Hyperthermostable mutants of Bacillus licheniformis {alpha}-amylase: multiple amino acid replacements and molecular modelling

We have identified previously two critical positions for thethermostability of the highly thermostable -amylase from Bacilluslicheniformis. We have now introduced all 19 possible aminoacid residues to these two positions, His 133 and Ala209. Themost favourable substitutions were to Ile and Val, respectively,which both increased the half-life of the enzyme at 80°Cby a factor of 3. At both positions a stabilizing effect ofhydrophobic residues was observed, although only in the caseof position 133 could a clear correlation be drawn between thehydrophobicity of the inserted amino acid and the gain in proteinstability. The construction of double mutants showed a cumulativeeffect of the most favourable and/or deleterious substitutions.Computer modelling was used to generate a 3-D structure of thewild-type protein and to model substitutions at position 209,which lies in the conserved (/ß)₈ barrel domain of-amylase; Ala209 would be located at the beginning of the thirdhelix of the barrel, in the bottom of a small cavity facingthe fourth helix. The model suggests that replacement by, forexample, a valine could fill this cavity and therefore increaseintra- and interhelical compactness and hydrophobic interactions.     

Plant pectin methylesterase and its inhibitor from kiwi fruit: Interaction analysis by surface plasmon resonance

Two surface plasmon resonance (SPR)-based interaction analysis methods were successfully implemented to explore the binding between plant PME and kiwi PMEI. In a first method, plant PMEs were immobilised on a chip surface via amine coupling. This experimental setup allowed studying the effect of pH and ionic strength on the PME–PMEI interaction kinetics. Strong binding was obtained at pH < 7 and at low salt concentrations, whereas both pH ? 8 and [NaCl] of ca. 1.0 M effectively caused dissociation. In a second method, kiwi PMEI was immobilised on a chip surface to which streptavidin had been covalently attached. Hereto, PMEI was biotinylated by means of a NHS-biotin reagent. With this immobilisation strategy, the effect of (partial) thermal or high pressure-induced denaturation of PME on its affinity towards PMEI was investigated. A notable degree of enzyme inactivation was required before interaction characteristics were significantly altered. Any incomplete inactivation of PME resulted in binding to the PMEI surface.     

State estimation and detection of changes in time interval models

This paper presents an estimation approach for Time Event Graphs such as P-Time Event Graphs and Time Stream Event Graphs. It is assumed that the nominal behavior is known and that transitions are partitioned as observable and unobservable transitions. The technique is applied to the detection of changes which are (possibly small) finite variations of dynamic models compared to this nominal behavior. The detected changes provide indications that can be used in future maintenance operations. Using the algebra of dioids, the approach uses a receding-horizon estimation of the greatest state and analyzes the consistency of the data.     

Experimental determination of self-heating in submicrometer MOStransistors operated in a liquid-helium ambient

Self-heating (SH) in submicrometer CMOS transistors operated at liquid-helium temperature and under different bias conditions was experimentally verified by measuring the temperature T_Si in the proximity of the device. T_Si was measured by using a silicon resistor, placed in the same bulk nearby the device under test, as a temperature sensor. It was found that the heat generated by the NMOS transistor of a CMOS inverter structure penetrates deep into the substrate and reduces very strongly the n-well impedance, giving rise to large variations in the kink of the I_drain -V_drain characteristics of the neighbor PMOS transistor. Experimental results confirm that SH must not be underestimated when characterizing and modeling low-temperature device operation     

Characterization of plasminogen activator inhibitor 1 mutants containing the P13 to P10 region of ovalbumin or antithrombin III: evidence that the P13 residue contributes significantly to the active to substrate transition

The serpin plasminogen activator inhibitor 1 (PAI-1) can occur, in vitro, in both an inhibitory and a non-inhibitory but cleavable substrate form. In the present study, we have evaluated the effect of replacing the P13 to P10 region of PAI-1 (Val-Ala-Ser-Ser), with the P13 to P10 region of either the non-inhibitory serpin ovalbumin (Glu-Val-Val-Gly; PAI-1-ovalbumin) or the inhibitory serpin antithrombin III (Glu-Ala-Ala-Ala; PAI-1-antithrombin III). In addition, we have replaced Val at position P13 with Glu (PAI-1-P13 (Val-->Glu)). Wild-type (wt) PAI-1 revealed specific activities of 80+/-9% (mean+/-S.D., n=4) of the theoretical maximum value towards t-PA. PAI-1-ovalbumin, PAI-1-antithrombin III and PAI-1-P13 (Val-->Glu) revealed specific activities of 86+/-15%, 77+/-11%, and 100+/-30% respectively, towards t-PA and similar inhibitory properties towards u-PA. Surprisingly, upon inactivation at 37 degreesC, the active conformation of the PAI-1 mutants converted partly into a substrate conformation (i.e. 52+/-5.2%, 55+/-8.2% and 46+/-4.6% for PAI-1-ovalbumin, PAI-1-antithrombin III and PAI-1-P13 (Val-->Glu), respectively) and partly into a latent conformation. This is in contrast to active wtPAI-1 which, as expected, is converted to the latent conformation (i.e. 86+/-1.0%). In conclusion, even though replacement of the P13 to P10 region of PAI-1 by the corresponding region of a non-inhibitory serpin or of an inhibitory serpin, does not directly affect its inhibitory properties, the nature of the amino acids in this region and of P13 in particular, contributes to its conformational transitions.     

Experimental results on fast surface states and 1/f noise in m.o.s. transistors

Experimental results for 1/f noise in m.o.s. transistors with very low surface state densities are presented. A direct relation between noise and surface state density is found, extending previously published data.     

Compromise approach for predictive control of Timed Event Graphs with specifications defined by P-time Event Graphs

In this paper, the aim is to make the predictive control of a plant described by a Timed Event Graph which follows the specifications defined by a P-time Event Graph. We propose a compromise approach between the ideal optimality of the solution and the on-line application of the computed solution when the relevant optimal control cannot be applied for a given computer. The technique is based on a reduction of the number of iterations of the fixed point algorithm such that the computed control remains causal. The analysis of the partial satisfaction of the specifications at each iteration of the algorithm defined in the (max, + ) algebra shows that a subset of constraints is guaranteed by the control computed at each iteration while another one is possibly satisfied.