Biomedical subjects

Karsten Dierks

Publications and source records attributed to Karsten Dierks.

5 recordsLinked to original sources

PubMed2005

Dimerization of the major birch pollen allergen Bet v 1 is important for its in vivo IgE-cross-linking potential in mice.

In type I allergy, the cross-linking of membrane IgE on B lymphocytes and of cytophilic IgE on effector cells by their respective allergens are key events. For cross-linking two IgE molecules, allergens need at least two epitopes. On large molecules, these could be different epitopes in a multivalent, or identical epitopes in a symmetrical, fashion. However, the availability of epitopes may be limited on small allergens such as Bet v 1, the major birch pollen allergen. The present work analyzes whether dimerization is required for the cross-linking capacity of this allergen. In immunoblots, murine monoclonal and polyclonal human Bet v 1-specific Abs detected, besides a Bet v 1 monomer of 17 kDa, a dimer of 34 kDa. In dynamic light scattering, Bet v 1 appeared as dimers and even multimers, but a single condition could be defined where it behaved exclusively monomerically. Small-angle x-ray scattering of the monomeric and dimeric samples resulted in diagrams agreeing with the calculated models. Circular dichroism measurements indicated that the structure of Bet v 1 was preserved under monomeric conditions. Skin tests in Bet v 1-allergic mice were positive with Bet v 1 dimer, but remained negative using the monomer. Furthermore, in contrast to dimeric Bet v 1, the monomer was less capable of activating murine memory B cells for IgE production in vivo. Our data indicate that the presentation of two identical epitopes by dimerized allergens is a precondition for cross-linking of IgE on mast cells and B lymphocytes.

Allergens↗

PubMed2004

Conformational states of the Rapana thomasiana hemocyanin and its substructures studied by dynamic light scattering and time-resolved fluorescence spectroscopy.

Hemocyanins are dioxygen-transporting proteins freely dissolved in the hemolymph of mollusks and arthropods. Dynamic light scattering and time-resolved fluorescence measurements show that the oxygenated and apo-forms of the Rapana thomasiana hemocyanin, its structural subunits RtH1 and RtH2, and those of the functional unit RtH2e, exist in different conformations. The oxygenated respiratory proteins are less compact and more asymmetric than the respective apo-forms. Different conformational states were also observed for the R. thomasiana hemocyanin in the absence and presence of an allosteric regulator. The results are in agreement with a molecular mechanism for cooperative dioxygen binding in molluscan hemocyanins including transfer of conformational changes from one functional unit to another.

Animals↗

PubMed2004

Interactions of the neurotoxin vipoxin in solution studied by dynamic light scattering.

The neurotoxin vipoxin is the lethal component of the venom of Vipera ammodytes meridionalis. It is a heterodimer of a basic toxic His-48 phospholipase A2 (PLA2) and an acidic nontoxic Gln-48 PLA2. The shape of the neurotoxin and its separated components in solution as well as their interactions with calcium, the brain phospholipid phosphatidylcholine, and two inhibitors, elaidoylamide and vitamin E, were investigated by dynamic light scattering. Calcium binding is connected with a conformational change in vipoxin observed as a change of the hydrodynamic shape from oblate ellipsoid to a shape closer to a sphere. The Ca2+-bound form of vipoxin, which is catalytically active, is more compact and symmetric than the calcium-free heterodimer. Similar changes were observed as a result of the Ca2+-binding to the two separated subunits. In the presence of aggregated phosphatidylcholine, the neurotoxic complex dissociates to subunits. It is supposed that only the toxic component binds to the substrate, and the other subunit, which plays a chaperone function, remains in solution. The inhibition of vipoxin with the synthetic inhibitor elaidoylamide and the natural compound vitamin E changes the shape of the toxin from oblate to prolate ellipsoid. The inhibited toxin is more asymmetric in comparison to the native one. Similar, but not so pronounced, effects were observed after the inhibition of the monomeric and homodimeric forms of the toxic His-48 PLA2. Circular dichroism measurements in the presence of urea, methylurea, and ethylurea indicate a strong hydrophobic stabilization of the neurotoxin. Hydrophobic interactions stabilize not only the folded regions but also the regions of intersubunit contacts.

Binding Sites↗

PubMed2004

Size and shape determination of proteins in solution by a noninvasive depolarized dynamic light scattering instrument.

Dynamic light scattering (DLS) is a well-known noninvasive technique for investigating interactions of protein molecules in solution. Unfortunately, DLS is not very sensitive to small size changes because covariables, such as temperature, viscosity, and refractive index, are not precisely known, or they vary as functions of an experiment run, making it difficult to resolve subtle size changes of only a few Angstrom. It is usually not possible, if these covariables are not systematically measured and brought into the DLS analysis, to separate monomers from dimers when both are present in solution. We present here measurements with a variant of DLS that determines rotational diffusion as well as translation diffusion. This technique, called depolarized dynamic light scattering (DDLS) is, like DLS, also an old method, but it is rarely used due to enormous practical difficulties. However, we have found that a combination of DLS with DDLS is very promising, because it allows for a rough shape determination of the molecule under study and it is more sensitive to subtle size changes. We built an instrument that overcomes some of the difficulties, and report measurements made with this instrument. One of the samples was Photosystem-I, a membrane protein for photosynthesis. Its dimensions were determined to be 9.6 nm thick and 26 nm in diameter, values that are in good agreement with the dimensions obtained from X-ray diffraction analysis of single crystals.

Biophysics↗

PubMed2002

Separating nucleation and growth in protein crystallization using dynamic light scattering.

A means of controlling crystallization is to separate the phases of nucleation and growth. Methods to achieve this, other than seeding, involve lowering the supersaturation by changing the temperature or diluting drops after incubating them for a given time at nucleation conditions. However, by the time nuclei or crystals are visible under the microscope too many nuclei will have formed. Dynamic Light Scattering was applied practically, to determine the most likely time for nucleation-growth decoupling to be performed successfully. The time at which DLS showed a significant change in the size-distribution of species in solution, corresponded to that optimal time.

Animals↗