^D40N in the absence and presence of an intermediate analog by combining ab initio path integral simulations and isotope effect experiments.