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During vacuum condensation of metals on frozen proteins, nanoclusters are preferentially formed at specific surface sites (decoration). Understanding the nature of metal/protein interaction is of interest for structure analysis and is also important in the fields of biocompatibility and sensor development. Studies on the interaction between metal and distinct areas on the protein which enhance or impede the probability for cluster formation require information on the structural details of the protein's surface underlying the metal clusters. On three enzyme complexes, lumazine synthase from Bacillus subtilis, proteasome from Thermoplasma acidophilum and GTP cyclohydrolase I from Escherichia coli, the decoration sites as determined by electron microscopy (EM) were correlated with their atomic surface structures as obtained by X-ray crystallography. In all three cases, decoration of the same protein results in different cluster distributions for gold and silver. Gold decorates surface areas consisting of polar but uncharged residues and with rough relief whereas silver clusters are preferentially formed on top of protein pores outlined by charged and hydrophilic residues and filled with frozen buffer under the experimental conditions. A common quality of both metals is that they strictly avoid condensation on hydrophobic sites lacking polar and charged residues. The results open ways to analyse the binding mechanism of nanoclusters to small specific sites on the surface of hydrated biomacromolecules by non-microscopic, physical-chemical methods. Understanding the mechanism may lead to advanced decoration techniques resulting in fewer background clusters. This would improve the analysis of single molecules with regard to their symmetries and their orientation in the adsorbed state and in precrystalline assemblies as well as facilitate the detection of point defects in crystals caused by misorientation or by impurities.

The riboflavin synthase/lumazine synthase complex of Bacillus subtilis catalyzes the last two steps in riboflavin biosynthesis. The protein comprises a capsid of 60 beta subunits with lumazine synthase activity and a core of three alpha subunits with riboflavin synthase activity. The beta subunits catalyze the formation of 6,7-dimethyl-8-ribityllumazine (3) from 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (1) and 3,4-dihydroxy-2-butanone 4-phosphate (2). Complexes of recombinant lumazine synthase (beta(60) capsids) with 6-trifluoromethyl-7-oxo-8-ribityllumazine (10) as well as 7S- or 7R-6,7-bistrifluoromethyl-8-ribityllumazine hydrate (11) were studied by (19)F NMR spectroscopy. Despite the large molecular weight of approximately 960 kDa of the protein, spectra with separated signals of free and bound ligand could be obtained. An unusually large shift difference of 8 ppm was observed between the 7-trifluoromethyl signals of free and bound ligand for epimer B of 11 and the enzyme. The signal is sensitive to the replacement of amino acid residues F22 and H88. Lumazine synthase catalyzes the elimination of the 7-trifluoromethyl group of R-diastereomer epimer A in a haloform-like reaction. The elimination reaction is also catalyzed by F22 mutants. The H88R mutant displays an opposite stereoselectivity for epimer B and a greatly enhanced reaction rate. From a model of the epimers in the active site of the protein, the main function of the side chain of F22 seems to be to keep the substrate ring in the correct position. H88 is in a position suited to act as proton acceptor in both the physiological as well as the haloform reaction. A different mechanism of the haloform-reaction is proposed in the case of the H88R mutant, initiated by hydrogen bonding of the 7-trifluorormethyl group and the guanidinium group of the arginine residue.

SecA is the peripheral membrane-associated subunit of the enzyme complex 'preprotein translocase' which assists the selective transport of presecretory proteins into and across bacterial membranes. The SecA protein acts as the molecular motor that drives the translocation of presecretory proteins through the membrane in a stepwise fashion concomitant with large conformational changes accompanying its own membrane insertion/retraction reaction cycle coupled to ATPase activity. The high flexibility of SecA causes a dynamic conformational heterogeneity which presents a barrier to growth of crystals of high diffraction quality. As shown by fluorescence spectroscopy, the T(m) of the endothermic transition of cytosolic SecA from Bacillus subtilis is shifted to higher temperatures in the presence of 30% glycerol, indicating stabilization of the protein in its compact membrane-retracted conformational state. High glycerol concentrations are also necessary to obtain three-dimensional crystals suitable for X-ray diffraction analysis, suggesting that stabilization of the conformational dynamics of SecA may be required for effective crystallization. The SecA crystals grow as hexagonal bipyramids in the trigonal space group P3(1)12; they possess unit-cell parameters a = 130.8, b = 130.8, c = 150.4 A at 100 K and diffract X-rays to approximately 2.70 A using a high-flux synchrotron-radiation source.

The peptide sequence B18, derived from the membrane-associated sea urchin sperm protein bindin, triggers fusion between lipid vesicles. It exhibits many similarities to viral fusion peptides and may have a corresponding function in fertilization. The lipid-peptide and peptide-peptide interactions of B18 are investigated here at the ultrastructural level by electron microscopy and x-ray diffraction. The histidine-rich peptide is shown to self-associate into two distinctly different supramolecular structures, depending on the presence of Zn(2+), which controls its fusogenic activity. In aqueous buffer the peptide per se assembles into beta-sheet amyloid fibrils, whereas in the presence of Zn(2+) it forms smooth globular clusters. When B18 per se is added to uncharged large unilamellar vesicles, they become visibly disrupted by the fibrils, but no genuine fusion is observed. Only in the presence of Zn(2+) does the peptide induce extensive fusion of vesicles, which is evident from their dramatic increase in size. Besides these morphological changes, we observed distinct fibrillar and particulate structures in the bilayer, which are attributed to B18 in either of its two self-assembled forms. We conclude that membrane fusion involves an alpha-helical peptide conformation, which can oligomerize further in the membrane. The role of Zn(2+) is to promote this local helical structure in B18 and to prevent its inactivation as beta-sheet fibrils.

A monoclinic crystal modification of GTP cyclohydrolase I (space group P2(1), a = 204.2 A, b = 210.4 A, c = 71.8 A, alpha = gamma = 90 degrees, beta = 95.8 degrees) was studied by freeze-etching electron microscopy and by Patterson correlation techniques. The freeze-etched samples were either shadowed with Pt/C or decorated with monolayers of gold, silver or platinum. Correlation averaged electron micrographs of decoration replicas indicated 5-fold molecular symmetry. In conjunction with the molecular mass of the active GTP cyclohydrolase I enzyme complex of about 210,000 Da, which had been reported in the literature, and a molecular mass of the protomers of 24,700 Da, the electron microscopic observation suggests that the enzyme is a decamer with 5-fold symmetry. The processed images of decorated crystal surfaces also showed that the four protein multimers in the crystal unit cell are related by 4-fold pseudosymmetry. A Patterson analysis of the X-ray data showed two non-crystallographic 5-fold axes, inclined at 12 degrees to each other, thus confirming and extending the electron microscopic findings. Additionally, local 2-fold axes were found in planes perpendicular to the 5-fold particle axes. Thus, the combined X-ray and electron microscope data indicate that GTP cyclohydrolase I is a decamer with D5 symmetry. A procedure for hkl assignments of the crystal planes observed in electron micrographs was developed. On this basis, it was possible to determine the approximate molecular positions in the ab plane. Independent information on the crystal packing was obtained by single isomorphous replacement and electron density averaging. The 5-fold averaged 6 A electron density shows that the GTP cyclohydrolase I decamer is torus-shaped with an approximate diameter of 100 A and a thickness of 65 A. The study demonstrates that the combination of freeze-etching electron microscopy with Patterson analysis of X-ray data is a powerful approach for the solution of complex crystallographic problems. The procedure for this analysis as well as possible pitfalls are discussed in detail.

Background: Tetrahydrobiopterin serves as the cofactor for enzymes involved in neurotransmitter biosynthesis and as regulatory factor in immune cell proliferation and the biosynthesis of melanin. The biosynthetic pathway to tetrahydrobiopterin consists of three steps starting from GTP. The initial reaction is catalyzed by GTP cyclohdrolase I (GTP-CH-I) and involves the chemically complex transformation of the purine into the pterin ring system., Results: The crystal structure of the Escherichia coli GTP-CH-I was solved by single isomorphous replacement and molecular averaging at 3.0 A resolution. The functional enzyme is a homodecameric complex with D5 symmetry, forming a torus with dimensions 65 A x 100 A. The pentameric subunits are constructed via an unprecedented cyclic arrangement of the four-stranded antiparallel beta-sheets of the five monomers to form a 20-stranded antiparallel beta-barrel of 35 A diameter. Two pentamers are tightly associated by intercalation of two antiparallel helix pairs positioned close to the subunit N termini. The C-terminal domain of the GTP-CH-I monomer is topologically identical to a subunit of the homohexameric 6-pyruvoyl tetrahydropterin synthase, the enzyme catalyzing the second step in tetrahydrobiopterin biosynthesis., Conclusions: The active site of GTP-CH-I is located at the interface of three subunits. It represents a novel GTP-binding site, distinct from the one found in G proteins, with a catalytic apparatus that suggest involvement of histidines and, possibly, a cystine in the unusual reaction mechanism. Despite the lack of significant sequence homology between GTP-CH-I and 6-pyruvoyl tetrahydropterin synthase, the two proteins, which catalyze consecutive steps in tetrahydrobiopterin biosynthesis, share a common subunit fold and oligomerization mode. In addition, the active centres have an identical acceptor site for the 2-amino-4-oxo pyrimidine moiety of their substrates which suggests an evolutionarily conserved protein fold designed for pterin biosynthesis.

The lumazine synthase/riboflavin synthase complex of Bacillus subtilis consists of an icosahedral capsid of 60 beta subunits enclosing a core of 3 alpha subunits. The preparation of reconstituted hollow capsids consisting of 60 beta subunits and their crystallization in a hexagonal (space group P6(3)22) and in a monoclinic (space group C2) modification have been described. The rotational and translational parameters of the protein molecules in both crystal forms were studied by electron microscopy of freeze-etch replicas and by Patterson correlation techniques. Decoration with silver and image processing provided images with the positions of the 3-fold and 5-fold molecular axes being labelled by metal clusters. This allowed the unequivocal determination of the orientation and translational position of the protein molecules with respect to the crystallographic axes in the hexagonal modification. From inspection of the decoration images it was immediately obvious that the hexagonal crystal forms of alpha 3 beta 60 and of beta 60 are isomorphous. In the monoclinic crystals, a local icosahedral 2-fold coincides with the crystallographic 2-fold axis. The exact solution of the particle orientation was determined by interpretation of Patterson self-rotation functions for the icosahedral symmetry axes. Rotational and translational parameters for the monoclinic modification are given. A rational procedure for the efficient application of freeze-etching techniques in order to elucidate the packing in crystals of large proteins is described.

On the surface of protein molecules the formation of metal clusters during vacuum condensation is controlled by topochemical features of the substrate and by specific properties of the decorating material. The resulting metal distribution (decoration pattern) can be mapped by electron microscopy in conjunction with image processing. We have applied this technique to freeze-etched crystals of the lumazine synthase-riboflavin synthase complex and its derivative obtained by binding of the heteropolytungstate (NaP5W30O110)(NH4)14.31 H2O. The decoration pattern of the free protein and its heteropolytungstate derivative showed marked differences. The correlation of these data with the X-ray structure of the protein showed an increased affinity of both gold and silver to the location of heteropolytungstate. Decoration sites can, but do not need to, be close to the protein surface. Actually, two of the observed decoration sites are located on a layer of ice as thick as 20 A, which apparently transmits underlying topochemical features. Preferential affinity of a site to a given metal must be seen as a property that depends on specific interaction with the decorating material but also on the differential affinities in adjacent areas.

Crystals of heavy riboflavin synthase from Bacillus subtilis were freeze-etched and vacuum-coated at normal incidence with 0.1 to 0.4 nm of gold and silver, respectively. This decoration technique was applied to probe the protein surface for preferential nucleation sites. Image processing of the electron micrographs revealed two particular decoration sites for silver and a different one for gold. According to X-ray crystallography, the riboflavin synthase molecules are spherical and smooth except for a surface corrugation of less than 1 nm, which can not be depicted by heavy-metal shadowing. Thus the decoration sites represent sites of specific physical-chemical interactions between the condensing metal and the protein. The decoration pattern correctly reflects the icosahedral symmetry of the almost spherical protein molecules. Owing to the molecule's symmetry, the position of these topochemical sites with respect to the symmetry axes can be localized within 5A. The packing of the molecules in the crystal can be directly observed on shadowed replicas. Only decoration, however, makes it possible to observe the exact orientation of the molecules within the crystal planes and to derive the true lattice constant along the 6-fold screw axis. This proves decoration to be a technique suitable for studying crystal packing and the molecular symmetry of protein complexes at high resolution. The technique can be applied to crystals that are not large enough or insufficiently ordered for X-ray crystallography.