Research in the field of molecular biophysics:



Studies of metal site structure, protein conformations and enzymatic mechanisms of zinc enzymes:
Fundamental work on the relation between metal coordination geometry and protein conformation have been performed for carboxypeptidase [10,21] and alcohol dehydrogenase [6]. In addition the metal coordination geometry for cadmium carboxypeptidase have been studied under steady state hydrolysis of peptides. Concerning carboxypeptidase the studies have revealed that the metal site structure are almost identical in solution and in the crystalline state. However, in the crystalline state one unique and rigid geometry is found in contrast to solution where several geometries are simultaneously present indicating protein flexibility. Under turnover conditions in solution rigidity is again obtained. For alcohol dehydrogenase a close correlation between the conformation of the enzyme in solution and the conformation derived for the crystalline state by X-ray diffraction was observed. Beta-lactamase [19,22,23] hydrolyze the beta-lactam ring in antibiotic. We have investigated the relation between the number of zinc ions bound to the enzyme and hydrolysis. An important discovery was that there are different mechanism for the mono and binuclear zinc-enzyme. Furthermore, mechanism for both were proposed. A striking feature is that the mixed cadmium,zinc enzyme is more active than the native zinc,zinc enzyme. A detailed study of the cadmium enzyme still ongoing have shown that the preference for the two metal sites in beta-lactamase is different for cadmium and zinc. The site thought of as the site performing hydrolysis is preferred by zinc and the other site by cadmium. This together with other evidence strongly suggest that the second site act as a cocatalytic site thus explaining why cadmium can both decrease and increase catalytic activity depending on which site cadmium occupies. Thus when a mononuclear cadmium enzyme is prepared very little activity occurs because the site of hydrolysis is weakly occupied whereas when zinc occupies this site together with cadmium in the second site increased activity arises because of the cocatalytic effect. This has been shown by the combination of kinetic measurements with PAC spectroscopy.

Studies of metal site structure, protein conformations and protein binding for blue copper proteins:
We have in the previous frame grant shown that for cadmium azurin the main contributions to the coordination geometry comes from a nearly planar and very rigid trigonal geometry. It was also previously shown that mutation of the conserved methionine 121 to other amino acids in general makes the geometry flexible and deviates from the planar geometry and at the same time opens up for the entrance of solvent ligands. Related to the work within the present grant we have shown that plastocyanin besides conforming to almost the same planar trigonal geometry as azurin it shows a planar restricted movements of the ligands [4]. A major question concerning type I copper proteins such as azurin and plastocyanin is coordination geometrical differences between Cu(I) and Cu(II) and its eventual influence on the redox potential. We have tried to investigate this by using two PAC isotopes 111Ag and 111mCd as probes for Cu(I) and Cu(II) respectively. In the case of native azurin we showed that the coordination geometries for Ag(I) and Cd(II) were different but the difference could be ascribed as a planar movement of one of the coordinating histidines by a few degrees and further that the Ag(I) geometry relaxes to the Cd(II) geometry within 100 ns [7]. In contrast, a methionine 121 to leucine mutation shows substantial differences between the coordination geometries of Ag(I) and Cd(II). Likewise in the methionine 121 to histidine mutation showed many difference in the number and type of coordination geometries present at various pH for Ag(i) and Cd(II) [11]. Finally we have initiated a study of the binding of Ag(I)-plastocyanin and Cd(II)-plastocyanin to photosystem I demonstrating that the binding affinity are different for the two mention species [28].

Calculations of NQI in cadmium complexes:
Previously only very simple molecular orbital methods were realistic to apply to the electron rich cadmium complexes. Such calculations using the idea behind the angular overlap model were shown to be rather successful in spite of the many approximations necessary (Bauer et al. 1988). However, with the invention of modern high speed cpu and parallel computing we have done ab initio calculations on two relatively large cadmium complexes [1,26,27]. The results achieved were shown to be accurate within 15%. Modeling the catalytic metal site of alcohol dehydrogenase and performing ab initio together with energy minimisation with respect to the coordination geometry showed that ab initio calculations have the potential of predicting the local metal coordination geometry in a metalloprotein [13]. It is nevertheless clear that for the vast majority of NQI determined for cadmium in a chemical and biological context there is a need for the simple AOM to calculate NQI. That this methods compare favorable with ab initio at the present state of accuracy is shown in [25].

Studies of protein structure in solution, protein-ligand and protein-protein interactions:
In the study of protein-protein, interaction is of importance. We have previously studied this for insulin at intermediate salt strength. This was now extended to the case of highly charged albumin at low salt content [14]. The influence of divalent metals on protein-protein complexes using antibodies were also studied [9].

Light scattering on a large system such as casein micelles ( 300 nm diameter) needs consideration about whether or not the usual Rayleigh-Debye-Gans theory also used by us [12] is applicable or one should make full us of Maxwell equations also known as Mie scattering. If the casein micelles are structured as a homogeneous sphere it would be necessary to use Mie scattering. However, our neutron studies on casein micelles shows that the micelles are heterogeneous object composed of microspheres with a diameter of about 20 nm thus validating the application of Rayleigh-Debye-Gans theory [2]. In order to go further and characterize the enzymatic aggregation and gelation of casein micelles and additional problem occurs namely that of multiple scattering. A way to solve this problem in turbidimetry was shown in [15]. However, in the same work it was also shown that the information content is so low in turbidimetry that a choice between different possible structures is impossible. For the initial state of casein aggregation we have shown that one can derive the weight average molar mass from turbidimetric measurement. This we have used to compare different kinetic models for the aggregation [18]. By using the same principle of correcting for multiple scattering a new instrument for light scattering of dense, turbid systems was used. The results showed for the first time a reliable fractal exponent for casein aggregation and gelation [24]. Beta-lactoglobulin exhibits a very interesting aggregation pattern when heated. It has been shown by circular dichroism measurements that the alfa-helix structure is broken whereas the extensive beta-sheet structure is kept upon heating to about 70 degree Celcius. This partly denatured protein aggregates and at high enough concentration also gelates. An extensive literature existst about the heat aggregation of beta-lactoglobulin. However, no consistency in the findings were revealed. We showed that performing experiments on well defined salt conditions achieved by dialysis consistent findings could be obtained [16]. This enabled us to study the mechanism of heat aggregation in greater detail such that intermediate oligomers in the process of aggregation could be derived [17]. This will make it possible to set up a minimal set of equations describing the increase and decrease of all molecules detected by simultaneous light scattering and size exclusion chromatography. We further characterized the state of oligomerisation before heat aggregation by small angle neutron scattering [20]. This will form an important basis for selecting different conditions such as pH and salt and protein concentration for future studies of heat aggregation of beta-lactoglobulin.

Studies of protein membrane interactions:
As a continuation of our work on multilamellar lipid bilayers we have made a detailed description of the extraction of information about the phase transition and bilayer structure for phospholipid with small angle neutron scattering [3]. In addition we have studied the effect of small amount of solutes on the bilayer structure [5]. In more detail, the effect of cholesterol in low percentage on the softness/rigidity of the bilayer structure were done [8]. This led to the suggestion that small amount of cholesterol could control the bending rigidity of the membrane surface enabling or disabling vesicle-budding.

Publications in the period 1996-1998:
1. L. Hemmingsen and Ulf Ryde (1996) Ab initio calculations of electric field gradients in cadmium complexes. J. Phys. Chem. 100:4803-4809.
2. Hansen, S., Bauer, R., Pedersen, J., S., Mortensen, K., Lomholt, S., B., Quist, K. B. (1996). Structure of casein micelles studied by small-angle neutron scattering. European Biophysics Journal 24:143-147.
3. Lemmich, J., Mortensen, K., Ipsen, J.,H., Hønger, T., Bauer, R., Jørgensen, K. and Mouritsen, O. (1996). Small-angle neutron scattering from multilamellar lipid bilayers: Theory, model, and experiment. Physical Review E 53:5169-5180.
4. W Troger, C Lippert, T Butz, K Sigfridsson, O Hansson, E Mclaughlin, R Bauer, E Danielsen, L Hemmingsen, MJ Bjerrum (1996). Small scale intramolecular flexibility in Cd-111m-plastocyanin. Zeitschrift Fur Naturforschung A 51:431-436.
5. Lemmich, J., Mortensen, K., Ipsen, J.,H., Hønger, T., Bauer, R., Jørgensen, K. and Mouritsen, O. (1996). Solutes in small amounts provide for lipid-bilayer softness: Cholesterol, short chain lipids and bola lipids. European Biophysics Journal 25:61-65.
6. L. Hemmingsen, R. Bauer, M. J. Bjerrum, M. Zeppezauer, H. W. Adolph, G. Formicka, E. Cedergren-Zeppezauer (1996) Catalytic site Cd-substituted horse liver alcohol dehydrogenase: Binary and ternary inhibitor complexes. European Journal of Biochemistry 241:546-551.
7. Rogert Bauer, Eva Danielsen, Lars Hemmingsen, Morten J. Bjerrum, Örjan Hansson and Kulwant Singh. (1997) Interplay between oxidation state and coordination geometry of metal ions in Azurin. Journal of the American Chemical Society, 119:157-163*.
8. Lemmich, J., Mortensen, K., Ipsen, J.,H., Hønger, T., Bauer, R., Jørgensen, K. and Mouritsen, O. (1997). Cholesterol in small amounts makes lipid bilayers softer in small amount whereas larger amounts provide for rigidity. European Biophysics Journal 25:293-304.
9. Bauer, R., Mueller, A., Richter, M., Schneider, K., Frey, J. and Engelhardt, W. (1997). Influence of heavy metal ions on antibody and immune complexes investigated by dynamic light scattering and enzyme-linked immunosorbent assay. Biochim. Biophys. Acta 1334:98-108.
10. Rogert Bauer, Eva Danielsen, Lars Hemmingsen, Marianne V. Sørensen, Jens Ulstrup, Esben P. Friis, David S. Auld and Morten J. Bjerrum (1997) Structure and dynamics of the metal site of cadmium substituted carboxypeptidase A in solution and crystalline states and under steady-state peptide hydrolysis Biochemistry 36:11514-1152436*.
11. Danielsen, E., Kroes, S.J., Canters, G.W., Bauer, R., Hemmingsen , L and Messerschmidt, A. (1997) Coordination geometries for monovalent and divalent metal ions in [His121]azurin. Studies using perturbed angular correlation of gamma rays. Eur. J. Biochem. 250:249-259.
12. Müller, J.J, Hansen, S. And Pürschel, H.V. (1997) The use of small angle scattering and the maximum-entropy method for shape-model determination from distance-distribution functions. Journal of Applied Crystallography 29:547-554.
13. Ulf Ryde and Lars Hemmingsen (1997) The active site structure of cadmium substituted alcohol dehydrogenase: A theoretical interpretation of perturbed angular correlation of gamma rays measurements. J. Biol. Inorg. Chemistry 2:567-579.
14. Sjöberg, B. and Mortensen, K. (1997) Structure and thermodynamics of nonideal solutions of colloidal particles: Investigation of salt-free solutions of human serium albumin by using small-angle neutron scattering and Monte Carlo simulation. Biophys. Chem. 65:75-83.
15. Worning, P., Bauer, R., Øgendal, L. and Lomholt, S. (1998) A novel approach to turbidimetry of dense systems: An investigation of the enzymatic gelation of casein micelles. Journal of Colloid and Interface Science 203:265-277.
16. Holt, C., Waninge, R., Sellers, P., Paulsson, M., Bauer, R., Øgendal, L., Roefs, S. P. F. M., Léonil, J and Maubois, J. L. (1998): Comparison of the Effects of Heating on the Thermal Denaturation of 9 Different Beta-Lactoglobulin Preparations of Genetic Variants A, B or A/B, as Measured by Microcalorimetry. International Dairy Journal 8:99-104.
17. Bauer, R., Hansen, S. and Øgendal, L. (1998): Detection of intermediate Oligomers, Important for the Formation of Heat Aggregates of Beta-Lactoglobulin. International Dairy Journal 8:105-112*.
18. Lomholt, S.B., Worning, P., Øgendal, L., Qvist, K.B., Hyslop, D.B. and Bauer, R. (1998) Kinetics of the renneting reaction followed by measurement of turbidity as a function of wavelength. Journal of Dairy Research, in press.
19. Paul Soto, R., Hernandez Valladares, M., Galleni, M., Bauer, R. Zeppezauer, M., Frere, J.M. and Adolph, H.W. (1998). Mono and binuclear Zn-beta-lactamase from Bacteroides Fragilis: Catalytic and structural roles of the zinc ions. FEBS Letter, accepted for publication.
20. Verheul, M., Pedersen, J.S., Roefs, S.P.F.M. and de Kruif, C.G. (1998) Association behaviour of native beta-lactoglobulin. Accepted for publication in Biopolymers.
21. Bukrinsky, J.T. Bjerrum, M.J. and Kadziola, A. Native carboxypeptidase A in a new crystal environment reveals a different position of the important tyrosine 248. Accepted for publication in Biochemistry.
22. Paul Soto, R., Zeppezauer, M., Adolph, H.W., Galleni, M., Frere, J.M., Wouters, J. Hemmingsen, L. Hansen, B. And Bauer R. Direct evidence of interacting metal sites in Cd-derivatives of beta-lactamase. Manuscript.
23. Paul Soto, R., Bauer, R., Frere, J.M., Galleni, M., Meyer-Klaucke, W. Nolting, H., Rossolini, G.M., de Seny, D., Hernandez-Valladares, M., Zeppezauer, M. and Adolph, H.W. Different mechanism for mono- and binuclear Zn-beta-lactamase: Role of the conserved cysteine. Submitted to Biochemistry*.
24. Lehner, D., Worning, P., Fritz, G., Øgendal, L, Bauer, R. and Glatter, O. Characterisation of enzymatically induced aggregation of Casein Micelles in natural concentration by in-situ static light scattering and ultra low shear viscosity. Submitted to Journal of colloid and interface science*.
25. Hemmingsen, L., Ryde, U. And Bauer, R. Nuclear quadrupole interactions in cadmium complexes: Semiempirical and ab initio calculations. Submitted to Journal of Physical Chemistry*.
26. Hemmingsen, L., Bauer, R., Bjerrum, M.J., Schwarz. K., Blaha, P. And Andersen, P. Structure, chemical bonding and nuclear quadrupole interactions of beta-Cd(OH)2: Experiment and first principles calculations. Manuscript.
27. Hansen, B., Bukrinsky, J.T., Hemmingsen, L., Bjerrum, M.J., Singh, K. And Bauer, R. Evidence of a change in coordination geometry in the nuclear transformation from Ag(I) to Cd(II) in a single crystal of Ag[111Ag](imidazole)2NO3 derived by ab initio quantum mechanical calculations applied to the experimentally determined nuclear quadrupole interaction tensor. Manuscript.
28. Danielsen, E., Scheller, H.V., Bauer, R., Hemmingsen, L., Bjerrum, M.J., Nielsen, H.L. and Hanson Ö. Plastocyanin binding to photosystem I as a function of charge state. Manuscript.