Self-assembled enzymatic monolayer directly bound to a gold surface: activity and molecular recognition force spectroscopy studies

Escherichia coli dihydrofolate reductase (ecDHFR) has one surface cysteine, C152, located opposite and distal to the active site. Here, we show that the enzyme spontaneously assembles on an ultraflat gold surface as a homogeneous, covalently bound monolayer. Surprisingly, the activity of the gold-immobilized ecDHFR as measured by radiographic analysis was found to be similar to that of the free enzyme in solution. Molecular recognition force spectroscopy was used to study the dissociation forces involved in the rupture of AFM probe-tethered methotrexate (MTX, a tight-binding inhibitor of DHFR) from the gold-immobilized enzyme. Treatment of the ecDHFR monolayer with free MTX diminished the interaction of the functionalized tip with the surface, suggesting that the interaction was indeed active-site specific. These findings demonstrate the viability of a simple and direct enzymatic surface-functionalization without the use of spacers, thus, opening the door to further applications in the area of biomacromolecular force spectroscopy.     

Effects of the donor-acceptor distance and dynamics on hydride tunneling in the dihydrofolate reductase catalyzed reaction

A significant contemporary question in enzymology involves the role of protein dynamics and hydrogen tunneling in enhancing enzyme catalyzed reactions. Here, we report a correlation between the donor-acceptor distance (DAD) distribution and intrinsic kinetic isotope effects (KIEs) for the dihydrofolate reductase (DHFR) catalyzed reaction. This study compares the nature of the hydride-transfer step for a series of active-site mutants, where the size of a side chain that modulates the DAD (I14 in E. coli DHFR) is systematically reduced (I14V, I14A, and I14G). The contributions of the DAD and its dynamics to the hydride-transfer step were examined by the temperature dependence of intrinsic KIEs, hydride-transfer rates, activation parameters, and classical molecular dynamics (MD) simulations. Results are interpreted within the framework of the Marcus-like model where the increase in the temperature dependence of KIEs arises as a direct consequence of the deviation of the DAD from its distribution in the wild type enzyme. Classical MD simulations suggest new populations with larger average DADs, as well as broader distributions, and a reduction in the population of the reactive conformers correlated with the decrease in the size of the hydrophobic residue. The more flexible active site in the mutants required more substantial thermally activated motions for effective H-tunneling, consistent with the hypothesis that the role of the hydrophobic side chain of I14 is to restrict the distribution and dynamics of the DAD and thus assist the hydride-transfer. These studies establish relationships between the distribution of DADs, the hydride-transfer rates, and the DAD's rearrangement toward tunneling-ready states. This structure-function correlation shall assist in the interpretation of the temperature dependence of KIEs caused by mutants far from the active site in this and other enzymes, and may apply generally to C-H→C transfer reactions.     

Monte Carlo simulation of ion beam-assisted solid phase epitaxy

Abstract

We have developed a computer simulation of ion beam-induced epitaxial crystallisation (IBIEC) based on the familiar kink-and-ledge model which describes thermally activated solid phase epitaxy. In the latter, kinks are assumed to form (via thermal activation) on ledges. Here, we assume that the deposited ion energy induces kink formation on the terraces as well as on the ledges. We then derive, with minimal restrictions on the growth laws, the interface morphology evolution. The latter is correlated to the growth speed, and an interpretation of the IBIEC temperature dependence is proposed. The power laws which characterise the simulated interface evolution are those of the Kardar-Parisi-Zhang universality class.     

Fourier Interferometry as Applied to Microspectrofluorometry and Fluorescence Imaging of Living Cells

Excitation-emission fluorescence spectroscopy and imaging are applied to studies of cellular metabolism and at the convergence of cellular differentiation, detoxification, transformation, and senescence. Metabolic activity, intracellular redox levels, and compartmentation are probed by coenzyme [NAD(P)H] transients and monochlorobimane for glutathione dehydrogenase. Gene expression or its failure in lysosomal disorders is identified with fluorogenic probes. The "multiorganelle detoxification complex" is visualized and investigated with cytotoxic agents. A kind of photoactivated "accelerated cellular senescence" is recognized by accumulation of Schiff bases. Conventional and novel mitochondrial probes are used to localize these organelles in Saccharomyces cerevisiae as a model for future studies in mammalian cells and to detect in these very cells the organelle interactions resulting from the action of mitochondria-toxic drugs. The potential of these studies for biotechnology and instrumentation relying on fibers and integrated optics is considerably enhanced by Fourier interferometry.     

Boundary conditions for the Swain-Schaad relationship as a criterion for hydrogen tunneling

Hydrogen quantum mechanical tunneling has been suggested to play a role in a wide variety of hydrogen-transfer reactions in chemistry and enzymology. An important experimental criterion for tunneling is based on the breakdown of the semiclassical prediction for the relationship among the rates of the three isotopes of hydrogen (hydrogen -H, deuterium -D, and tritium -T). This is denoted the Swain-Schaad relationship. This study examines the breakdown of the Swain-Schaad relationship as criterion for tunneling. The semiclassical (no tunneling) limit used hereto (e.g., 3.34, for H/T to D/T kinetic isotope effects), was based on simple theoretical considerations of a diatomic cleavage of a stable covalent bond, for example, a C-H bond. Yet, most experimental evidence for a tunneling contribution has come from breakdown of those relationship for a secondary hydrogen, that is, not the hydrogen whose bond is being cleaved but its geminal neighbor. Furthermore, many of the reported experiments have been mixed-labeling experiments, in which a secondary H/T kinetic isotope effect was measured for C-H cleavage, while the D/T secondary effect accompanied C-D cleavage. In experiments of this type, the breakdown of the Swain-Schaad relationship indicates both tunneling and the degree of coupled motion between the primary and secondary hydrogens. We found a new semiclassical limit (e.g., 4.8 for H/T to D/T kinetic isotope effects), whose breakdown can serve as a more reliable experimental evidence for tunneling in this common mixed-labeling experiment. We study the tunneling contribution to C-H bond activation, for which many relevant experimental and theoretical data are available. However, these studies can be applied to any hydrogen-transfer reaction. First, an extension of the original approach was applied, and then vibrational analysis studies were carried out for a model system (the enzyme alcohol dehydrogenase). Finally, the effect of complex kinetics on the observed Swain-Schaad relationship was examined. All three methods yield a new semiclassical limit (4.8), above which tunneling must be considered. Yet, it was found that for many cases the original, localized limit (3.34), holds fairly well. For experimental results that are between the original and new limits (within statistical errors), several methods are suggested that can support or exclude tunneling. These new and clearer criteria provide a basis for future applications of the Swain-Schaad relationship to demonstrate tunneling in complex systems.     

Andreev reflections on Y(1-x)Ca(x)Ba(2)Cu(3)O(7-delta): evidence for an unusual proximity effect

We have measured Andreev reflections between an Au tip and Y(1-x)Ca(x)Ba(2)Cu(3)O(7-delta) thin films in the in-plane orientation. The conductance spectra are best fitted with a pair potential having the d(x2-y2)+is symmetry. We find that the amplitude of the is component is enhanced as the contact transparency is increased. This is an indication for an unusual proximity effect that modifies the pair potential in the superconductor near the surface with the normal metal.     

A programmable electronic circuit for modelling CO2 laser dynamics

We introduce a programmable electronic circuit implementing the rich dynamics of CO2 laser models. The design and the implementation of the circuit are accomplished by using a programmable analog device, which permits an experimental characterization of the laser dynamics. The experimental results shown in the paper demonstrate that the circuit exhibits homoclinic chaos typical of CO2 laser with feedback modulation of cavity losses. Moreover, experimental results showing that noise regularizes the dynamical time scales of the system are reported.     

Production and characterization of polyclonal antibodies to sulfamethazine and their potential use in immunoaffinity chromatography for urine sample pre-treatment

An immunoaffinity chromatographic (IAC) method for isolating sulfamethazine (SMZ) from incurred urine samples was developed. This was achieved by (i) generating polyclonal antibodies that recognize equally well SMZ and its major urinary metabolites, (ii) evaluating in an ELISA procedure the influence of methanol, salt and pH on the antigen-antibody interaction in order to determine the optimum conditions for IAC and (iii) covalent coupling of the IgG fractions of anti-SMZ to CNBr activated Sepharose for the preparation of re-usable immunoaffinity columns, having a high capacity for SMZ (1900 ng SMZ mL-1 gel). For desorbing SMZ from the immunoaffinity column, different elution modes were evaluated, with 40% MeOH-0.1 mol L-1 HOAc-0.5 mol L-1 NaCl being the most efficient combination. Using the IAC column for processing SMZ spiked urine samples resulted in high recoveries, ranging from 92 to 100%. Because of the high cross-reactivity with the major metabolites of SMZ present in urine of treated animals, the antibodies show excellent properties for use in both IAC and ELISA. For the isolation and concentration of the parent drug and its major metabolites, the urine could be applied directly to the IAC column, without the time-consuming step of deconjugation. Moreover, the use of IAC prior to ELISA for the analysis of incurred urine samples showed good efficiency for the elimination of matrix interferences. Owing to the urine-tissue relationship, the urine concentrations can be used to predict the presence of the parent drug in tissues and so possible violations of the maximum residue limit (MRL) can be controlled.