scholarly journals Characterization of the solution properties and conformation of pneumolysin, the membrane-damaging toxin of Streptococcus pneumoniae

1993 ◽  
Vol 296 (3) ◽  
pp. 671-674 ◽  
Author(s):  
P J Morgan ◽  
P G Varley ◽  
A J Rowe ◽  
P W Andrew ◽  
T J Mitchell
Keyword(s):  
Streptococcus Pneumoniae ◽  
Tertiary Structure ◽  
Analytical Ultracentrifugation ◽  
Sedimentation Coefficient ◽  
Spectroscopic Methods ◽  
Solution Conformation ◽  
Structure Information ◽  
Self Association ◽  
Secondary And Tertiary Structure

Pneumolysin is a membrane-damaging toxin produced by Streptococcus pneumoniae. In order to understand fully the mode of action of this toxin, it is necessary to have an appreciation of the size, self-association behaviour and solution conformation of pneumolysin. A combination of analytical ultracentrifugation methodologies has shown that pneumolysin lacks self-association behaviour in solution and has provided a weight-average M(r) (M omega) of 52,000 +/- 2000, which was in agreement with that derived from the amino acid sequence. By determining a sedimentation coefficient (S20,w0) of 3.35 +/- 0.10 S, it was possible to suggest a model for the gross solution conformation of pneumolysin monomers. Spectroscopic methods provide additional secondary and tertiary structure information.

Masking of the Fc region in human IgG4 by constrained X-ray scattering modelling: implications for antibody function and therapy

2010 ◽  
Vol 432 (1) ◽  
pp. 101-114 ◽  
Author(s):  
Yuki Abe ◽  
Jayesh Gor ◽  
Daniel G. Bracewell ◽  
Stephen J. Perkins ◽  
Paul A. Dalby
Keyword(s):  
Concentration Dependence ◽  
Solution Structure ◽  
Analytical Ultracentrifugation ◽  
Sedimentation Coefficient ◽  
Radius Of Gyration ◽  
X Ray ◽  
X Ray Scattering ◽  
Starting Point ◽  
Self Association ◽  
Ray Scattering

Of the four human IgG antibody subclasses IgG1–IgG4, IgG4 is of interest in that it does not activate complement and exhibits atypical self-association, including the formation of bispecific antibodies. The solution structures of antibodies are critical to understand function and therapeutic applications. Thus IgG4 was studied by synchrotron X-ray scattering. The Guinier X-ray radius of gyration RG increased from 5.0 nm to 5.1 nm with an increase of concentration. The distance distribution function P(r) revealed a single peak at 0.3 mg/ml, which resolved into two peaks that shifted to smaller r values at 1.3 mg/ml, even though the maximum dimension of IgG4 was unchanged at 17 nm. This indicated a small concentration dependence of the IgG4 solution structure. By analytical ultracentrifugation, no concentration dependence in the sedimentation coefficient of 6.4 S was observed. Constrained scattering modelling resulted in solution structural determinations that showed that IgG4 has an asymmetric solution structure in which one Fab–Fc pair is closer together than the other pair, and the accessibility of one side of the Fc region is masked by the Fab regions. The averaged distances between the two Fab–Fc pairs change by 1–2 nm with the change in IgG4 concentration. The averaged conformation of the Fab regions appear able to hinder complement C1q binding to the Fc region and the self-association of IgG4 through the Fc region. The present results clarify IgG4 function and provide a starting point to investigate antibody stability.

scholarly journals RNA Multimerisation in the DNA Packaging Motor of Bacteriophage φ29

2005 ◽  
Vol 6 (2) ◽  
pp. 127-134 ◽  
Author(s):  
Jonathan P. A. Wood ◽  
Stephanie A. Capaldi ◽  
Mark A. Robinson ◽  
Andrew J. Baron ◽  
Nicola J. Stonehouse
Keyword(s):  
Analytical Ultracentrifugation ◽  
Molecular Level ◽  
Viral Assembly ◽  
Partial Specific Volume ◽  
Wild Type ◽  
Rna Molecules ◽  
Dna Packaging Motor ◽  
Self Association

The use of bacteriophages as experimental tools allows the investigation of interactions between components at the molecular level that are often not possible in more complex virus systems. The bacteriophage φ29 acts as a molecular machine to package its own genomic DNA during viral assembly. Self-associating RNA molecules, called pRNA, have an essential role in the function of this machine. This paper reports the characterization of this self-association (which leads to multimerisation of wild-type and truncated variant pRNAs) by analytical ultracentrifugation (including determination of the partial specific volume of the pRNA), together with an investigation into the domains of the molecule important for multimerisation by the use of complementary DNA probes.

scholarly journals Characterization of the type I dehydroquinase from Salmonella typhi

1993 ◽  
Vol 295 (1) ◽  
pp. 277-285 ◽  
Author(s):  
J D Moore ◽  
A R Hawkins ◽  
I G Charles ◽  
R Deka ◽  
J R Coggins ◽  
...  
Keyword(s):  
Tertiary Structure ◽  
Salmonella Typhi ◽  
Type I ◽  
Active Site Residues ◽  
X Ray ◽  
E Coli ◽  
Product Mixture ◽  
Secondary And Tertiary Structure ◽  
Nabh4 Reduction

The type I dehydroquinase from the human pathogen Salmonella typhi was overexpressed in an Escherichia coli host and purified to homogeneity. The S. typhi enzyme was characterized in terms of its kinetic parameters, important active-site residues, thermal stability and c.d. and fluorescence properties. In all important respects, the enzyme from S. typhi behaves in a very similar fashion to the well-characterized enzyme from E. coli, including the remarkable conformational stabilization observed on reduction of the substrate/product mixture by NaBH4. This gives confidence that the information from X-ray studies on the S. typhi enzyme [Boys, Fawcett, Sawyer, Moore, Charles, Hawkins, Deka, Kleanthous and Coggins (1992) J. Mol. Biol. 227, 352-355] can be applied to other type I dehydroquinases. Studies of the quenching of fluorescence of the S. typhi enzyme by succinimide show that NaBH4 reduction of the substrate/product imine complex involves a dramatic decrease in the flexibility of the enzyme, with only very minor changes in the overall secondary and tertiary structure.

CHARACTERIZATION OF BETA-GALACTOSIDASE FROM SACCHAROMYCES FRAGILIS1

1971 ◽  
Vol 34 (6) ◽  
pp. 300-306 ◽  
Author(s):  
W. L. Wendorff ◽  
C. H. Amundson
Keyword(s):  
Tertiary Structure ◽  
Lactose Hydrolysis ◽  
Buffer Solution ◽  
Enzymatic Function ◽  
Competitive Inhibitors ◽  
Optimum Ph ◽  
Physical And Chemical ◽  
Beta Galactosidase ◽  
Secondary And Tertiary Structure

Studies were made on physical and chemical characteristics of β-galactosidase in cell-free extracts of Saccharomyces fragilis Y-1109. The enzyme was stable at pH 6.0–7.0. When frozen in buffer solution, it was stable for more than 3 months but at 51 C, it lost 96% of the activity in 10 min. The optimum pH for lactose hydrolysis at 37 C was 6.5. The enzyme was activated by K+ while Mn++ served as the cofactor for the enzyme. Manganese appeared to be important in maintaining the integrity of the secondary and tertiary structure of the enzyme molecule. Inactivation of the enzyme by urea indicated the importance of secondary and tertiary structure in the enzymatic function of the yeast β-galactosidase. Beta-galactosidase appeared to be a sulfhydryl enzyme since heavy metals, p-chloromercuribenzoate, and iodoacetate inhibited the enzyme. Cysteine and galactose were competitive inhibitors of the enzyme, whereas glucose and various amines were non-competitive inhibitors.

scholarly journals Methylmalonyl-CoA mutase from Propionibacterium shermanii: characterization of the cobalamin-inhibited form and subunit-cofactor interactions studied by analytical ultracentrifugation

1993 ◽  
Vol 290 (2) ◽  
pp. 551-555 ◽  
Author(s):  
E N Marsh ◽  
S E Harding
Keyword(s):  
Analytical Ultracentrifugation ◽  
Guanidine Hydrochloride ◽  
Sedimentation Coefficient ◽  
Order Structure ◽  
High Concentration ◽  
Subunit Dissociation ◽  
Denatured Protein ◽  
Inactive Form ◽  
Partial Unfolding

A large proportion of adenosylcobalamin-dependent methylmalonyl-CoA mutase from Propionibacterium shermannii is isolated in an inactive form which contains a tightly bound cobalamin. Even when the enzyme was denatured in 5.0 M guanidine hydrochloride the cobalamin remained associated with the protein. However, when dithiothreitol was added to the denatured protein, the pink inhibitor was rapidly converted into a yellow-brown compound which could be removed by dialysis. Enzyme activity could be recovered after removal of the denaturant, although surprisingly this did not depend on prior treatment with dithiothreitol. The interaction between the protein and inhibitor was investigated by using analytical ultracentrifugation under denaturing conditions. The sedimentation coefficient s20,w was measured in various concentrations of guanidine hydrochloride. A complicated picture emerged in which at low denaturant concentrations subunit dissociation, partial unfolding and aggregation occur, whereas at high concentration the protein behaves as a monodisperse species. No major differences in sedimentation were observed between the enzyme-cobalamin complex and the cobalamin-free enzyme, suggesting that the inhibitor does not significantly stabilize higher-order structure within the protein.

scholarly journals Characterization of the Self Association ofAvian SarcomaVirus Integrase by Analytical Ultracentrifugation

1999 ◽  
Vol 274 (46) ◽  
pp. 32842-32846 ◽  
Author(s):  
Jacqueline Coleman ◽  
Steven Eaton ◽  
George Merkel ◽  
Anna Marie Skalka ◽  
Thomas Laue
Keyword(s):  
Analytical Ultracentrifugation ◽  
The Self ◽  
Self Association

scholarly journals Hyaluronan: the absence of amide–carboxylate hydrogen bonds and the chain conformation in aqueous solution are incompatible with stable secondary and tertiary structure models

2006 ◽  
Vol 396 (3) ◽  
pp. 487-498 ◽  
Author(s):  
Charles D. Blundell ◽  
Paul L. Deangelis ◽  
Andrew Almond
Keyword(s):  
Aqueous Solution ◽  
Hydrogen Bonds ◽  
Tertiary Structure ◽  
Amide Proton ◽  
Solution Conformation ◽  
Tertiary Structures ◽  
Nmr Data ◽  
Carboxylate Groups ◽  
Correlation Spectrum ◽  
Secondary And Tertiary Structure

Contradictory descriptions for the aqueous solution conformation of the glycosaminoglycan hyaluronan (HA) exist in the literature. According to hydrodynamic and simulation data, HA molecules are stiffened by a rapidly interchanging network of transient hydrogen bonds at the local level and do not significantly associate at the global level. In marked contrast, models derived from NMR data suggest that the secondary structure involves persistent hydrogen bonds and that strong associations between chains can occur to form vast stable tertiary structures. These models require an extended 2-fold helical conformation of the HA chain and specific hydrogen bonds between amide and carboxylate groups. To test these descriptions, we have used 15N-labelled oligosaccharides and high-field NMR to measure pertinent properties of the acetamido group. The amide proton chemical shift perturbation and carboxylate group pKa value are inconsistent with a highly populated hydrogen bond between the amide and carboxylate groups. Amide proton temperature coefficients and chemical exchange rates confirm this conclusion. Comparison of oligomer properties with polymeric HA indicates that there is no discernible difference in amide proton environment between the centre of octasaccharides and the polymer, inconsistent with the formation of tertiary structures. A [1H-1H-15N] NOESY-HSQC (heteronuclear single-quantum correlation) spectrum recorded on an HA octasaccharide revealed that amide groups in the centre are in a trans orientation and that the average solution conformation is not an extended 2-fold helix. Therefore the two key aspects of the secondary and tertiary structure models are unlikely to be correct. Rather, these new NMR data agree with descriptions from hydrodynamic and simulations data.

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