Dimer–monomer equilibrium of human thymidylate synthase monitored by fluorescence resonance energy transfer

An ad hoc bioconjugation/fluorescence resonance energy transfer (FRET) assay has been designed to spectroscopically monitor the quaternary state of human thymidylate synthase dimeric protein. The approach enables the chemoselective engineering of allosteric residues while preserving the native protein functions through reversible masking of residues within the catalytic site, and is therefore suitable for activity/oligomerization dual assay screenings. It is applied to tag the two subunits of human thymidylate synthase at cysteines 43 and 43′ with an excitation energy donor/acceptor pair. The dimer–monomer equilibrium of the enzyme is then characterized through steady‐state fluorescence determination of the intersubunit resonance energy transfer efficiency.     

Factors determining the reconstructive denaturation of proteins in sodium dodecyl sulfate solutions. Further circular dichroism studies on structural reorganization of seven proteins

The factors determining the onset and extent of reconstructive denaturation of proteins were considered by comparing circular dichroism (CD) data of seven proteins and previously published findings. The effects of sodium dodecyl sulfate (SDS) on the conformation of the following proteins were tested: lysozyme, the mitogens fromPhytolacca americana (fractions Pa2 and Pa4), lectin fromWistaria floribunda, ovine lutropin, a Bence Jones protein, and histone H2B. While the helix content of lysozyme was raised by SDS slightly, in the Bence Jones protein andW. floribunda lectin it increased from near zero to about 25–30%. In histone H2B the helix content was raised by SDS even to about 48%. However, no clear indication of helix formation could be observed in the mitogens and lutropin, even at low pH or 2.0–2.5. The tertiary structure of the proteins was perturbed by SDS. It was concluded that the reorganization of secondary structure of the proteins was favored by the following factors: (1) presence of helicogenic amino acid sequences in the protein, (2) availability of positively charged sites of the basic amino acids for interactions with the dodecyl ion, (3) absence of a large surplus of negatively charged sites on the surface of protein, and (4) absence of extensive disulfide cross-linking within the macromolecule. Both hydrophobic and electrostatic interactions occur in reconstructive denaturation, and the newly formed helices are stabilized by hydrophobic shielding by the alkyl chains of the alkyl sulfate.     

Molecular characterization of human platelet glycoproteins IIIa and IIb and the subunits of the latter

Sedimentation equilibrium and low-angle laser-light scattering were used to determine the molar mass of the glycoprotein moieties in the complexes of sodium dodecyl sulphate with the human platelet membrane glycoproteins IIb (GPIIb), IIIa (GPIIIa), and the (GPIIb) and (GPIIb) subunits of GPIIb. The values obtained by both procedures, except those for GPIIb, agree within experimental error with those calculated from their chemical composition: GPIIb (114,000 g mol^-1), GPIIb (22,200 g mol^-1), and GPIIIa (91,500 g mol^-1). The molar mass of GPIIb determined by light scattering (142,000 g mol^-1) and sedimentation equilibrium at different solvent densities (134,000 g mol^-1) also agree, within experimental error, with the values calculated either from its chemical composition (136,500 g mol^-1) or from the sum of the molar masses of its subunits. However the molar mass determined by sedimentation equilibrium at constant solvent density, is consistently underestimated (116,000 g mol^-1).High-performance size-exclusion chromatography in sodium dodecyl sulphate solutions overestimates the molar mass of these glycoproteins and their Stokes radii, and therefore the maximal frictional ratios derived from them.Abbreviations GPIIb glycoprotein IIb - GPIIIa glycoprotein IIIa - GPIIb and GPIIb and subunits of GPIIb, respectively - CM-GPIIb CM-GPIIb, and CM-GPIIIa, totally reduced and carboxymethylated forms of GPIIb, GPIIb, and GPIIIa, respectively - SDS sodium dodecyl sulphate - eosin-ITC eosin-5-isothiocyanate     

Chromosome aberration and ploidy equilibrium of Vicia faba in tissue culture

Summary Different phytohormone concentrations induced different fequencies of various chromosome aberrations in calli of Vicia faba. NAA 10 ppm plus KT 2.5 ppm produced more haploids and NAA 30 ppm plus NAA 7.5 ppm produced more tetraploids and breakage. The relationship among the aberrations was analyzed. The hypothesis of ploidy equilibrium was established. The chromosome doubling rate and reduction rate of each treated group were calculated in relation to the observed data and the hypothesis. The frequency of tetraploids and breakage are correlated with each other. The frequency of total aberrations is linearly correlated with that of micronucleus formation. The regression equation is x=31.92+ 10.67 y.     

Physicochemical characterization of alpha-crystallins from bovine lenses: hydrodynamic and conformational properties

A detailed investigation of hydrodynamic and conformational behavior has been made of the HM-crystallin and -crystallins of bovine lens. Results from this study indicated that HM (high-molecular-weight -crystallin) and (low-molecular-weight -crystallin) possess considerable size and charge heterogeneities in their native structures and subunit polypeptides, respectively. Sedimentation velocity showed a heterogeneous polydisperse system of HM with an average sedimentation coefficient of about 50 S and a more homogeneous system of -crystallin of 20 S. Viscosity and circular dichroism studies pointed to a compact and globular shape of dominant -sheet conformation for -crystallin, yet a highly asymmetrical and aggregated form for HM. The conformational stability of -crystallin was investigated in the presence of various denaturants. The evidence presented shows that hydrogen bonding is the main force in maintaining the quaternary structure of compact native -crystallin. Conformational flexibility of -crystallin demonstrated in the equilibrium unfolding study indicated a multistep transition that made the extraction of thermodynamic data from the heat denaturation study difficult. Temperature perturbation on -crystallin suggested the possible involvement of hydrophobic interaction in the aggregation process, leading to the formation of HM from -crystallin. The comparison of conformational properties between HM and -crystallin strongly indicated that HM is a denatured form of -crystallin.     

Conformational change of bovine serum albumin by heat treatment

The thermal denaturation of bovine serum albumin (BSA) was studied at pH 2.8 and 7.0 in the range of 2–65°C. The relative proportions of -helix, -structure, and disordered structure in the protein conformation were determined as a function of temperature, by the curve-fitting method of circular dichroism spectra. With the rise of temperature at pH 7.0, the proportion of -helix decreased above 30°C and those of -structure and disordered structure increased in the same temperature range. The structural change was reversible in the temperature range below 45°C. However, the structural change was partially reversible upon cooling to room temperature subsequent to heating at 65°C. On the other hand, the structural change of BSA at pH 2.3 was completely reversible in the temperature range of 2–65°C, probably because the interactions between domains and between subdomains might disappear due to the acid expansion. The secondary structure of disulfide bridges-cleaved BSA remained unchanged during the heat treatment up to 65°C at pH 2.8 and 7.0.     

Structural similarities among the high molecular weight protein fractions of oilseeds

Data on the physico-chemical properties of proteins from soybean, groundnut, sesame seed, sunflower seed, safflower seed, mustard seed, rapeseed and cotton seed are fairly extensive. An examination of the available data on high molecular weight proteins suggests that there are similarities in many of their properties. In this report the similarity in amino acid composition, size and shape, molecular weight, secondary structure, subunit composition, association-dissociation at high and low pH, stability towards denaturants, hydrolysis by enzymes and quaternary structure of the high molecular weight proteins is discussed. Based on these similarities a model has been proposed for the associationdissociation, denaturation and reassociation behaviour of the high molecular weight proteins of oilseeds.     

Metal ion stabilization of the conformation of a recombinant 19-kDa catalytic fragment of human fibroblast collagenase.

A recombinant 19-kDa human fibroblast collagenase catalytic fragment modeled on a naturally occurring proteolytic product was purified from E. coli inclusion bodies. Following renaturation in the presence of zinc and calcium, the fragment demonstrated catalytic activity with the same primary sequence specificity against small synthetic substrates as the full-length collagenase. Unlike the parent enzyme, it rapidly cleaved casein and gelatin but not native type I collagen. Intrinsic fluorescence of the three tryptophan residues was used to monitor the conformational state of the enzyme, which underwent a 24-nm red shift in emission upon denaturation accompanied by quenching of the fluorescence and loss of catalytic activity. Low concentrations of denaturant unfolded the fragment while the full-length enzyme displayed a shallow extended denaturation curve. Calcium remarkably stabilized the 19-kDa fragment, zinc less so, while together they were synergistically stabilizing. Among divalent cations, calcium was the most effective stabilizer, EC50 approximately 60 microM, and similar amounts were required for substrate hydrolysis. Catalytic activity was more sensitive to denaturation than was tryptophan fluorescence. Least sensitive was the polypeptide backbone secondary structure assessed by CD. These observations suggest that the folding of the 19-kDa collagenase fragment is a multistep process stabilized by calcium.     

Prospective theoretical and experimental study towards electrochemically grafted poly(N-vinyl-2-pyrrolidone) films on metallic surfaces

Quantum chemistry calculations of equilibrium geometry, atomic charges, dipole moment, and frontier orbital energies are carried out on model N-vinyl-2-pyrrolidone, its radical anion and cation, and three protonated derivatives. Attempts to obtain poly(N-vinyl-2-pyrrolidone) films grafted on nickel and platinum electrodes, respectively by cathodic and anodic polarizations, are reported. A thin, covering and insulating film is obtained on the Pt anode, as indicated by surface characterizations carried out with UPS and IRRAS spectroscopies. The fact that the film remains adherent to the metallic surface in spite of a permanent contact with an acetonitrile solution in which poly(N-vinyl-2-pyrrolidone) is soluble suggests that chemisorption has indeed been achieved.     

人肌肌酸激酶胍变性时的失活与构象变化的比较研究

应用二阶导数光谱、紫外差吸收光谱和荧光光谱等监测手段,研究了人肌肌酸激酶在盐酸胍溶液中的构象变化。二阶导数光谱结果表明,若以6M盐酸胍中肌酸激酶酪氨酸残基的暴露程度为100％,则天然酶酪氨酸残基的暴露程度只有2％。而紫外差吸收光谱和荧光光谱的变化与兔肌肌酸激酶的结果相似。比较不同胍浓度下人肌肌酸激酶的失活与构象变化,表明酶的失活先于构象变化。同时还测定了不同浓度胍溶液中人肌酶的失活与构象变化的速度常数。结果表明以几种方法测定的构象变化均为单相的一级过程,而酶的失活却呈现了由快慢两相组成的一级反应过程。比较同浓度胍溶液中的失活速度与构象变化速度,发现酶失活的快相反应速度常数比构象变化的速度常数大1—2个数量级,慢相速度常数与构象变化速度常数相近。上述结果进一步支持了酶的活性部位构象柔性的观点。