Promod R. Pratap

Neural system for structural health monitoring

This is an overview paper that discusses the concept of an embeddable structural health monitoring system for use in composite and heterogeneous material systems. The sensor system is formed by integrating groups of autonomous unit cells into a structure, much like neurons in biological systems. Each unit cell consists of an embedded processor and a group of distributed sensors that gives the structure the ability to sense damage. In addition, each unit cell periodically updates a central processor on the status of health in its neighborhood. This micro-architectured synthetic nervous system has an advanced sensing capability based on new continuous sensor technology. This technology uses a plurality of serially connected piezoceramic nodes to form a distributed sensor capable of measuring waves generated in structures by damage events, including impact and crack propagation. Simulations show that the neural system can detect faint acoustic waves in large plates. An experiment demonstrates the use of a simple neural system that was able to measure simulated acoustic emissions that were not clearly recognizable by a single conventional piezoceramic sensor.

An Artificial Neural Receptor System for Structural Health Monitoring

The artificial neural receptor system is a new approach that greatly simplifies data acquisition, and hence may enable practical Structural Health Monitoring for large structures. The benefits of the system involve reducing data acquisition channels while maintaining the ability to extract substantial structural response information. The approach presented uses an array of piezoelectric sensors wired to mimic the basic receptor connectivity of the biological nervous system. The method of solving for array outputs from individual sensor strains is demonstrated using a 3-by-3 sensor array. For an N-by-N array of sensors, the number of channels of data acquisition is reduced from N 2 in a conventional system to 2N in the artificial neural receptor system. The first few microseconds of signal output from the array rows and columns can determine the location of the excitation. An artificial neural network analysis can be used to extract excitation locations and individual sensor strains from the array. Acoustic emissions and dynamic strains are measured by the array.

FTIR study of ATP-induced changes in Na+/K+-ATPase from duck supraorbital glands

The Na+/K+-ATPase uses energy from the hydrolysis of ATP to pump Na+ ions out of and K+ ions into the cell. ATP-induced conformational changes in the protein have been examined in the Na+/K+-ATPase isolated from duck supraorbital salt glands using Fourier transform infrared spectroscopy. Both standard transmission and attenuated total internal reflection sample geometries have been employed. Under transmission conditions, enzyme at 75 mg/ml was incubated with dimethoxybenzoin-caged ATP. ATP was released by flashing with a UV laser pulse at 355 nm, which resulted in a large change in the amide I band. The absorbance at 1659 cm(-1) decreased with a concomitant increase in the absorbance at 1620 cm(-1). These changes are consistent with a partial conversion of protein secondary structure from alpha-helix to beta-sheet. The changes were approximately 8% of the total absorbance, much larger than those seen with other P-type ATPases. Using attenuated total internal reflection Fourier transform infrared spectroscopy, the decrease in absorbance at approximately 1650 cm(-1) was titrated with ATP, and the titration midpoint K0.5 was determined under different ionic conditions. In the presence of metal ions (Na+, Na+ and K+, or Mg2+), K0.5 was on the order of a few microM. In the absence of these ions, K0.5 was an order of magnitude lower (0.1 microM), indicating a higher apparent affinity. This effect suggests that the equilibrium for the ATP-induced conformational changes is dependent on the presence of metal ions.

Fluorescence measurements of nucleotide association with the Na(+)/K(+)-ATPase.

The Na(+)/K(+)-ATPase, a membrane-associated ion pump, uses energy from the hydrolysis of ATP to pump 3 Na(+) ions out of and 2 K(+) into cells. The dependence of ATP hydrolysis on ATP concentration was measured using a fluorescence coupled-enzyme assay. The dependence on concentration of nucleotide association with the ATPase was examined using ADP and ATP-induced quenching of the fluorescence of ATPase labeled with Cy3-maleimide (Cy3-ATPase) or Alexa Fluor 546 carboxylic acid, succinimidyl ester (AF-ATPase). The kinetics of ATP hydrolysis in the presence of Na(+) and K(+) exhibited negative cooperativity with a Hill coefficient (n(H)) of 0.66 and a half-maximal concentration (K(0.5)) of 61 microM; in the absence of K(+), n(H) was 0.58 and K(0.5) was 13 microM. Nucleotide-induced fluorescence quenching exhibited negative cooperativity with an n(H) of 0.3-0.5. These results suggest that negative cooperativity observed in ATP hydrolysis is attributable to negative cooperativity in nucleotide association to the ATPase. Interaction between AF-ATPase and ATP labeled with Alexa Fluor 647 (AF-ATP) showed significant Förster resonance energy transfer (FRET). These results indicate that the ATPase exists as oligoprotomeric complexes in this preparation, and that this aggregation has significant effects on enzyme function.

Artificial nerve system for structural monitoring

Recent structural health monitoring techniques have focused on developing global sensor systems that can detect damage on large structures. The approach presented here uses a piezoelectric sensor array system that mimics the biological nervous system architecture to measure acoustic emissions and dynamic strains in structures. The advantage of this approach is that the number of channels of data acquisition used for an N-by-N sensor array can be reduced from N2 to 2N. For large arrays the number of data acquisition channels is tremendously reduced. When transient damage events occur on the structure, the array output time histories can be recorded and the location of the excitation can be accurately determined using combinatorial logic. A trade-off is the difficulty of extracting individual sensor time histories from the array outputs without a neural network or a regressive technique. Only the sums of the sensor strains of each row and column can be exactly calculated using the voltage outputs of the array. The array approach allows efficient use of data acquisition instrumentation for structural health monitoring. Applications for the sensor array include crack and delamination detection, dynamic strain measurement, impact detection, and localization of damage on large complex structures.

NUCLEOTIDE BINDING TO IAF-LABELLED NA+/K+-ATPASE MEASURED BY STEADY STATE FLUORESCENCE QUENCHING BY TNP-ADP

Nucleotide binding to 5-iodoacetamidofluorescein (IAF) labelled Na+/K(+)-ATPase was measured by steady state fluorescence quenching of the fluorescein label via energy transfer to trinitrophenyl (TNP) labelled nucleotide. TNP-nucleotides are valuable probes of nucleotide binding to ATPases. Interpretation of these and other experiments in our laboratory using TNP-nucleotides with the Na+/K(+)-ATPase rely on having a good model for the interaction of TNP-nucleotide with the enzyme. Sets of fluorescence quenching curves obtained by titrating the enzyme with TNP-ADP in the presence of various concentrations of ADP could not be adequately modelled using a simple model with a single nucleotide binding site. Therefore, we compare various models which allow for additional TNP-nucleotide binding to the enzyme. In the two-site model, the additional binding is to a second specific site for which TNP-nucleotide and unlabelled nucleotide compete. In two other models, the additional binding (in one case saturable, and in the other case non-saturable) of TNP-nucleotide is not blocked by or affected by unlabelled nucleotide, and is, therefore, referred to as non-specific binding of the TNP-nucleotide. The goal of this work is to determine which of the distinctly different physical pictures associated with these models most accurately describes the interaction of TNP-nucleotide with the enzyme. We find that the interaction of TNP-ADP with IAF-labelled Na+/K(+)-ATPase is best described by a model in which there are two classes of binding: TNP-ADP and ADP compete for a specific binding site with dissociation binding constants of 0.13 microM for TNP-ADP and 2.0 microM for ADP; and non-saturable non-specific binding of TNP-ADP.

Kinetics of conformational changes associated with potassium binding to and release from Na+K+-ATPase

The Na+/K(+)-ATPase functions in cells to couple energy from the hydrolysis of ATP to the transport Na+ out and K+ in. The fluorescent probe IAF (iodoacetamidofluorescein) covalently binds to this enzyme, reporting conformational changes without inhibiting enzyme activity. This paper describes experiments using dog kidney enzyme labeled with IAF to examine kinetics of conformational changes resulting from added Na+ and K+, measured in terms of steady-state and stopped-flow fluorescence changes. Kinetics of these fluorescence changes were examined as a function of temperature from two initial conditions: (a) enzyme in the high-fluorescence form (E(high)) was rapidly mixed with varying [K+]; and (b) enzyme in the low-fluorescence form (E(low)) was rapidly mixed with varying [ATP]. These experiments showed: (1) The rate constant for the fluorescence change from E(high) to E(low) was much larger than that for the opposite transition, E(low) to E(high); (2) the apparent free energy of activation (Ea(app)) for the two transitions were different (as estimated from Arrhenius plots); (3) under steady-state conditions, IAF fluorescence did not change when ATP was added to E(low)(K+) in the absence of Na+; (4) the apparent free energy of activation was independent of [K+] for the E(high) to E(low) transition (at 16.4 kcal/mol) but increased with [ATP] for the E(low) to E(high) transition; (5) Ea(app) for the E(low) to E(high) transition with 1 mM ATP was approximately the same as that in the absence of ATP (34 kcal/mol). These results can be interpreted as: (i) in the transition from E(low) to E(high), IAF reported a conformational change that occurred after K+ release to the intracellular side and which is involved in Na+ binding; (ii) Ea(app) increased with [ATP], while increasing the entropy of the transition state. Thus, ATP appeared to destabilize the enzyme during the transition from E(low) to E(high).

TRANSIENT KINETICS OF SUBSTRATE BINDING TO NA+/K+-ATPASE MEASURED BY FLUORESCENCE QUENCHING

This paper examines the transient kinetics of substrate binding to the Na+/K(+)-ATPase labelled with iodoacetamidofluorescein (IAF) using fluorescence quenching by trinitrophenyl-ATP (TNP-ATP). Earlier work (E.H. Hellen, P.R. Pratap, 1996, Fluorescence quenching of IAF-Na+/K(+)-ATPase via energy transfer to TNP-labelled nucleotide, Proceedings of the VIIIth International Conference on the Na+/K(+)-ATPase, in press) has shown that TNP-nucleotide binds to specific sites (from which unlabelled nucleotide can displace it) and nonspecific sites (from which unlabelled nucleotide cannot displace it). Under stopped-flow conditions, quenching of IAF-enzyme fluorescence was well described by a stretched exponential (F(t) = F infinity + delta F exp[-Bt alpha]). Physically, this function may be interpreted in terms of its inverse Laplace transform phi (k), which describes a distribution of rate-constants; alpha reflects the width of this distribution. As TNP-ATP concentration increased, alpha decreased, reflecting TNP-ATP binding to sites with higher energy barriers. alpha decreased by about the same amount with increasing [TNP-ATP] in the presence of saturating ATP, indicating that the distribution of rate-constants is largely associated with the nonspecific binding sites. However, alpha was significantly less than 1 for ATP-induced fluorescence recovery in the presence of TNP-ATP, indicating that rate-constants associated with specific binding site are also distributed. The distribution of rate-constants for binding to the specific site indicates a distribution in the energy of the transition state for substrate binding. These results suggest that the specific binding site (in either the empty or the full state) may exist in a series of conformations separated by small energy barriers. However, the energy barriers for binding associated with these conformations are significantly distributed.

Transient kinetics and thermodynamics of anthroylouabain binding to Na/K-ATPase

The Na/K-ATPase is an integral membrane protein enzyme which uses energy derived from hydrolysis of ATP to pump Na+ out of and K+ into the cell. Ouabain belongs to a class of drugs known as cardiac glycosides, which are useful for treating congestive heart failure. Therapeutic value is achieved when these drugs bind to and inhibit the Na/K-ATPase of cardiac muscle. We gain insight into this important interaction by measuring the thermodynamics of the interaction of anthroylouabain (AO), a fluorescent derivative of ouabain, with the Na/K-ATPase. AO has the useful property that its fluorescence intensity is greatly enhanced (approximately 10x) when it binds to the enzyme. Using this enhancement, we measure temperature dependence of transient kinetics for the association and dissociation of AO interacting with membrane fragments of Na/K-ATPase purified from dog kidney. Using a standard Eyring analysis, we find that the overall association of AO with the enzyme is driven by substantial contributions from both enthalpy and entropy, and that in an energy diagram for the association pathway, the free energy change is quite similar to that of ouabain deduced from previously published results [E. Erdmann, W. Schoner, BBA 307 (1973) 386]. However, in the transition state, there are substantial differences for the enthalpy and entropy, presumably due to the presence of the anthracene moiety.

The voltage-sensitive dye RH421 detects a Na+,K+-ATPase conformational change at the membrane surface

RH421 is a voltage-sensitive fluorescent styrylpyridinium dye which has often been used to probe the kinetics of Na+,K+-ATPase partial reactions. The origin of the dyes response has up to now been unclear. Here we show that RH421 responds to phosphorylation of the Na+,K+-ATPase by inorganic phosphate with a fluorescence increase. Analysis of the kinetics of the fluorescence response indicates that the probe is not detecting phosphorylation itself but rather a shift in the proteins E1/E2 conformational equilibrium induced by preferential phosphate binding to and phosphorylation of enzyme in the E2 conformation. Molecular dynamics simulations of crystal structures in lipid bilayers indicate some change in the proteins hydrophobic thickness during the E1-E2 transition, which may influence the dye response. However, the transition is known to involve significant rearrangement of the proteins highly charged lysine-rich cytoplasmic N-terminal sequence. Using poly-l-lysine as a model of the N-terminus, we show that an analogous response of RH421 to the E1→E2P conformational change is produced by poly-l-lysine binding to the surface of the Na+,K+-ATPase-containing membrane fragments. Thus, it seems that the prime origin of the RH421 fluorescence response is a change in the interaction of the proteins N-terminus with the surrounding membrane. Quantum mechanical calculations of the dyes visible absorption spectrum give further support to this conclusion. The results obtained indicate that membrane binding and release of the N-terminus of the Na+,K+-ATPase α-subunit are intimately involved in the proteins catalytic cycle and could represent an effective site of regulation.