Markus Häner

Two-dimensional crystal packing of matrix porin. A channel forming protein in Escherichia coli outer membranes

Two-dimensional crystalline porin sheets were obtained by reconstitution of monodisperse protein trimers and phospholipids (dimyristoylphosphatidylcholine) by detergent dialysis, analogous to the reconstitution method used for functional tests (Schindler & Rosenbusch, 1981). Three different packing arrangements were observed: two were hexagonal (with p3 symmetry and lattice constants of 9.3 nm and 7.9 nm), and one rectangular (a = 7.9 nm, b = 13.9 nm). The different crystals could be correlated to phospholipid-to-protein weight ratios of 0.16 to 0.72. At the higher ratio, large hexagonal lattices predominated. Higher lipid ratios did not reveal other crystal forms. The packing arrangement of the large hexagonal form appears very similar to the hexagonal habit of three-dimensional crystal forms (Garavito et al., 1983). The shape of the stain-penetrated triplet indentations appeared conserved in the crystal forms to a resolution of 2.2 nm. The mass distribution between triplets, however, were significantly different. They are likely to correspond primarily to lipids. Mass determinations of unstained porin by scanning transmission electron microscopy showed that unit cells consisted of single trimers. The mass found (100,000 daltons) is in good agreement with the value obtained by sedimentation equilibrium analysis.

Formulation of sustained release aqueous Zn–hirudin suspensions ☆

Sustained release formulations for recombinant hirudin (rHir), an anticoagulant thrombin-specific inhibitor, were developed. Zn-rHir suspensions were formed by precipitation with zinc salts at neutral pH. Characterization of protein precipitation was by UV analysis, capillary electrophoresis (CE), zinc analysis, light and electron microscopy, and particle size analysis. The precipitation of aqueous rHir solution with ZnCl(2) solution at neutral pH resulted in Zn-rHir suspensions. Optimum yields of pelletized Zn-rHir were obtained between pH 7.0 and 7.4. For complete precipitation ( approximately 100%) a molar ratio of zinc to rHir of >28 was necessary. As shown by electron microscopy, the smallest resolvable unit of Zn-rHir suspensions was 20 nm. Agglomerates of up to 200 microm were observed by light microscopy. Zinc salt-induced precipitation phenomena were also investigated using ZnBr(2), ZnI(2), Zn(NO(3))(2) and ZnSO(4) instead of ZnCl(2). ZnSO(4) showed the lowest precipitation efficiency. All other salts behaved similar to ZnCl(2). Upon storage the pelletized protein content of the ZnCl(2) based precipitates was stable ( approximately 95% rHir after 1 year at room temperature), whereas the pelletized protein content of ZnSO(4) based precipitates dropped sharply after precipitation (2% remaining after 13 days at room temperature). This indicates a transition of the ZnSO(4) based precipitates to hexagonal basic zinc sulfate plates and free rHir. The driving force is the lower aqueous solubility of basic zinc sulfate as compared to the higher solubility of basic zinc chloride.

Structure and Assembly of Neurofilaments (NF): Analysis of Native NF and of Specific NF Subunit Combinations

A large body of information on the primary and secondary structure of intermediate filaments (IF) is now available [Weber et al., 1983; Steinert et al., 1985]. In the quest to ultimately understand IF structure, function and interaction at the molecular level, next steps include: (i) establishing the rules by which IF polypetides assemble into supramolecular structures; (ii) determining the three-dimensional (3-D) molecular architecture of IF; and (iii) establishing correlations between the molecular structure and function of the various IF. Because it has not been possible to obtain crystalline arrays of IF proteins in a consistent manner - which is a prerequisite for X-ray diffraction analysis of proteins - investigators have sought alternative techniques to approach these questions. In our labs we have begun to apply in vitro reconstitution of filaments and scanning transmission electron microscopy (STEM) to the study of neurofilaments (NF). This is a preliminary report on our work with NF, including effective and reproducible separation of the three NF polypeptides, in vitro reconstitution of NF from individual as well as specific combinations of subunits, and STEM mass measurements of native and reconstituted NF. We have observed that in vitro reassembly of NF takes place within strict limits of temperature, pH and salt concentration. In addition, STEM mass measurements disclose a marked polymorphism both of native and reconstituted NF.