Oxidation-reduction properties of two engineered redox-sensitive mutant Escherichia coli malate dehydrogenases.

Redox potentials for two inactivating intrasubunit disulfides that link helix-5 and helix-9 in mutant Escherichia coli malate dehydrogenases have been determined. The Em is -285 mV when cysteines are at positions 121 and 305 and -295 mV when the cysteines are at positions 122 and 305. Oxidation to the disulfide affects kcat but not Km values. In the single V121C and N122C mutants, the Cys in helix-5 affects the Km for oxalacetate. The pH optimum in the direction of malate formation is affected by the redox state of the enzyme. Clearly, a disulfide bond can and does form between Cys residues substituted into positions 121 or 122 in the nucleotide binding domain and 305 in the carbon substrate binding domain of this NAD-dependent malate dehydrogenase. Apparently, crosslinking the domains interferes with catalysis.

Oxidation-reduction properties of disulfide-containing proteins of the Rhodobacter capsulatus cytochrome c biogenesis system.

Oxidation-reduction titrations for the active-site disulfide/dithiol couples of the helX- and ccl2-encoded proteins involved in cytochrome c biogenesis in the purple non-sulfur bacterium Rhodobacter capsulatus have been carried out. The R. capsulatus HelX and Ccl2 proteins are predicted to function as part of a dithiol/disulfide cascade that reduces a disulfide on the apocytochromes c so that two cysteine thiols are available to form thioether linkages between the heme prosthetic group and the protein. Oxidation-reduction midpoint potential (E(m)) values, at pH 7.0, of -300 +/- 10 and -210 +/- 10 mV were measured for the HelX and Ccl2 (a soluble, truncated form of Ccl2) R. capsulatus proteins, respectively. Titrations of the disulfide/dithiol couple of a peptide designed to serve as a model for R. capsulatus apocytochrome c(2) have also been carried out, and an E(m) value of -170 +/- 10 mV was measured for the model peptide at pH 7.0. E(m) versus pH plots for HelX, Ccl2, and the apocytochrome c(2) model peptide were all linear over the pH range from 5.0 to 8.0, with the -59 mV/pH unit slope expected for a reaction in which two protons are taken up for each disulfide that is reduced. These results provide thermodynamic support for the proposal that HelX reduces Ccl2 and that reduced Ccl2, in turn, serves as the reductant for the production of the two thiols of the CysXxxYyyCysHis heme-binding motif of the apocytochromes.

Oxidation-reduction properties of chloroplast thioredoxins, ferredoxin:thioredoxin reductase, and thioredoxin f-regulated enzymes.

Oxidation-reduction midpoint potentials were determined, as a function of pH, for the disulfide/dithiol couples of spinach and pea thioredoxins f, for spinach and Chlamydomonas reinhardtii thioredoxins m, for spinach ferredoxin:thioredoxin reductase (FTR), and for two enzymes regulated by thioredoxin f, spinach phosphoribulokinase (PRK) and the fructose-1,6-bisphosphatases (FBPase) from pea and spinach. Midpoint oxidation-reduction potential (Em) values at pH 7.0 of -290 mV for both spinach and pea thioredoxin f, -300 mV for both C. reinhardtii and spinach thioredoxin m, -320 mV for spinach FTR, -290 mV for spinach PRK, -315 mV for pea FBPase, and -330 mV for spinach FBPase were obtained. With the exception of spinach FBPase, titrations showed a single two-electron component at all pH values tested. Spinach FBPase exhibited a more complicated behavior, with a single two-electron component being observed at pH values >/= 7.0, but with two components being present at pH values <7.0. The slopes of plots of Em versus pH were close to the -60 mV/pH unit value expected for a process that involves the uptake of two protons per two electrons (i. e., the reduction of a disulfide to two fully protonated thiols) for thioredoxins f and m, for FTR, and for pea FBPase. The slope of the Em versus pH profile for PRK shows three regions, consistent with the presence of pKa values for the two regulatory cysteines in the region between pH 7.5 and 9.0.

Deformation measurement with object-induced dynamic phase shifting.

A simple, yet powerful, means of computing the phase of fringe patterns depicting dynamic phenomena is presented. It is shown that the basic principle of the phase-shifting methods can be extended to the case of dynamic situations. The crux is to recognize that the phenomenon under examination can itself provide the necessary incremental phase shifts. This new method possesses a very wide range of applications in the field of deformation measurement.

Phase shifting of whole-field fringes in dual-plate speckle photography.

New developments of a phase-shifting readout system for the analysis of whole-field fringes in dual-plate speckle photography are presented.

Phase shifting of whole field speckle photography fringes.

A phase shifting readout system is used to analyze whole field fringes in dual plate speckle photography and preliminary results obtained with a first prototype are presented.

Measurement of difference deformation using speckle interferometry.

An application of speckle interferometry is presented that allows one to compare the resistance to stress of two nominally identical specimens. The interference pattern gives contours of equal differences in displacements of the two stressed specimens. The method is briefly developed, and its experimental feasibility is demonstrated by comparing the deflections of two square plates clamped along the edges and subjected to centrally concentrated loads.

Mechanical testing of the external fixator by holographic interferometry.

Tests carried out on the Hoffmann- Vidal external fixator by means of holographic interferometry are reported. These tests refer exclusively to the quality of functioning of the joints connecting transfixing pins and side bars. It is a question of 1) evaluating the behavior of the ball joint subjected to realistic loads, 2) verifying the rigidity of a lightened joint bowl as compared to a heavier one, and 3) comparing the respective resistance of two similar joint models to a rotation of the bar around its axis. The main findings are the remarkable elastic behavior of the ball joint, the effectiveness of the lightened bowl, and the fact that two cousin models may behave quite differently with respect to their resistance to axial slipping rotation of the bar. The interferometric technique is thus expected to have further applications in similar evaluations.

Speckle motions induced by rigid-body movements in freespace geometry: an explicit investigation and extension to new cases.

In line with the current interest in speckle motion in free-space propagation, it is proposed to investigate new aspects of the induced speckle motion due to rigid-body movements of a model using as a starting point the well-known concept of homology, first stated in holographic interferometry, the importance of which has only recently been realized in speckle metrology. At the outset, stating the homology conditions leads to a general expression relating the speckle shift to the geometrical parameters at the recording and to the six degrees of movement of the object surface. The expression presented in this explicit form is found to be true for both opaque and thin transparent diffusing models and for different conditions of illumination and observation. The study is then extended to (1) when the object is at rest, subjecting the illuminating source to a small displacement and (2) when the wavelength of the illuminating beam is changed between the two exposures. The case of the rigid-body displacement of the object is experimentally verified and the results are found to be in good agreement with theory. The viability of the method to make measurements is assessed, several applications are envisaged, and some advantages are pointed out.