Loes M. J. Kroon-Batenburg

An intensity evaluation method: EVAL-14

A reflection intensity integration method is presented based upon ab initio calculation of three-dimensional (x, y, ω) reflection boundaries from a few physical crystal and instrument parameters. It is especially useful in challenging circumstances, such as the case of a crystal that is far from spherical, anisotropic mosaicity, α1α2 peak splitting, interference from close neighbours, twin lattices or satellite reflections, and the case of streaks from modulated structures, all of which may frustrate the customary profile-learning and -fitting procedures. The method, called EVAL-14, has been implemented and extensively tested on a Bruker Nonius KappaCCD diffractometer.

EVAL15: a diffraction data integration method based on ab initio predicted profiles

A novel diffraction data integration method is presented, EVAL15, based upon ab initio calculation of three-dimensional (x, y, ω) reflection profiles from a few physical crystal and instrument parameters. Net intensities are obtained by least-squares fitting the observed profile with the calculated standard using singular value decomposition. This paper shows that profiles can be predicted satisfactorily and that accurate intensities are obtained. The detailed profile analysis has the additional advantage that specific physical properties of the crystal are revealed. The EVAL15 method is particularly useful in circumstances where other programs fail, such as regions of reciprocal space with weak scattering, crystals with anisotropic shape or anisotropic mosaicity, Kα1/Kα2 peak splitting, interference from close neighbours, twin lattices, or satellite reflections of modulated structures, all of which may frustrate the customary profile learning and fitting procedures. EVAL15 allows the deconvolution of overlapping reflections.

Tissue-Type Plasminogen Activator Is a Multiligand Cross-β Structure Receptor

Abstract Tissue-type plasminogen activator (tPA) regulates fibrin clot lysis by stimulating the conversion of plasminogen into the active protease plasmin [1]. Fibrin is required for efficient tPA-mediated plasmin generation and thereby stimulates its own proteolysis. Several fibrin regions can bind to tPA [1], but the structural basis for this interaction is unknown. Amyloid β (Aβ) is a peptide aggregate that is associated with neurotoxicity in brains afflicted with Alzheimers disease [2]. Like fibrin, it stimulates tPA-mediated plasmin formation [3–5]. Intermolecular stacking of peptide backbones in β sheet conformation underlies cross-β structure in amyloid peptides [6]. We show here that fibrin-derived peptides adopt cross-β structure and form amyloid fibers. This correlates with tPA binding and stimulation of tPA-mediated plasminogen activation. Prototype amyloid peptides, including Aβ and islet amyloid polypeptide (IAPP) (associated with pancreatic β cell toxicity in type II diabetes [7]), have no sequence similarity to the fibrin peptides but also bind to tPA and can substitute for fibrin in plasminogen activation by tPA. Moreover, the induction of cross-β structure in an otherwise globular protein (endostatin) endows it with tPA-activating potential. Our results classify tPA as a multiligand receptor and show that cross-β structure is the common denominator in tPA binding ligands.

A 1H-NMR and MD study of intramolecular hydrogen bonds in methyl β-cellobioside

Abstract The existence of an HO-3⋯O-5′ intramolecular hydrogen bond in methyl β-cellobioside in solution in Me 2 SO- d 6 and H 2 O-CD 3 OD (4:1 w/w) was studied by 500-MHz 1 H-NMR spectroscopy and MD simulations. Temperature coefficients for the chemical shift of the hydroxyl resonances in these solvents were determined and the rates of proton exchange in the latter solvent were obtained from NOE data. With H 2 O-CD 3 OD as the solvent, the HO-3⋯O-5′ hydrogen bond was insignificant, but its presence in Me 2 SO- d 6 was confirmed.

The crystal and molecular structures of cellulose I and II

The paper describes molecular dynamics (MD) simulations on the crystal structures of the Iβ and II phases of cellulose. Structural proposals for each of these were made in the 1970s on the basis of X-ray diffraction data. However, due to the limited resolution of these data some controversies remained and details on hydrogen bonding could not be directly obtained. In contrast to structure factor amplitudes in X-ray diffraction, energies, as obtained from MD simulations, are very sensitive to the positions of the hydroxyl hydrogen atoms. Therefore the latter technique is very suitable for obtaining such structural details. MD simulations of the Iβ phase clearly shows preference for one of the two possible models in which the chains are packed in a parallel orientation. Only the parallel-down mode (in the definition of Gardner and Blackwell (1974) J Biopolym 13: 1975-2001) presents a stable structure. The hydrogen bonding consists of two intramolecular hydrogen bonds parallel to the glycosidic linkage for both chains, and two intralayer hydrogen bonds. The layers are packed hydrophobically. All hydroxymethyl group are positioned in the tg conformation. For the cellulose II form it was found that, in contrast to what seemed to emerge from the X-ray fibre diffraction data, both independent chains had the gt conformation. This idea already existed because of elastic moduli calculations and 13C-solid state NMR data. Recently, the structure of cellotetraose was determined. There appear to be a striking similarity between the structure obtained from the MD simulations and this cellotetraose structure in terms of packing of the two independent molecules, the hydrogen bonding network and the conformations of the hydroxymethyl group, which were also gt for both molecules. The structure forms a 3D hydrogen bonded network, and the contribution from electrostatics to the packing is more pronounced than in case of the Iβ structure. In contrast to what is expected, in view of the irreversible transition of the cellulose I to II form, the energies of the Iβ form is found to be lower than that of II by 1 kcal mol-1 per cellobiose.

Recombinant endostatin forms amyloid fibrils that bind and are cytotoxic to murine neuroblastoma cells in vitro

Endostatin is a fragment of collagen XVIII that acts as an endogenous inhibitor of tumor angiogenesis and tumor growth. Anti‐tumor effects have been described using both soluble and insoluble recombinant endostatin. However, differences in endostatin structure are likely to cause differences in bioactivity. In the present study we have investigated the structure and cellular effects of insoluble endostatin. We found that insoluble endostatin shows all the hallmarks of amyloid aggregates. Firstly, it binds Congo red and shows the characteristic apple‐green birefringe when examined under polarized light. Secondly, electron microscopy shows that endostatin forms short unbranched fibrils. Thirdly, X‐ray analysis shows the abundant presence of cross‐β sheets, the tertiary structure that underlies fibrillogenesis. None of these properties was observed when examining soluble endostatin. Soluble endostatin can be triggered to form cross‐β sheets following denaturation, indicating that endostatin is a protein fragment with an inherent propensity to form amyloid deposits. Like β‐amyloid, found in the brains of patients with Alzheimers disease, amyloid endostatin binds to and is toxic to neuronal cells, whereas soluble endostatin has no effect on cell viability. Our results demonstrate a previously unrecognized functional difference between soluble and insoluble endostatin, only the latter acting as a cytotoxic amyloid substance.

CROSREL: Full relaxation matrix analysis for NOESY and ROESY NMR spectroscopy

SummaryA method is proposed for quantitative analysis of ROESY peak intensities, to which corrections are applied for their offset dependence and for direct HOHAHA effects. Additionally the effects of anisotropic and internal motion can be assessed. This method has been implemented for full relaxation matrix analysis in the CROSREL program. Although CROSREL is applicable to NOESY data, its use for ROESY peak intensities has been evaluated here, because of its innovative character in this respect. The agreement between calculated and experimental intensities is expressed by a weighted residual Rw factor, similar to X-ray crystallography. The merits of the program have been tested on methyl(d3) β-cellobioside, for which a ROESY buildup series has been acquired, and for which extensive MD simulations have been performed. It is concluded that correction for direct HOHAHA effects is obligatory for the analysis of ROESY data. Extension of the model for methyl β-cellobioside with internal and anisotropic motion, as was derived from MD data, did not improve the results obtained for assumed isotropic tumbling of a rigid model. It has been shown that ROESY peak intensities can be analysed successfully by the CROSREL program.

Supramolecular hydrogels formed by β-cyclodextrin self-association and host–guest inclusion complexes

Supramolecular hydrogels are highly interesting for drug delivery and tissue engineering applications, especially those systems that display a combination of tunable properties, high mechanical strength and easy preparation from well-available and biocompatible building blocks. In the present paper, we show that the combination of free β-cyclodextrin (βCD) and 8-arm or linear cholesterol-derivatized poly(ethylene glycol) (PEG–chol) in aqueous solution resulted in the formation of almost fully elastic gels with storage moduli in the range of 10–500 kPa. X-Ray diffraction measurements demonstrated the presence of crystalline βCD domains in the hydrogel networks. Rheological experiments further proved that hydrogel formation is based on inclusion complex formation between these βCD clusters and cholesterol coupled to the terminal end of PEG. The observation that the gels were weakened by addition of the competitive βCD–guest molecule adamantanecarboxylic acid (ACA) supported the proposed gelation mechanism. The gel mechanical properties were dependent on temperature, concentration of cholesterol-derivatized PEG and/or βCD, PEGs molecular weight and its architecture. This hydrogel system can be considered as an excellent candidate for future applications in the biomedical and pharmaceutical fields.

Characterization of poly(l-lactic acid) microspheres loaded with holmium acetylacetonate

Holmium-loaded PLLA microspheres are useful systems in radioembolization therapy of liver metastases because of their low density, biodegradability and favourable radiation characteristics. Neutron activated Ho-loaded microspheres showed a surprisingly low release of the relatively small holmium complex. In this paper factors responsible for this behaviour are investigated, in particular by the use of differential scanning calorimetry, scanning electron microscopy, infrared spectroscopy and X-ray diffraction. The holmium complex is soluble in PLLA up to 8% in films and 17% in microspheres. Interactions between carbonyl groups of PLLA, and the Ho-ion in the HoAcAc complex, explain very satisfactorily the high stability of holmium-loaded microspheres.

Conformational analysis of methyl β-cellobioside by ROESY NMR spectroscopy and MD simulations in combination with the CROSREL method

Methyl beta-cellobioside has been studied extensively by molecular dynamics (MD) simulations in water and by ROESY NMR spectroscopy in order to establish its solution structure. The MD simulations were started with four significantly different minimal energy conformations. The MD trajectories were analysed with respect to interproton distances and mobility, in order to find models for application in the analysis of NMR data. The ROESY spectra were analysed by using the CROSREL method, which allows quantitative analysis of ROESY spectra through correction for the offset dependence and incorporation of HOHAHA transfer estimates. These results were compared with data obtained from an initial rate analysis of the ROESY data and with the MD data. It is concluded that methyl beta-cellobioside in aqueous solution is in the same extended conformation that is also found in the solid state.