Multipoint binding in metal-affinity chromatography II. Effect of pH and imidazole on chromatographic retention of engineered histidine-containing cytochromes c.

Protein binding in immobilized metal affinity chromatography (IMAC) was studied using a set of Saccharomyces cerevisiae iso-1-cytochrome c variants which differed only in their histidine content and placement. Elution with an imidazole gradient enabled separation of cytochrome c variants based on their histidine multiplicity. Millimolar concentrations of imidazole dramatically decreased protein partitioning to the IMAC support as measured by the chromatographic capacity factors under isocratic conditions. Fitting the partitioning data to the "stoichiometric displacement" model indicates that cytochrome c variants containing from one to four surface histidines each displaced approximately three equivalents of imidazole upon adsorption. Therefore even a protein with a single surface histidine appears to coordinate to multiple copper sites on the IMAC support at neutral pH. The effect of pH on the capacity factors of these variants measured in the absence of imidazole further supports this interpretation. Although the presence of a surface histidine was required for retention at neutral pH, a variant with no surface histidines still partitioned strongly to the IMAC support at higher pH (pH > 7.5). These results indicate the contribution of additional protein-metal-coordinating groups, presumably surface amines, to chromatographic retention in IMAC.

A monoclonal antibody to lateral element proteins in synaptonemal complexes of Lilium longiflorum.

To identify synaptonemal complex (SC) proteins in Lilium longiflorum (lily), monoclonal antibodies were generated using mice immunized with isolated pachytene nuclei. While most of the resulting monoclonal antibodies recognized nucleolar or chromatin proteins, one monoclonal antibody (anti-LE) was found that binds to lateral elements. Anti-LE bound more to lateral elements of SCs digested with DNase than to lateral elements that had not been digested with DNase. The opposite pattern of labeling was observed using monoclonal antibodies to lily chromatin and nucleolar proteins. These results indicate that anti-LE is specifically recognizing lateral element proteins and not chromatin or nucleolar proteins surrounding the lateral elements. On immunoblots, anti-LE binds to three pachytene nuclear proteins (Mr 60000, 66000 and 70000), two tetrad (early microspore) nuclear proteins (Mr 60000 and 70000), and two root tip nuclear proteins (Mr 52000 and 60000). However, anti-LE does not bind to proteins from leaf nuclei. Of these four tissues, leaf is the only one that does not have actively dividing cells. This observation suggests that at least some SC proteins are related to nuclear proteins from mitotically active cells.

Multiple-site binding interactions in metal-affinity chromatography. I. Equilibrium binding of engineered histidine-containing cytochromes c.

Mechanisms of protein retention in immobilized metal-affinity chromatography (IMAC) have been probed using a set of Saccharomyces cerevisiae iso-1-cytochrome c histidine variants constructed by site-directed mutagenesis. Proteins containing a single accessible histidine exhibit Langmuir-type isotherms with maximum protein binding capacities between 5 and 10% of the maximum copper loading and the capacity of the support to bind imidazole. A simple model that assumes that the copper sites are densely packed and can be blocked by protein adsorption yields binding constants for single-histidine proteins that are similar to the binding constant for free imidazole. Proteins containing multiple accessible histidines do not exhibit simple Langmuir-type behavior; they appear to interact with the support by simultaneous coordination to more than one metal ion, the result of which is to increase the apparent binding affinity by as much as a factor of 1000. The protein binding constant depends on the availability of copper sites: binding is significantly weaker at low surface concentrations of copper that presumably cannot support multiple-site interactions. The protein binding capacity drops to zero at copper loadings less than one-half the maximum, indicating that immobilized iminodiacetic acid ligands are sufficiently close together that two can coordinate a single copper ion, which precludes its interaction with a protein. Protein adsorption via multiple-site coordination has important consequences for the optimization of IMAC separations and the design of new IMAC supports.

Inherent asymmetry of the structure of F1-ATPase from bovine heart mitochondria at 6.5 A resolution.

ATP synthase, the assembly which makes ATP in mitochondria, chloroplasts and bacteria, uses transmembrane proton gradients generated by respiration or photosynthesis to drive the phosphorylation of ADP. Its membrane domain is joined by a slender stalk to a peripheral catalytic domain, F1-ATPase. This domain is made of five subunits with stoichiometries of 3 alpha: 3 beta: 1 gamma: 1 delta: 1 epsilon, and in bovine mitochondria has a molecular mass of 371,000. We have determined the 3-dimensional structure of bovine mitochondrial F1-ATPase to 6.5 A resolution by X-ray crystallography. It is an approximately spherical globule 110 A in diameter, on a 40 A stem which contains two alpha-helices in a coiled-coil. This stem is presumed to be part of the stalk that connects F1 with the membrane domain in the intact ATP synthase. A pit next to the stem penetrates approximately 35 A into the F1 particle. The stem and the pit are two examples of the many asymmetric features of the structure. The central element in the asymmetry is the longer of the two alpha-helices in the stem, which extends for 90 A through the centre of the assembly and emerges on top into a dimple 15 A deep. Features with threefold and sixfold symmetry, presumed to be parts of homologous alpha and beta subunits, are arranged around the central rod and pit, but the overall structure is asymmetric. The central helix provides a possible mechanism for transmission of conformational changes induced by the proton gradient from the stalk to the catalytic sites of the enzyme.

Crystallization of F1-ATPase from bovine heart mitochondria.

Crystals of the F1-ATPase sector of the ATP synthase complex from bovine heart mitochondria have been grown from solutions containing polyethylene glycol 6000. The crystals diffract to 2.9 A resolution on a laboratory X-ray source. They are orthorhombic and belong to the space group P2(1)2(1)2(1). The unit cell axes are a = 285 A, b = 108 A, c = 140 A. There is one molecule of F1-ATPase in the asymmetric unit.

Crystal structure of a chimeric Fab' fragment of an antibody binding tumour cells.

The crystal structure of a chimeric Fab' fragment of a monoclonal antibody is presented. The Fab' comprises the murine light chain and heavy chain variable domains of the carcinoma-binding antibody B72.3 fused to the constant domain of human kappa, and the first constant domain and hinge domain of human gamma 4, respectively. A model for the Fab' has been determined by molecular replacement and refined to a resolution of 3.1 A with an R-factor of 17.6%. The additional residues that distinguish a Fab' from a Fab fragment are seen to be disordered in the crystals. The H3 hypervariable loop is short and adopts a sharp hairpin turn in a conformation that results from an interaction between the lysine side-chain of H93 and the main-chain carbonyl group of H96. The remaining hypervariable loops display conformations similar to those predicted from the canonical structures approach, although loop H2 is apparently displaced by a salt-bridge formed between H55 Asp and the neighbouring H73 Lys. These and other features of the structure likely to be important in grafting the hypervariable loops to an otherwise human framework are discussed.

Visualization of Trichoderma reesei Cellobiohydrolase I and Endoglucanase I on Aspen Cellulose by Using Monoclonal Antibody-Colloidal Gold Conjugates.

Monoclonal antibodies (MAbs) specific for cellobiohydrolase I (CBH I) and endoglucanase I (EG I) were conjugated to 10- and 15-nm colloidal gold particles, respectively. The binding of CBH I and EG I was visualized by utilizing the MAb-colloidal gold probes. The visualization procedure involved immobilization of cellulose microfibrils on copper electron microscopy grids, incubation of the cellulose-coated grids with cellulase(s), binding of MAb-colloidal gold conjugates to cellulase(s), and visualization via transmission electron microscopy. CBH I was seen bound to apparent crystalline cellulose as well as apparent amorphous cellulose. EG I was seen bound extensively to apparent amorphous cellulose with minimal binding to crystalline cellulose.

Protein stabilization by engineered metal chelation.

A ligand can shift a protein's folding/unfolding equilibrium by binding with higher affinity to the native state. A metal-chelating site consisting of two histidines separated by three residues (His-X3-His) engineered into an alpha-helix provides a general and easily-implemented means for protein stabilization by this mechanism. We have tested this approach with the iso-1-cytochrome c of Saccharomyces cerevisiae substituted with histidine at positions 4 and 8 in its N-terminal alpha-helix. One mM Cu(II) complexed to iminodiacetate stabilizes the cytochrome c variant by ca. 1 kcal/mol, as determined by guanidinium chloride-induced unfolding. The protein's folding/unfolding equilibrium is shifted by a free energy equal to that calculated from the metal ion's preferential binding to the native protein. Given the ubiquity of surface alpha-helices and the additional possibility of inserting di-histidine chelating sites into turns and beta-structures, we conclude that this is a useful method for protein stabilization.

Crystallization and preliminary X-ray diffraction study of a chimaeric Fab' fragment of antibody binding tumour cells.

Crystals have been obtained of a chimaeric Fab' fragment that binds to a tumour-associated mucin-like glycoprotein TAG72. The Fab' fragment comprises the variable heavy and light-chain domains of a murine monoclonal antibody, B72.3, coupled to human gamma 4 and kappa constant regions. The crystals are orthorhombic and belong to the space group P2(1)2(1)2(1), with unit cell dimensions a = 67.9 A, b = 94.2 A and c = 208.8 A. Diffraction to 2.6 A resolution was observed using synchrotron radiation. Despite the acute radiation sensitivity of the crystals a full native data set has been collected using the Weissenberg camera at the Photon Factory synchrotron. These data will be used for molecular replacement calculations in an attempt to elucidate the structure of this chimaeric Fab' fragment.

Cu(II)-binding properties of a cytochrome c with a synthetic metal-binding site: His-X3-His in an alpha-helix.

A metal-binding site consisting of two histidines positioned His-X3-His in an alpha-helix has been engineered into the surface of Saccharomyces cerevisiae iso-1-cytochrome c. The synthetic metal-binding cytochrome c retains its biological activity in vivo. Its ability to bind chelated Cu(II) has been characterized by partitioning in aqueous two-phase polymer systems containing a polymer-metal complex, Cu(II)IDA-PEG, and by metal-affinity chromatography. The stability constant for the complex formed between Cu(II)IDA-PEG and the cytochrome c His-X3-His site is 5.3 x 10(4) M-1, which corresponds to a chelate effect that contributes 1.5 kcal mol-1 to the binding energy. Incorporation of the His-X3-His site yields a synthetic metal-binding protein whose metal affinity is sensitive to environmental conditions that alter helix structure or flexibility.

Cross-reactive and specific monoclonal antibodies against cellobiohydrolases I and II and endoglucanases I and II of Trichoderma reesei.

Splenocytes derived from mice inoculated with a commercial cellulase preparation or purified cellulases were fused with a stable myeloma cell line (SP2/0). Specific monoclonal antibodies to cellobiohydrolases I and II and endoglucanases I and II were established. In addition to specific monoclonal antibodies, we were also able to establish stable hybridoma cell lines which produced monoclonal antibodies that recognized similar epitopes possessed by two or more of the above cellulases. By obtaining monospecific antibodies for all four individual cellulases, the role and function of the individual cellulases can thus be studied in greater detail.

Structural aspects of proton-pumping ATPases.

ATP synthase is found in bacteria, chloroplasts and mitochondria. The simplest known example of such an enzyme is that in the eubacterium Escherichia coli; it is a membrane-bound assembly of eight different polypeptides assembled with a stoichiometry of alpha 3 beta 3 gamma 1 delta 1 epsilon 1 a1b2c10-12. The first five of these constitute a globular structure, F1-ATPase, which is bound to an intrinsic membrane domain, F0, an assembly of the three remaining subunits. ATP synthases driven by photosynthesis are slightly more complex. In chloroplasts, and probably in photosynthetic bacteria, they have nine subunits, all homologues of the components of the E. coli enzyme; the additional subunit is a duplicated and diverged relation of subunit b. The mammalian mitochondrial enzyme is more complex. It contains 14 different polypeptides, of which 13 have been characterized. Two membrane components, a (or ATPase-6) and A6L, are encoded in the mitochondrial genome in overlapping genes and the remaining subunits are nuclear gene products that are translated on cytoplasmic ribosomes and then imported into the organelle. The sequence of the proteins of ATP-synthase have provided information about amino acids that are important for its function. For example, amino acids contributing to nucleotide binding sites have been identified. Also, they provide the basis of models of secondary structure of membrane components that constitute the transmembrane proton channel. An understanding of the coupling of the transmembrane potential gradient for protons, delta mu H+, to ATP synthesis will probably require the determination of the structure of the entire membrane bound complex. Crystals have been obtained of the globular domain, F1-ATPase. They diffract to a resolution of 3-4 A and data collection is in progress. As a preliminary step towards crystallization of the entire complex, we have purified it from bovine mitochondria and reconstituted it into phospholipid vesicles.

Tertiary structural similarity between a class A beta-lactamase and a penicillin-sensitive D-alanyl carboxypeptidase-transpeptidase.

beta-Lactam antibiotics--the penicillins, cephalosporins and related compounds--act by inhibiting enzymes that catalyse the final stages of the synthesis of bacterial cell walls. Recent crystallographic studies of representative enzymes are beginning to reveal the structural bases of antibiotic specificity and mechanism of action, while intensive efforts are being made to understand the beta-lactamase enzymes that are largely responsible for bacterial resistance to these antibiotics. It has been suggested that the beta-lactamases and beta-lactam target enzymes may be evolutionarily related and some similarity of amino-acid sequence around a common active-site serine residue supports this idea. We present here the first evidence from a comparison of three-dimensional structures in support of this hypothesis: the structure of beta-lactamase I from Bacillus cereus is similar to that of the penicillin-sensitive D-alanyl-D-alanine carboxypeptidase-transpeptidase from Streptomyces R61.

Cimetidine prophylaxis for gastrointestinal bleeding in an intensive care unit.

The efficacy of cimetidine in the prevention of gastrointestinal haemorrhage in a general intensive care unit was evaluated in 221 patients by a placebo controlled double blind randomised study. Criteria for bleeding were (i) haematemesis or gastric aspirate greater than 50 ml fresh blood, (ii) melaena or fresh blood per rectum with an upper source verified by endoscopy if the gastric aspirate was clear, (iii) a fall in haemoglobin level greater than 2 g/dl in a 24 hour period associated with either 4+ occult blood in stools, or coffee ground gastric drainage of at least 100 ml. The drug and placebo groups were similar for age, sex, duration of study and risk factors. One hundred and fourteen received cimetidine and 107 placebo. Only 8% of the patients bled with no significant difference between the two groups (6/114 cimetidine, 11/107 placebo; p = 0.16). There was no correlation between the frequency of bleeding and either the number of risk factors per patient or the duration of intensive care unit stay. Thirteen patients died in each study group, resulting in overall mortality of 12%. The low incidence of haemorrhage, the lack of statistical benefit from cimetidine and the similar mortality all argue against the routine use of this drug in intensive care unit patients.