Structure and functional complementation of engineered fragments from yeast phosphoglycerate kinase

Previous studies have shown that, although the isolated structuraldomains of yeast phosphoglycerate kinase recover a quasi-nativestructure in vitro as well as in vivo, they do not reassociatenor generate a functional enzyme. The aim of this work was firstto study the folding of complementary fragments different fromstructural domains and second to determine the requirementsfor their reassociation and functional complementation. Themethod used for producing rigorously defined fragments consistsof the introduction of a unique cysteinyl residue in the proteinfollowed by a specific cleavage by 5'5'-dithiobis(2-nitrobenzoate)/potassiumcyanide at this residue. Two pairs of complementary fragmentswere thus obtained, 1–96/97–415 and 1–248/249–415.The structure and stabilities of the different fragments werestudied. The short fragments, i.e. 1–96 and 249–415were found to contain some secondary structure, but to havea low stability. Each large fragment has a high structural contentand a stability close to that of the corresponding domain. Incontrast to that observed with the isolated domains, a weakbut significant complementation was observed for the two pairsof fragments; the pair of fragments 1–248/249–415recovered 8% of the activity of the native enzyme upon complementation.An independent refolding of the complementary fragments beforereassociation decreased the yield of complementation for thepair of fragments 1–96/97–415, but did not affectthe complementation for the other pair (1–248/249–415).From the present data and previous work on the isolated domains,it appears that the correct folding of the isolated fragmentsis not a prerequisite for their complementation.     

Structure-based design of inhibitors specific for bacterial thymidylate synthase

Thymidylate synthase is an attractive target for antiproliferative drug design because of its key role in the synthesis of DNA. As such, the enzyme has been widely targeted for anticancer applications. In principle, TS should also be a good target for drugs used to fight infectious disease. In practice, TS is highly conserved across species, and it has proven to be difficult to develop inhibitors that are selective for microbial TS enzymes over the human enzyme. Using the structure of TS from Lactobacillus casei in complex with the nonsubstrate analogue phenolphthalein, inhibitors were designed to take advantage of features of the bacterial enzyme that differ from those of the human enzyme. Upon synthesis and testing, these inhibitors were found to be up to 40-fold selective for the bacterial enzyme over the human enzyme. The crystal structures of two of these inhibitors in complex with TS suggested the design of further compounds. Subsequent synthesis and testing showed that these second-round compounds inhibit the bacterial enzyme at sub-micromolar concentrations, while the human enzyme was not inhibited at detectable levels (selectivities of 100-1000-fold or greater). Although these inhibitors share chemical similarities, X-ray crystal structures reveal that the analogues bind to the enzyme in substantially different orientations. Site-directed mutagenesis experiments suggest that the individual inhibitors may adopt multiple configurations in their complexes with TS.     

Investigation of ultrasonic wave scattering by a randomly rough solid-solid interface

An imperfect interface between two dissimilar materials is modeled by a random interface profile. A theoretical study of the interaction of ultrasonic waves with the rough solid-solid interface is presented. The reflection and transmission coefficients for longitudinal and shear coherent waves are calculated as a function of the angle of incidence within the framework of a second order perturbation theory. The effects of the statistical interface parameters, as well as the interface spectral density on the scattered fields, are investigated. These results are used to determine the roughness-induced attenuation of the coherent fields as a function of the above parameters. In addition, the relation between the incoherent part of the scattering cross-section, and interface roughness is examined.     

Discriminating surface-breaking from subsurface cracks in pressurized water pipes by means of a parametric modulation technique

The discrimination of surface-breaking cracks from cracks that are entirely embedded within the material is paramount to the assessment of the integrity of pressurized water pipes. This work addresses this issue investigating the use of a parametric modulation technique towards this end. Tests are conducted on 1D surface-breaking cracks that are either dry or contain water. The response of these defects is investigated as a function of crack depth and closure, as well as of amplitude and frequency of the modulation. The ensuing results suggest that fluid-filled and dry cracks can be discriminated from each other. The origin of the different response of these defects is discussed in terms of both crack's stiffness and modulation-induced dynamics.     

Folding, heterodimeric association and specific peptide recognition of a murine alphabeta T-cell receptor expressed in Escherichia coli

In a systematic study of the murine T-cell receptor UZ3-4, expressed and refolded from inclusion bodies in Escherichia coli, it was found that functional molecules can be obtained only under a very narrow set of conditions. The refolded T-cell receptor UZ3-4 specifically recognizes its cognate peptide (from mycobacterial Hsp60) in the context of H-2Db, but not another peptide bound to H-2Db, and the dissociation constant was determined by BIAcore as 10(-4) M. Using T-cell receptor constructs comprising all extracellular domains (ValphaCalpha and VbetaCbeta), found to be necessary for stability of the final product, significant amounts of native molecules were obtained only if the intermolecular Calpha-Cbeta disulfide bridge bond was deleted, even though the interaction between the complete alpha and beta-chain was determined to be very weak and fully reversible (KD approximately 10(-7) to 10(-6) M). Fusion of Jun and Fos to the constant domains also decreased the folding yield, because of premature association of intermediates leading to aggregation. Furthermore, only in a very narrow set of concentrations of oxidized and reduced glutathione, native disulfide bonds dominated. This shows that T-cell receptor domains are very prone to aggregation and misassociation during folding, compounded by incorrect disulfide bond formation. Once folded, however, the heterodimeric molecule is very stable and could be concentrated to millimolar concentration.     

Acoustic microscopy and dispersion of leaky Rayleigh waves onrandomly rough surfaces: a theoretical study

A theoretical investigation of the dispersion of leaky Rayleigh waves propagating along one-dimensional (1-D) rough fluid-solid interfaces was carried out by simulating the measurement process of a line-focus beam acoustic microscope. The interface profiles were described in terms of their rms, also known as the roughness of the profile, autocorrelation length, and autocorrelation function. The reflectivity of the interfaces was calculated by using a second-order perturbation approach in the profile roughness. Theoretical V(z) curves were generated and analyzed to yield values of the phase velocity of the Rayleigh waves. The dependence of the Rayleigh wave velocity on the profile and material parameters was examined. Significant variations of the phase velocity were found for values of the roughness which are small compared to the shortest of the wavelengths involved in the scattering. The dispersion relations also showed considerable sensitivity to changes in mechanical properties typical of materials of engineering interest. In the low-frequency range, simulations indicated the dispersion of Rayleigh waves to be rather insensitive to the spectral content of the profile     

Characterizing Particle Flow by Acoustic Emission

A simple theoretical model which captures the statistical nature of an acoustic emission (AE) signal generated by a stream of solid particles impinging on a flat solid surface is presented. It rests on well-known results dating back to the fundamental work by Hertz, which assumes the impact to be elastic, the particle to be spherical, and the surface on which the particle bounces perfectly flat. The average power of the signal is evaluated in two limits. In the first one, the frequency content of the signal is identical to that of the forces developed during impact. In the second limit, only the low frequency part of the source function contributes to the spectrum of the recorded signal, the transfer function of the measuring system acting as a low-pass filter. In this case, it is shown that the result has an immediate physical interpretation, although its practical relevance is still limited. Additional progress is accomplished by experimentally proving that the statistical average of the particles’ momentum may be replaced with the average flow velocity, a quantity that may be directly measured in real life situations. It is suggested that this simplified version of the theoretical result may provide a useful tool to characterize particle flow in systems of industrial interest.     

Dairy maturation of milk used in the manufacture of Parmigiano-Reggiano cheese: effects on physico-chemical characteristics, rennet-coagulation aptitude and rheological properties

The aim of this research was to study the effects of dairy maturation on the physico-chemical characteristics and technological properties of milk used for Parmigiano-Reggiano cheese manufacture. Three different operating conditions (CF1, CF2 and CF3) were considered. Full cream milk from the evening milking was stored on the farm and delivered to the cheese factory in churns (CF1) or in thermoregulated tanks at a temperature not lower than 18 degrees C (CF2 and CF3). The natural creaming (10-12 h overnight) was performed in a traditional large flat vat containing 10-12 hl (CF1 and CF2) or in thermoregulated large flat vats containing 60 hl at about 15 degrees C (CF3). Twenty-four, 24 and 22 maturation trials were performed in CF1, CF2, and CF3, respectively, during 2 consecutive years. A significant increase (P相似文献   

Forced Nonlinear Vibrations of a One-Dimensional Bar with Arbitrary Distributions of Hysteretic Damage

An analytical model is presented which predicts the forced, nonlinear response of a bar with arbitrarily distributed damage. Damage, which is either described by quadratic hysteresis, or due to dislocations interacting with point defects distributed along the dislocations’ glide planes, is considered. The wave equation is solved by means of a perturbation approach. Resonance frequency shift caused by damage-induced material softening, nonlinear attenuation, and higher harmonics’ generation are evaluated. For damage which is described by quadratic hysteresis, this model recovers the well-known dependence of the three acoustic quantities mentioned above on the source’s strength. On the other hand, for damage due to dislocations, both frequency shift and nonlinear attenuation present a distinctive nonlinear behavior the origin of which resides in the stress dependence of the fraction of dislocations breaking away from the point defects. Furthermore, different distributions of damage having the same integrated intensity are shown to generate nonlinear effects of increasing magnitude as their spatial extent decreases. Finally, it is suggested that, once the effect of the source’s strength is removed, spectral features may be used to assess the spatial extent of damage.     

Ultrasonic wave scattering from rough,imperfect interfaces. Part II. Incoherent and coherent scattered fields

A theoretical model for ultrasonic wave scattering for geometrically irregular and imperfectly bonded interfaces is presented. Part I presents the stochastic interface characterization and a model for its mechanical response based on a micromechanics model of asperity contact. Part II uses this interface representation to write the well used quasi-static boundary conditions for scattering from a.flat imperfect interface¹ directly on the irregular interface profile. The boundary conditions are then expanded in an asymptotic series in the roughness parameter (standard deviation of the surface height) which is small compared to wavelength. The slope of the profile must also be everywhere small. These equations are solved exactly for the zero-th and second order terms, which are the flat coherent solution and its' first coherent correction, and the first order term, which is the first term in the expansion for the incoherently scattered solution. Results for obliquely incident longitudinal and shear waves show a strong dependence on the roughness in both the coherent and incoherent reflected fields, but little if any dependence on the roughness in the transmitted fields. In particular, the reflected coherent fields show markedly increasing attenuation compared to the flat compliant interface with increasing roughness and increasing ultrasonic frequency, the latter result being in qualitative agreement with results for scattering from an inhomogeneous array of individual scatterers.² There is evidence in the incoherent reflected fields for the existence of an incoherent leaky interface disturbance which manifests itself as a bulk incoherent shear wave at a scattering angle equal to the critical longitudinal angle. A coherent true interface wave is also supported by the rough interface which is shown to further attenuate the coherent reflected fields compared to the flat compliant interface solution.