Research is performed in the area of experimental molecular biophysics. Specific topics are:
Participants: Bauer, R.; Bjerrum, M.; Danielsen, E.; Hemmingsen, L., Butz, T. (Leipzig Univ.); Tröger, W. (Leipzig Univ.); Canters, G.W. (Leiden Univ.); Messerschmidt, A. (Max-Planck-Institut, Martinsried); Karlsson, G. (Göteborgs Univ.)
The structure of the metal-center in wild type azurin, met121x-mutants of azurin and the his117gly-mutant of azurin has been studied using Cd-111m-PAC where copper has been substituted by radioactive 111Cd. The met121x-mutants have been compared to wild-type stellacyanin. Also the effect of exogeneous ligands on the structure of the metal-site in the his117gly-mutant has been studied.
Participants: Bauer, R.; Bjerrum, M.; Danielsen, E.; Hemmingsen, L.; Scheller, H., Butz, T. (Leipzig Univ.); Tröger, W. (Leipzig Univ.); Hansson, Ö. (Göteborgs Univ.)
The aim of the project is to study the interaction of plastocyanin with photosystem I and cytochrome f respectively. The difference between bound and non-bound plastocyanin will be determined using IIImCd-PAC to monitor the rotational diffusion of plastocyanin.
The so-called catalytic site zinc ion in alcohol dehydrogenase plays a crucial role in the process of reversible oxidation of alcohols. The zinc ion is replaced by Cd-111m. Measuring perturbed angular correlation of the PAC-spectrum of Cd-111m in enzyme and the enzyme-coenzyme complexes the pKa`s of the metalbound water, and possibly conformational changes accompanying the binding of coenzyme, can be deduced. Measurements on ternary inhibitor complexes and on the active, substrate-connecting enzyme will provide information on the coordination geometry during the catalytic cycle, and on which of the elementary steps is rate limiting. Measurements on crystalline (powder and/or single crystals) alcohol dehydrogenase will provide information about the applicability of structures determined by x-ray crystallography to structure in solution.
Participants: Bauer, R., Mouritsen, O.; Hønger, T.; Lemmich, J.; Ipsen, J. (alle Fysisk-Kemisk Inst., DTH); Mortensen, K. (Fysikafd., Forskningscenter Risø)
Small angle neutron scattering has been used to study the structure and dynamic of the bilayer membran. Fluctuation in the elasticity of the bilayer is observed for the pure bilayer system close to the temperature characterising the transition from a solid like to a fluid like phase.
Participants: Bauer, R.; Øgendal, L., Marcussen, J. and Maja Bojko (Danisco)
Starches can adopt different structures in solution, many of them characterised by a large element of randomness. We have performed dynamic light scattering on various starches in order to study their structure in solution and their ability to form gels, important for their food technological use. The results show, among other features, the importance of heat and alkaline treatment.
Participants: Bauer, R.; Danielsen, E.; Hemmingsen, L.; Andersen, M.L., Carvalho, E.; Goethe, P.O. (begge Göteborgs Univ.)
In some cases elevated blood pressure can be reduced by inhibiting the angiotensin I converting enzyme (ACE). ACE contains 2 zinc atoms per enzyme. Whether or not the two corresponding metal sites are different is investigated by perturbed angular correlation of gamma rays.
Partiipants: Bauer, R., Hemmingsen, L., Ryde, U. (Lund Univ.)
Cd-111m PAC has led to important information about the coordination geometry of cadmium in small model compounds and in zinc enzyme metal centers. This project aims at (1) improving the angular overlap model, used until now for interpretation of the PAC results, by so-called ab initio quantum chemical calculations, and (2) extracting further information about the coordination geometry of zinc and cadmium.
Participants: Bauer, R.; Hansen, S., Pedersen, J.S. (Fysikafd., Forskningscenter Risø); Kaarsholm, N. (Novo Nordisk)
The aggregation behaviour of several mutants of insulin is studied by small-angle neutron scattering, as a function of protein concentration, pH and ionic strength. The aim is to elucidate both fundamental protein-protein interactions and the behaviour of insulin used for treatment as a monomer. Data are analyzed (1) by indirect Fourier transformation, and (2) by a thermodynamic equilibrium model.
Participants: Bauer, R., Hansen, M.B.; Hansen, S.; Worning, P.; Øgendal, L.; Horne, D. (Hannah Res. Inst., Ayr, Scotland), Lomholt, S.; Quist, K. (begge Inst. for Mejeriforskning, KVL)
Enzymatic aggregation of casein micelles is the core of cheesemaking. We initiate the process with chymosin and follow it in time using dynamic and static light scattering and turbidimetry. An instrument is being designed to overcome the difficulties, caused by multiple scattering, in scattering studies of undiluted milk. Turbidimetric measumerents on undiluted milk in a very narrow cuvette eventually leads to determination of typical sizes and distances in the sample.
Participants: Bauer, R.; Bjerrum, M.; Danielsen, E.; Hemmingsen, L., Butz, T. (Leipzig Univ.); Tröger, W. (Leipzig Univ.); Hansson, Ö. (Göteborgs Univ.)
Among the blue copper proteins responsible for electron transport in biosystems are plastocyanin and azurin. Cd-111m studies show that in both proteins cadmium is coordinated to two histidines and one cysteine in a planar structure, but the PAC spectra of the two are quite different. The simplest explanation is that the metal-site structure in azurin is rigid, whereas there is in-plane mobility of the plastocyanin ligands. This is studied through the temperature and viscosity dependence of the PAC spectra.
Participants: Bauer, R.; Bjerrum, M.; Danielsen, E.; Hemmingsen, L., Friis, E., Hammerstad, J., Pedersen, M.V.; Ulstrup, J. (DTU)
Carboxypeptidase A is a zinc-containing digestive enzyme cleaving peptides into separate aminoacids. We perform PAC studies of the structure of the active site during active catalysis and compare with similar studies with inhibition. Results show a drastic change in coordination geometry of the metal-site during catalysis when compared to the resting enzyme. By the angular overlap model, specific suggestions are given for the new structure.
Participants: Hansen, S.; Hemmingsen, L.
Essential information can be extracted from PAC-spectra using Bayes' method. We can (1) eliminate parameters of little interest and thereby determine the interesting parameters more accurately, (2) among several models, decide which is the most appropriate when fitting the experimental data.
Participants: Hansen, S.; Müller, J. (Max-Delbrück-Centrum für Molekulare Medizin, Berlin)
The maximum entropy method for data analysis is applied to the problem of desmearing small-angle X-ray scattering curves. This is done through real-space modelling of the distance distribution function for the scatterer. The determination of parameters like e.g. the overall size of the scatterer or the noise level of the experiment is done by evaluation of their evidence.
Participants: Hansen, S.; Steenstrup, S. (Niels Bohr Instituttet, KU); Wilkins, S. (CSIRO, Melbourne)
Advantages and limitations of maximum entropy and Bayesian methods for data analysis are investigated. Problems studied include e.g. model comparison, Bayesian parameter estimation, error analysis and the ability of maximum entropy to estimate distributions without any a priori positivity constraint. The work is done with a general perspective, but with examples taken from the field of small-angle scattering.
Participants: Hansen, S.; Knott, R. (Ansto, Sydney)
Two model conformations have been suggested for the macro structure of serine proteinase inhibitor. The structure consists of 6kDa subunits and theoretical calculations show that the proposed structures have significantly different X-ray scattering profiles.
Development of an x-ray focusing EDXRF excitation technique for trace element analysis of small thin-film specimens
Participants: Laursen, J., Beckhoff, B.; Kanngiesser, B. (Universität Bremen)
X-ray focusing and monochromatising devices for photon energies above 5keV can be designed out of Highly Oriented Pyrolytic Graphite (HOPG) crystals. Besides the excellent integral reflectivity, the special shape of the doubly-curved crystals ensures a high-incident solid angle acceptance together with the capability to focus the beam down to about 1 mm FWHM spot sizes.
Participants: Laursen, J.; Henriksen, C.; Andersen. J.O.; Kølster, P. (de 3 sidstnævnte Inst.f. Jordbrugsvidenskab, KVL)
The aims of the project are 1) development of standardized procedures for producing crystallization pictures (IMF, KVL), 2) development of procedures for quantifying the pictures, including computerized image processing (in cooperation with I'M, DATU) and 3) application of the method in connection with examining the effects of growth and storage conditions (in cooperation with SP, Årslev).
Participants: Laursen, J.; Saaby, H.J., Mulvad, G.; Pedersen, H.S. (Lægeklinikken, Nuuk); Hansen, J.C. (CAM, Århus Univ.); Milman, N. (Hørsholm Sygehus)
The objective of the project is to provide supplementary information on heavy metal concentrations in human organs in Greenland. By including selenium and the composition of the stable isotopes C-13 and N-15 we attempt at evaluating the parameters measured at biomarkers of traditional/marine food intake and relating them to indices of coronary heart diseases.
Participants: Tingstad, A.; Laursen, J.; Henriksen, C.; Bengt-Pedersen, C. (Kemisk Inst., KVL), Kirkeby, O. F. (Inst. for Politik, Ledelse og Filosofi, HHK); Balzer-Graf, U. (Forschungslabor UBG, Wetzikon, Schweiz)
The aims of the project are 1) production of specific capillary dynamolysis pictures of selected carrot material (FL UBG), 2) development of procedures for quantifying the pictures, including computerized image processing and 3) application of the method in connection with examining the effects of growth, fertilizing and storage conditions.
Participants: Saaby, H.J.
Optical analysis of stable isotopes (Optical Emission Spectrometry: OES) is carried out. The optical analysis is developed by analysis of molecular spectra from: N2, CN, C2, CO, CO+, etc. As an application of the method, isotope ratios are measured on a mass spectrometer in order to improve the analysis of natural variations of stable isotopes (C-13, N-15,O-18, S-34, etc.) in small biological samples.
Participants: Høgh-Schmidt, K.; Hansen, J.M.
Construction of a general dynamic microclimate/photosynthesis model. The purpose of the model is to optimize plant photosynthesis in a greenhouse by dynamic control of the microclimate.
I.. Bauer, R.; Hansen, M.; Hansen, S.; Øgendal, L.; Lomholt, S.; Qvist, K.; Horne, D. (1995): The structure of casein aggregates during renneting studied by indirect Fourier transformation and inverse Laplace tranformation of static and dynamic light scattering data respectively. Journal of Chemical Physics 103:2725-2737.
II.. Bruun-Jensen, L.; Skovgaard, I.M.; Madsen, E.A.; Skibsted, L.H.; Bertelsen, G. (1995): The combined effect of tocopherols, L-ascorbyl palmitate, and L-ascorbic acid on the development of warmed-over flavour in cooked, minced turkey. Food Chemistry (in press).
III.. Carvalho, E.; Goethe, P.; Bauer, R.; Danielsen, E.; Hemmingsen, L. (1995): Effect of inhibition in the coordination geometries for cadmium at the metal sites in the angiotensin I converting enzyme. European Journal of Biochemistry 234:780-785.
IV.. Danielsen, E.; Bauer, R.; Hemmingsen, L.; Andersen, M.; Bjerrum, M.; Butz, T.; Tröger, W.; Canters, G.W.; Hoitink, C.W.G.; Karlsson, G.; Hansson, Ö.; Messerschmidt, A. (1995): Structure of metal site in azurin, met121 mutants of azurin and stellacyanin investigated by Cd-111m-PAC. Journal of Biological Chemistry 270:573-580.
V.. Danielsen, E.; Bauer,R.; Hemmingsen, L.; Bjerrum, M.; Butz, T.; Tröger, W.; Canters, G.W.; den Blaauwen, T.; van Pouderoyen, G. (1995): Structure of metal site in Cd-substituted His117Gly mutant of azurin with and without addition of imidazole derivatives. European Journal of Biochemistry 233:554-560.
VI.. Hemmingsen, L; Hansen, S. (1995): Bayes' method applied to selection of models in PAC. Zeitschrift für Naturforschung part A (in press).
VII.. Hemmingsen, L.; Bauer, R.; Bjerrum, M.J.; Zeppezauer, M; Adolph, H.W.; Formicka, G.; Cedergren-Zeppezauer, E. (1995): Cd-substituted horse liver alcohol dehydrogenase: Catalytic site coordination geometry and protein conformation. Biochemistry 34:7145-7153.
VIII..Knott, R.B.; Capel, M.; Hansen, S.; Handschumacher, R.E. (1995): A small-angle scattering study of the binding of cyclosporin-A to cyclophilin. Journal of Applied Crystallography 28:546-552.
IX.. Lemmich, J.; Mortensen, K.; Ipsen, J.H.; Hønger, T.; Bauer, R.; Jørgensen, K.; Mouritsen, O. (1995): Soft and repulsive: Relationship between lipid membrane in-plane fluctuations, bending rigidity, and repulsive undulation forces. Modern Physics Letter B 8:1803-1814.
X.. Lemmich, J.; Mortensen, K.; Ipsen, J.H.; Hønger, T.; Bauer, R.; Mouritsen, O. (1995): Pseudocritical behaviour and unbinding of phospholipids. Physical Review Letters 75:3958-xxxx (in press).
XI.. Müeller, J.J.; Hansen, S.; Lukowski, G.; Gast, K. (1995): Multimodal particle size distribution or fractal surface of acrylic acid copolymer nanoparticles: A small-angle X-ray scattering study using direct Fourier and indirect maximum entropy methods. Journal of Applied Crystallography 28:1-8.
XII.. Pedersen, J.S.; Hansen, S.; Bauer, R. (1994): Scaling in the aggregation behaviour of zinc-free insulin studied by small-angle neutron scattering. Il Nuovo Cimento 16D:1447-1455.
XIII..Pedersen, J.S.; Hansen, S.; Bauer, R. (1995): Aggregation behavior of zinc-free insulin studied by small-angle neutron scattering: analysis by use of a thermodynamic equilibrium model. Progress in Colloid and Polymer Science 98:215-218.
XIV.. Samso, M.; Daban, J-R.; Hansen, S.; Jones, G.R. (1995): Evidence for sodium dodecyl sulfate-protein complexes adopting a necklace structure in conditions that attempt to simulate those for gel electrophoresis. European Journal of Biochemistry 232:818-824.
XV.. Bauer, R.; Danielsen, E.; Hemmingsen, L.; Bjerrum, M.J.; Pedersen, M.V.; Ulstrup, J. (1995): Interplay between protein structure and metal coordination. Protein fold: A distance based approach. Eds. Bohr, H.; Brunak, S. CRC Press, Boca Raton, Florida. 31-42.