In vivo labeling: a glimpse of the dynamic proteome and additional constraints for protein identification

Identities ascribed to the intact protein ions detected in MALDI-MS of whole bacterial cells or from other complex mixtures are often ambiguous. Isolation of candidate proteins can establish that they are of correct molecular mass and sufficiently abundant, but by itself is not definitive. An in vivo labeling strategy replacing methionine with selenomethionine has been employed to deliver an additional constraint for protein identification, i.e., number of methionine residues, derived from the shift in mass of labeled versus unlabeled proteins. By stressing a culture and simultaneously labeling, it was possible to specifically image the cells' response to the perturbation. Because labeled protein is only synthesized after application of the stress, it provides a means to view dynamic changes in the cellular proteome. These methods have been applied to identify a 15,879 Da protein ion from E. coli that was induced by an antibacterial agent with an unknown mechanism of action as SpY, a stress protein produced abundantly in spheroplasts. It has also allowed us to propose protein identities (and eliminate others from consideration) for many of the ions observed in MALDI (and ESI-MS) whole cell profiling at a specified growth condition.     

In situ soft X-ray dynamic microscopy of electrochemical processes

We report a pilot study in in situ electrochemical X-ray dynamic microscopy, based on a model system: the anodic and cathodic behaviour of Ag in neutral NaCl and (NH₄)₂SO₄ aqueous solutions. In situ X-ray imaging highlighted mesoscopic features related to corrosion processes, yielding soluble (NH₄⁺ solution) and insoluble (Cl⁻ solution) corrosion products, as well as cathodic growth morphologies. X-ray absorption and phase contrast images were collected, confirming the feasibility of soft X-ray microscopic measurements with lateral resolution down to a few tens of nm in electrochemical cells for operation in a high vacuum environment in presence of liquid electrolytes.     

In situ video-STM studies of dynamic processes at electrochemical interfaces

Atomic-scale dynamic processes during Cu(100) dissolution/deposition in pure and Cu-containing 0.01 M HCl solution were studied in situ by high-speed electrochemical STM (video-STM). Direct observations of the equilibrium fluctuations at atomic kinks in the steps on the crystal surface due to the local removal/addition of atoms reveal the same anisotropic behavior found previously in Cu-free electrolytes, caused by the influence of the ordered (2 x 2) Cl adlayer on the kink structure. A first quantitative analysis of these fluctuations and interpretation in terms of a local current exchange density was attempted. In addition, observations on the nucleation of vacancy- or ad-rows at terrace corners and within the Cu steps are presented and the relevance of these processes for the macroscopic current density is discussed.     

In situ dynamic measurements of the enhanced SERS signal using an optoelectrofluidic SERS platform

A novel active surface-enhanced Raman scattering (SERS) platform for dynamic on-demand generation of SERS active sites based on optoelectrofluidics is presented in this paper. When a laser source is projected into a sample solution containing metal nanoparticles in an optoelectrofluidic device and an alternating current (ac) electric field is applied, the metal nanoparticles are spontaneously concentrated and assembled within the laser spot, form SERS-active sites, and enhance the Raman signal significantly, allowing dynamic and more sensitive SERS detection. In this simple platform, in which a glass slide-like optoelectrofluidic device is integrated into a conventional SERS detection system, both dynamic concentration of metal nanoparticles and in situ detection of SERS signal are simultaneously possible with only a single laser source. This optoelectrofluidic SERS spectroscopy allows on-demand generation of 'hot spots' at specific regions of interest, and highly sensitive, reliable, and stable SERS measurements of the target molecules in a tiny volume (～500 nL) of liquid sample without any fluidic components and complicated systems.     

In situ evaluation of lipase performances through dynamic asymmetric cyanohydrin resolution

A dynamic resolution process based on multiple reversible cyanohydrin formation coupled to lipase-mediated transesterification is demonstrated. The resulting process resulted in the efficient evaluation of complex lipase performances in asymmetric cyanohydrin acylate synthesis. Dynamic systems were generated and resolved in situ, and the effects of the reaction conditions could be directly monitored for the overall system. By this concept, the enzyme activity, chemo- and stereoselectivity for all involved substrates could be simultaneously evaluated.     

In situ kinetics of layer-by-layer assembled nonlinear-optical-active amphiphiles from dynamic surface force measurements

We report the synthesis and layer-by-layer (LBL) deposition of a class of azo-benzene surfactants with the polycation poly(ethylenimine) (PEI). The different surfactants of the type X-azo-(CH2)10-SO3-, where X = -NO2, -CN, and -COCH3 in the azo-benzene moiety, have decreasing electron-withdrawing strengths. We use dynamic surface force measurements to study the in situ kinetics of adsorption of the amphiphiles onto PEI. Ex situ kinetics data obtained by adsorption-paused UV-visible spectroscopy validate the surface force results. These measurements describe the first application of dynamic force measurements to follow adsorption in LBL systems. UV-visible spectroscopy, second harmonic generation (SHG), and single-wavelength ellipsometry were also used to characterize the films. The observed blue shift upon adsorption of the amphiphiles suggests H-type aggregation within the multilayer. Two of the surfactants studied within the LBL films follow Langmuir adsorption behavior with equilibrium adsorption times under 200 s. The SHG results are consistent with the expected trends in the hyperpolarizabilities of the amphiphiles.     

Covalent labeling of a chromatin reader domain using proximity-reactive cyclic peptides

Chemical probes for chromatin reader proteins are valuable tools for investigating epigenetic regulatory mechanisms and evaluating whether the target of interest holds therapeutic potential. Developing potent inhibitors for the plant homeodomain (PHD) family of methylation readers remains a difficult task due to the charged, shallow and extended nature of the histone binding site that precludes effective engagement of conventional small molecules. Herein, we describe the development of novel proximity-reactive cyclopeptide inhibitors for PHD3—a trimethyllysine reader domain of histone demethylase KDM5A. Guided by the PHD3–histone co-crystal structure, we designed a sidechain-to-sidechain linking strategy to improve peptide proteolytic stability whilst maintaining binding affinity. We have developed an operationally simple solid-phase macrocyclization pathway, capitalizing on the inherent reactivity of the dimethyllysine ε-amino group to generate scaffolds bearing charged tetraalkylammonium functionalities that effectively engage the shallow aromatic ‘groove’ of PHD3. Leveraging a surface-exposed lysine residue on PHD3 adjacent to the ligand binding site, cyclic peptides were rendered covalent through installation of an arylsulfonyl fluoride warhead. The resulting lysine-reactive cyclic peptides demonstrated rapid and efficient labeling of the PHD3 domain in HEK293T lysates, showcasing the feasibility of employing proximity-induced reactivity for covalent labeling of this challenging family of reader domains.

We describe the development of covalent cyclic peptide ligands which target a chromatin methylation reader domain using a proximity-reactive sulfonyl fluoride moiety.     

In situ measurements of nanotube dimensions in suspensions by depolarized dynamic light scattering

We show that the dimensions of carbon nanotubes (CNTs) in suspension can be characterized by depolarized dynamic light scattering. Taking advantages of this in situ technique, we investigate in detail the influence of sonication procedures on the length and diameter of CNTs in surfactant solutions. Sonication power is shown to be particularly efficient at unbundling nanotubes, whereas a long sonication time at low power can be sufficient to cut the bundles with limited unbundling. We finally demonstrate the influence of CNT dimensions on the electrical properties of CNT fibers. Slightly varying the sonication conditions, and thereby the suspended nanotube dimensions, can affect the fibers conductivity by almost 2 orders of magnitude.     

In situ polymerization of the 4-vinylbenzenesulfonic anion in Ni-Al-layered double hydroxide and its molecular dynamic simulation

This paper describes a systematic study on the thermal polymerization of both pristine 4-vinylbenzenesulfonic anion (VBS) and intercalated VBS in the two-dimensional (2D) gallery of Ni-Al layered double hydroxide (VBS/Ni-Al-LDH), by virtue of combining experimental and theoretical investigations. In situ FT-IR, in situ high-temperature X-ray diffraction (HT-XRD), UV-vis absorption spectroscopy, TG-DTA and elemental analysis were used to study the polymerization process, and it was found that the polymerization of VBS/Ni-Al-LDH occurs at ca. 150-170 degrees C, at least 40 degrees C lower than that of the pristine VBS, indicating that the layered structure of LDH is favorable for thermal polymerization of VBS. Therefore, this layered inorganic material may have potential application as a "molecular reactor" for enhancing the efficiency of polymerization reaction. Furthermore, the sheet-like polymerization product was obtained with the LDHs lamella as template. For better understanding the structure and arrangement of intercalated VBS and the polymerization product between the layers of Ni-Al-LDH, molecular dynamics (MD) simulation method was employed. The simulation results of hydration energies show that there are two relatively stable stages upon the increase of the number of interlayer water molecules. VBS molecules exhibit a tendency from tilted to vertical orientation with respect to the layers as the interlayer water content increases. Compared with the experimental results, the calculated interlayer spacing is more severely affected by interlayer water content. Finally, a typical tetramer product of VBS intercalated LDH was studied and the simulated equilibrium interlayer spacing is consistent with the experimental result of in situ HT-XRD. Based on the combination of experimental and theoretical studies on the interlayer polymerization system, the aim of this work is to deeply investigate the differences in thermal polymerization process between pristine monomers and intercalated ones in the gallery of LDHs, and to give detailed information of the arrangement and swelling behavior of guest molecules confined between the sheets of host layers.     

Stochastic dynamic simulation of a protein

The internal motions of a small protein, the bovine pancreatic trypsin inhibitor (BPTI) in solution, are investigated in the framework of the Langevin equation. In this approach, the effects of the solvent molecules are incorporated by suitably defining the friction and random forces. The friction coefficients are determined from a molecular dynamics simulation. The details of the rapid fluctuations of protein atoms obtained by stochastic and molecular dynamics simulation techniques are compared by calculating the generalized density of states obtained via an incoherent neutron scattering. Presently, our stochastic dynamics simulation is one order of magnitude faster than the molecular dynamics simulation with the explicit inclusion of the water molecules. Generalizations of the present stochastic dynamics approach for studying the large-scale motion in proteins are briefly outlined and the probability of a further speedup by an additional order of magnitude is discussed.     

Lunasin: a novel cancer preventive seed peptide that modifies chromatin

Lunasin is a novel cancer preventive peptide whose efficacy against chemical carcinogens and oncogenes has been demonstrated in mammalian cells and a skin cancer mouse model. In contrast, constitutive expression of the lunasin gene in mammalian cells leads to arrest of cell division and cell death. Isolated and characterized in soy, lunasin peptide is also documented in barley and wheat and is predicted to be present in many more seeds because of its possible role in seed development. Initial studies show that lunasin is bioavailable in mice when orally ingested. Lunasin internalizes into mammalian cells within minutes of exogenous application, and localizes in the nucleus after 18 h. It inhibits acetylation of core histones in mammalian cells but does not affect the growth rate of normal and established cancer cell lines. An epigenetic mechanism of action is proposed whereby lunasin selectively kills cells being transformed or newly transformed cells by binding to deacetylated core histones exposed by the transformation event, disrupting the dynamics of histone acetylation-deacetylation.     

Application of capillary electrophoresis to the analysis of soluble chromatin

Capillary electrophoresis (CE) has been applied to study DNA-protein complexes using as the test system soluble chromatin from chicken erythrocytes and rapidly proliferated cultured Chinese hamster fibroblast-like cells B11-dii-FAF-28. Separation was performed with home-made CE apparatus, using a regulated high-voltage power supply, UV-detector and fused silica capillaries with inner diameter 75 microm. The heterogeneity of nucleosomal particles with different DNA lengths after micrococcal nuclease digestion was detected.     

Applying a dynamic method to the measurement of ion mobility

A dynamic method is applied to measure the mobility of gas-phase ions in the dual ion funnel interface of the electrospray source of a quadrupole orthogonal time-of-flight mass spectrometer. In a new operational mode, a potential barrier was formed in the second ion funnel of the mass spectrometer and then progressively increased. In this region, a flow of gas drags the ions into the mass spectrometer while the electric force applied by the potential barrier decelerates them. Ions with lower mobility can be carried by the gas flow more easily than those with high mobility. Thus, electrical forces can block the more mobile ions more easily. Hence, the electric barrier formed in the ion funnel permits only ions below a certain mobility threshold to enter the mass spectrometer. When the barrier voltage is increased, this threshold moves from high to low mobilities. Ions with mobilities above the threshold cannot enter the mass spectrometer, and their signal decreases to zero. Thus, in a barrier voltage scan, mass spectrometric signals of ions sequentially disappear. Differentiation of these decreasing ion signal curves produces peaks from which an ion mobility spectrum can be reconstructed. Blocking voltages, i.e., the positions of the peaks on the barrier voltage scale are directly related to the mobility of these ions. An internal calibration using ions with known mobility values helps determine the unknown ion mobilities and allows calculation of ionic cross sections.     

Superstructures of wet inactive chromatin and the chromosome surface.

Superpacking of chromatin and the surface features of metaphase chromosomes have been studied by SiO replication of wet, unstained, and unfixed specimens in an exceedingly thin (less than or equal to nm) aqueous layer, keeping them wet. Hydrophilic Formvar substrates allow controlled thinning of the aqueous layer covering the wet specimens. Whole mounts of chromatin and chromosomes were prepared by applying a microsurface spreading method to swollen nuclei and mitotic cells at metaphase. The highest level of nucleosome folding of the inactive chromatin in chicken erythrocytes and rat liver nuclei is basically a second-order superhelical organization (width 150--200 nm, pitch distance 50--150 nm) of the elementary nucleosome filament. In unfavorable environments (as determined by ionic agents, fixative, and dehydrating agetns) this superstructure collapses into chains of superbeads and beads. Formalin (10%) apparently attacks at discrete sites of chromatin, which are then separated into superbeads. The latter consist of 4--6 nucleosomes and seemingly correspond to successive turns of an original solenoidal coil (width 30--35 nm), which forms the superhilical organization. When this organization is unfolded, eg, in 1--2 mM EDTA, DNAse-sensitive filaments (diameter 1.7 nm) are seen to be wrapped around the nucleosomes. The wet chromosomes in each metaphase spread are held to each other by smooth microtubular fibers, 20--20 nm in diameter. Before they enter into a chromsome, these fibers branch into 9--13 protofilaments, each 5 nm wide. The chromosome surface contains a dense distribution of subunits about 10--25 nm in diameter. This size distribution corresponds to that of nucleosomes and their superbeads. Distinct from this beaded chromosome surface are several smooth, 23--30-nm-diameter fibers, which are longitudinal at the centromere and seem to continue into the chromatid structure. The surface replicas of dried chromosomes do not show these features, which are revealed only in wet chromosomes.     

Histone modification analysis by chromatin immunoprecipitation from a low number of cells on a microfluidic platform

Histone modifications are important epigenetic mechanisms involved in eukaryotic gene regulation. Chromatin immunoprecipitation (ChIP) assay serves as the primary technique to characterize the genomic locations associated with histone modifications. However, traditional tube-based ChIP assays rely on large numbers of cells as well as laborious and time-consuming procedures. Here we demonstrate a novel microfluidics-based native ChIP assay which dramatically reduces the required cell number and the assay time by conducting cell collection, lysis, chromatin fragmentation, immunoprecipitation, and washing on a microchip. Coupled with real-time PCR, our assay permits the analysis of histone modifications from as few as ~50 cells within 8.5 h. We envision that our method will provide a new approach for the analysis of epigenetic regulations and protein-DNA interactions in general, based on scarce cell samples such as those derived from animals and patients.     

Kerr effect as a tool for the investigation of dynamic heterogeneities

We propose a dynamic Kerr effect experiment for the distinction between dynamic heterogeneous and homogeneous relaxations in glassy systems. The possibility of this distinction is due to the inherent nonlinearity of the Kerr effect signal. We model the slow reorientational molecular motion in supercooled liquids in terms of noninertial rotational diffusion. The Kerr effect response, consisting of two terms, is calculated for heterogeneous and for homogeneous variants of the stochastic model. It turns out that the experiment is able to distinguish between the two scenarios. We furthermore show that exchange between relatively "slow" and "fast" environments does not affect the possibility of frequency-selective modifications. It is demonstrated how information about changes in the width of the relaxation-time distribution can be obtained from experimental results.     

Metallohosts with a heart of carbon

Single-crystal X-ray diffraction studies have confirmed that Ni(3)S(3)-based molecular bowls prepared in one-pot reactions capture either CH(2)Cl(2) or C(60). The nature of the pendant substituents (naphthalen-2-ylmethyl, benzyl, or ethyl) around the rim of the bowl dictates the formation of a 1:1 (bowl host-C(60) guest) or 2:1 (capsule host-C(60) guest) architecture. In CDCl(3), the trimeric complexes were found to be in equilibrium with dimeric analogues. For the naphthalen-2-ylmethyl-substituted host, NMR spectroscopic titration data confirmed a 1:1 host-C(60) guest complex in 1,2-Cl(2)C(6)D(4) solution.     

Templating a polymer-scaffolded dynamic combinatorial library

A water soluble polymer-scaffolded dynamic combinatorial library whose members can interconvert through acylhydrazone exchange was prepared and shown to re-equilibrate in the presence of macromolecular templates.     

Activation by a cytokinin-binding protein of the phosphorylation reaction; Synthesis of RNA and protein in cottonplant chromatin

The dependence of protein kinase and RNA polymerase activities, and also of the synthesis of protein in nuclear chromatin, on the addition of benzylaminopurine and a cytokinin-binding protein to the incubation mixture has been investigated. It has been shown that all three processes are hormone-dependent. It has been found that the activity of protein kinase C is regulated by a cytokinin.A. S. Sadykov Institute of Bioorganic Chemistry, Academy of Sciences of the Republic of Uzbekistan, Tashkent, fax (3712) 62 70 71. Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 745–749, September–October, 1997.