Anti-opsonic properties of staphylokinase

Recently we described a novel bacteriophage-encoded pathogenicity island in Staphylococcus aureus that harbors a number of virulence factors that are all involved in the evasion of innate immunity. Here we describe a mechanism by which staphylokinase (SAK), frequently present on this pathogenicity island, interferes with innate immune defenses: SAK is anti-opsonic. By activating human plasminogen (PLG) into plasmin (PL) at the bacterial surface, it creates bacterium-bound serine protease activity that leads to degradation of two major opsonins: human immunoglobulin G (IgG) and human C3b. Incubation of opsonized bacteria with PLG and SAK resulted in removal of anti-staphylococcal IgGs and C3b from the bacterial surface. In phagocytosis assays this proved to be a very efficient mechanism to reduce the opsonic activity of human IgG and serum. The fact that SAK activates human PLG at the bacterial surface and removes IgG as well as C3b makes this protein a unique anti-opsonic molecule.     

Physical properties of rod-shaped molecules in solution

Several theories describing the physical properties of solutions containing rod-shaped molecules are reviewed. The results of the various theories are presented in a convenient form for implementation on a calculator or microcomputer. Expressions are derived for light scattering from thin rods using rational approximations for the sine and cosine integrals. These expressions apply to turbidity, Rayleigh, and dynamic light-scattering measurements. Accurate approximating equations are given for the various diffusional motions involved in dynamic light scattering. For shear-dependent viscosity and flow birefringence, empirically derived interpolation formulas are given. These provide values of the Simha factor, extinction angle and orientation function for a wide range of axial ratios and velocity gradients with relative errors less than 1%. We demonstrate how these equations can be applied to predict or model the properties of a solution containing thin rods of various lengths.     

The conformational properties of sucrose in aqueous solution: intramolecular hydrogen-bonding

A detailed analysis is presented of the nuclear (¹H and ¹³C) magnetic resonance (n.m.r.) properties of sucrose, using both D₂O and dimethyl sulfoxide-d₆ as solvents, based on measurements of coupling constants, chemical shifts, T₁ relaxation times, and nuclear Overhauser enhancements. Molecular modelling (HSEA calculations) suggests a strong conformational preference about the glycosidic linkages that is near to that for sucrose in the crystalline state, and this conformational rigidity is fully supported by the n.m.r. data, in terms of lack of influence of changes in concentration and temperature on the relevant n.m.r. parameters. The restricted rotation for the 1-hydroxymethyl group of the fructose residue is related to the persistence of the intramolecular hydrogen-bond between O-1^f and O-2^g. The presence of this bond was established for solutions in (CD₃)₂SO by the observation of isotopic chemicashifts on partial deuteration of the hydroxyl groups. The orientation of the 6-hydroxyl methyl group of the fructose residue is not that present in the crystalline state but, in (CD₃)₂SO, it may be intramolecularly hydrogen-bonded, as was demonstrated by titration of the hydroxyl groups with CD₃OD. Observations are made regarding hydrophobic topographies common to sucrose, saccharin, and 1-chloro-1-deoxysucrose, which may have a bearing on sweetness.     

The determination of the conformational properties of nucleic acids in solution from NMR data

A program, NUCFIT, has been written for simulating the effects of conformational averaging on nuclear Overhauser enhancement (NOE) intensities for the spin systems found in nucleic acids. Arbitrary structures can be generated, and the NOE time courses can be calculated for truncated one-dimensional NOEs, two-dimensional NOE and rotating frame NOE spectroscopy (NOESY and ROESY) experiments. Both isotropic and anisotropic molecular rotation can be treated, using Woessner's formalism (J. Chem. Phys. (1962) 37, 647-654). The effects of slow conformational averaging are simulated by taking population-weighted means of the conformations present. Rapid motions are allowed for by using order parameters which can be supplied by the user, or calculated for specific motional models using the formalism of Tropp (J. Chem. Phys. (1980) 72, 6035-6043). NOE time courses have been simulated for a wide variety of conformations and used to determine the quality of structure determinations using NMR data for nucleic acids. The program also allows grid-searching with least-squares fitting of structures to experimental data, including the effects of spin-diffusion, conformational averaging and rapid internal motions. The effects of variation of intra and internucleotide conformational parameters on NOE intensities has been systematically explored. It is found that (i) the conformation of nucleotides is well determined by realistic NOE data sets, (ii) some of the helical parameters, particularly the base pair roll, are poorly determined even for extensive, noise-free data sets, (iii) conformational averaging of the sugars by pseudorotation has at most second-order influence on the determination of other parameters and (iv) averaging about the glycosidic torsion bond also has, in most cases, an insignificant effect on the determination of the conformation of nucleotides.     

CD spectrum and conformational distribution of cyclotuftsin in solution

CD spectra for low-energy conformations of the tuftsin cycloanalogue, , were calculated. A theoretical spectrum obtained as the weighted average of calculated spectra for individual peptide backbone conformers is qualitatively consistent with an experimental CD spectrum in aqueous solution. The conformational distribution allows one to achieve agreement between calculated and experimental values of structural parameters of the cyclotuftsin molecule investigated by NMR spectroscopy.

CD spectrum calculation Theoretical conformational analysis Tuftsin cycloanalog Peptide conformation     

A potentiometric and CD investigation on the conformational properties of poly (L-p-aminophenylalanine) in aqueous solution

The conformational properties of poly(L -p-aminophenylalanine) have been investigated by potentiometric and circular dichroism (CD) techniques. It has been found that the polymer in the charge-free state can assume two ordered conformations, depending upon temperature conditions. At room temperature the polymer assumes the right-handed helical form by described Goodman and Peggion.¹ At temperatures higher than 40°C a new ordered conformation has been found, whose slow rate of formation and ir absorption properties are typical of the β-structure. The thermodynamic parameters relative to the coil-β transition, determined by potentiometric titration techniques, revealed that the thermodynamic stability of the β-structure is mainly due to enthalpic factors. The formation of this structure is unfavored on a kinetic basis.     

Persistent conformational heterogeneity of triosephosphate isomerase: separation and characterization of conformational isomers in solution

Subunit dissociation of dimeric rabbit muscle triosephosphate isomerase (TIM) by hydrostatic pressure has previously been shown not to follow the expected dependence on protein concentration [Rietveld and Ferreira (1996) Biochemistry 35, 7743-7751]. This anomalous behavior was attributed to persistent conformational heterogeneity (i.e., the coexistence of long-lived conformational isomers) in the ensemble of TIM dimers. Here, we initially show that subunit dissociation/unfolding of TIM by guanidine hydrochloride (GdnHCl) also exhibits an anomalous dependence on protein concentration. Dissociation/unfolding of TIM by GdnHCl was investigated by intrinsic fluorescence and circular dichroism spectroscopies and was found to be a highly cooperative transition in which the tertiary and secondary structures of the protein were concomitantly lost. A procedure based on size-exclusion chromatography in the presence of intermediate (0.6 M) GdnHCl concentrations was developed to isolate two conformational isomers of TIM that exhibit significantly different stabilities and kinetics of unfolding by GdnHCl. Complete unfolding of the two isolated conformers at a high GdnHCl concentration (1.5 M), followed by refolding by removal of the denaturant, completely abolished the differences in their unfolding kinetics. These results indicate that such differences stem from conformational heterogeneity of TIM and are not related to any chemical modification of the protein. Furthermore, they add support to the notion that long-lived conformational isomers of TIM coexist in solution and provide a basis for the interpretation of the persistent heterogeneity of this protein.     

Probing protein hydration and conformational states in solution.

The addition of polyethylene glycol (PEG), of various molecular weights, to solutions bathing yeast hexokinase increases the affinity of the enzyme for its substrate glucose. The results can be interpreted on the basis that PEG acts directly on the protein or indirectly through water activity. The nature of the effects suggests to us that PEG's action is indirect. Interpretation of the results as an osmotic effect yields a decrease in the number of water molecules, delta Nw, associated with the glucose binding reaction. delta Nw is the difference in the number of PEG-inaccessible water molecules between the glucose-bound and glucose-free conformations of hexokinase. At low PEG concentrations, delta Nw increases from 50 to 326 with increasing MW of the PEG from 300 to 1000, and then remains constant for MW-PEG up to 10,000. This suggests that up to MW 1000, solutes of increasing size are excluded from ever larger aqueous compartments around the protein. Three hundred and twenty-six waters is larger than is estimated from modeling solvent volumes around the crystal structures of the two hexokinase conformations. For PEGs of MW > 1000, delta Nw falls from 326 to about 25 waters with increasing PEG concentration, i.e., PEG alone appears to "dehydrate" the unbound conformation of hexokinase in solution. Remarkably, the osmotic work of this dehydration would be on the order of only one k T per hexokinase molecule. We conclude that under thermal fluctuations, hexokinase in solution has a conformational flexibility that explores a wide range of hydration states not seen in the crystal structure.     

Concentration-dependent conformational transition of alpha-elastin in aqueous solution

Circular dichroism measurements on aqueous solutions of alpha-elastin have given evidence of beta-bend structure at elevated concentrations. When Ca2+ was added, the concentration-dependent conformational transition was somewhat inhibited and the binding of the metal ion was shown, by means of equilibrium dialysis, to be essentially independent from alpha-elastin concentration in the range of 1 to 10 mg/ml. The results obtained are discussed in connection with the elasticity and calcification of elastin.     

Activation and conformational changes of adenylate kinase in urea solution

Ａｄｅｎｙｌａｔｅｋｉｎａｓｅ(ＥＣ2.7.4.3)ｃａｔａｌｙｚｅｓｔｈｅｉｎｔｅｒｃｏｎｖｅｒｓｉｏｎｏｆａｄｅｎｉｎｅｎｕｃｌｅｏｔｉｄｅｓａｃｃｏｒｄｉｎｇｔｏ:ＡＤＰ+ＭｇＡＤＰＡＭＰ+ＭｇＡＴＰ.Ｉｔｉｓｕｂｉｑｕｉｔｏｕｓａｎｄｐａｒｔｉｃｕｌａｒｌｙａｂｕｎｄａｎｔｉｎｔｉｓｓｕｅｓｗｉｔｈｈｉｇｈｅｎｅｒｇｙｔｕｒｎｏｖｅｒ.Ｒａｂｂｉｔｍｕｓｃｌｅａｄｅｎｙｌａｔｅｋｉｎａｓｅｉｓａｍｏｎｏｍｅｒｅｎｚｙｍｅｗｉｔｈｔｗｏｓｔｒｕｃｔｕｒｅｄｏｍａｉｎｓｃｏｍｐｏｓｅｄｏｆ194ａｍｉｎｏａ…     

The conformational study of chitin and chitosan oligomers in solution

The inter-residual dihedral angles phi and phip of chitin and chitosan oligomers were determined from experimental 3J(C-H) constants and ROESY cross peaks.     

X-Ray conformational study of the DNA duplex in solution

Earlier x-ray studies on dissolved linear DNA molecules were interpreted on the assumption that the molecules scattered as rigid rods. Improvement in equipment and advances in theory of the scattering from randomly oriented helices prompted us into a reinvestigation of this problem. Careful measurements were made on the scattering from both linear calf thymus DNA and from circular plasmid C0P608 superhelical DNA. Contrary to the earlier work, we find that the scattering patterns show a helical character, with maxima corresponding to those of a helix with pitch angle of 62°, close to that of the C-W helix. The patterns for both types of DNA, although similar, show a 5% displacement of the maximum in the superhelical form, just that expected when the C-W helix is superimposed on a superhelix axis. Introduction of intercalators (PtTS) causes a progressive extension of the helix, as shown by a shift to larger angles and a fading out of the maximum. In the concentration range of 40 mg/mL, interfernce peaks develop, the result of an apparent stacking of the chains, with an interchain distance of ～35 Å.     

Proteolytic modification of staphylokinase

There are three types of staphylokinase of different isoelectric points (6.7, 6.1 and 5.7). Staphylokinase of pI 6.7 was converted to that of pI 6.1 and then to that of pI 5.7 by the treatment with trypsin.Heterogeneity of staphylokinase might be the result of post-translational modification by proteolytic enzyme.     

重组葡激酶的分离纯化

在构建出高表达,高活性的葡激酶工程菌株的基础上,对表达产物进行了分离纯化研究。经离子交换纯化后,纯度达８５％－９０％,回收率为５０％－６０％,经凝胶过滤后,纯度达９９％以上,回收率为６０％,经ＳＤＳ－ＰＡＧＥ测定,相对分子质量为１５．５＊１０＾３,比活为１．０＊１０＾５,Ｎ端氨基酸序列与献报道的相符。     

Spectral properties of conformational motion in proteins

The conformational dynamics of large fragments of protein structure within the framework of a generalized model of bounded diffusion (GMBD) have been considered. This model is a development of the known ordinary model of bounded diffusion (MBD). The latter model assumes that the driving force of motion, which is at the same time a source of friction, has negligibly small correlation time (white noise). In contrast to the MBD, the GMBD takes into account the finite character of friction correlation time, i.e. memory friction effects. Two different mechanisms of friction for the fragment are considered: (1) friction by rapid density fluctuations due to the harmonic vibrational motion of atoms and (2) friction by slow encounters with surrounding fragments of protein structure. The present theory shows that at high frequencies the power decay of the spectrum which takes place in the MBD is replaced by an exponential one. In the narrow intermediate frequency range the results of the GMBD coincide with those of the MBD. At low frequencies the results of the two models differ quantitatively, though their qualitative behaviour is similar.