Diffraction cartography: applying microbeams to macromolecular crystallography sample evaluation and data collection.

Crystals of biological macromolecules often exhibit considerable inter-crystal and intra-crystal variation in diffraction quality. This requires the evaluation of many samples prior to data collection, a practice that is already widespread in macromolecular crystallography. As structural biologists move towards tackling ever more ambitious projects, new automated methods of sample evaluation will become crucial to the success of many projects, as will the availability of synchrotron-based facilities optimized for high-throughput evaluation of the diffraction characteristics of samples. Here, two examples of the types of advanced sample evaluation that will be required are presented: searching within a sample-containing loop for microcrystals using an X-ray beam of 5 microm diameter and selecting the most ordered regions of relatively large crystals using X-ray beams of 5-50 microm in diameter. A graphical user interface developed to assist with these screening methods is also presented. For the case in which the diffraction quality of a relatively large crystal is probed using a microbeam, the usefulness and implications of mapping diffraction-quality heterogeneity (diffraction cartography) are discussed. The implementation of these techniques in the context of planned upgrades to the ESRF's structural biology beamlines is also presented.

Lanthanide-based fluorogenic peptide substrate for the highly sensitive detection of thermolysin.

A new fluorogenic, lanthanide-based oligopeptide substrate for the detection of the zinc-dependent endoprotease thermolysin is described. Using time-resolved fluorescence measurement, a highly sensitive assay for thermolysin was developed with a 50pM detection limit (3.5fmol).

Characterization of collagenase blend enzymes for human islet transplantation.

BACKGROUND: Efficient islet isolation represents a necessary requirement for successful islet transplantation as a treatment for type 1 diabetes. The choice of collagenase for pancreas digestion is critical for the isolation outcome, and Liberase is the most widely used enzyme, although large intra-batched variability in activity and efficiency has been observed. METHODS: The aim of this study was to characterize Liberase components and their relative role in pancreas digestion. Liberase batches were characterized by microelectrophoresis. RESULTS: By means of microelectrophoresis, we identified three main proteins each with different prevalences between batches. Two proteins were found to correspond to class I (CI) and one to class II (CII) collagenase. In a series of 163 islet isolations, we observed that the CII correlated with islet yield (P<0.001) and digestion time (P<0.001); additionally, CI directly correlated with purity (P=0.028). Finally, when CII and one of the CI isoforms were >50 percentile, 15 of 36 preparations were transplanted, with 27 of 127 transplanted in the other cases (P=0.013). CONCLUSION: These results represent an important step toward the characterization of enzymes, with the final aim of identifying key components for a standardized product.

Developing photoactive affinity probes for proteomic profiling: hydroxamate-based probes for metalloproteases.

The denaturing aspect of current activity-based protein profiling strategies limits the classes of chemical probes to those which irreversibly and covalently modify their targeting enzymes. Herein, we present a complimentary, affinity-based labeling approach to profile enzymes which do not possess covalently bound substrate intermediates. Using a variety of enzymes belonging to the class of metalloproteases, the feasibility of the approach was successfully demonstrated in several proof-of-concept experiments. The design template of affinity-based probes targeting metalloproteases consists of a peptidyl hydroxamate zinc-binding group (ZBG), a fluorescent reporter tag, and a photolabile diazirine group. Photolysis of the photolabile unit in the probe effectively generates a covalent, irreversible linkage between the probe and the target enzyme, rendering the enzyme distinguishable from unlabeled proteins upon separation on a SDS-PAGE gel. A variety of labeling studies were carried out to confirm that the affinity-based approach selectively labeled metalloproteases in the presence of a large excess of other proteins and that the success of the labeling reaction depends intimately upon the catalytic activity of the enzyme. Addition of competitive inhibitors proportionally diminished the extent of enzyme labeling, making the approach useful for potential in situ screening of metalloprotease inhibitors. Using different probes with varying P(1) amino acids, we were able to generate unique "fingerprint" profiles of enzymes which may be used to determine their substrate specificities. Finally, by testing against a panel of yeast metalloproteases, we demonstrated that the affinity-based approach may be used for the large-scale profiling of metalloproteases in future proteomic experiments.

SOMCD: method for evaluating protein secondary structure from UV circular dichroism spectra.

This article presents SOMCD, an improved method for the evaluation of protein secondary structure from circular dichroism spectra, based on Kohonen's self-organizing maps (SOM). Protein circular dichroism (CD) spectra are used to train a SOM, which arranges the spectra on a two-dimensional map. Location in the map reflects the secondary structure composition of a protein. With SOMCD, the prediction of beta-turn has been included. The number of spectra in the training set has been increased, and it now includes 39 protein spectra and 6 reference spectra. Finally, SOM parameters have been chosen to minimize distortion and make the network produce clusters with known properties. Estimation results show improvements compared with the previous version, K2D, which, in addition, estimated only three secondary structure components; the accuracy of the method is more uniform over the different secondary structures.

Addition of a methyl group changes both the catalytic velocity and thermostability of the neutral protease from Bacillus stearothermophilus.

Specific activity was compared between wild-type (WT) neutral protease from Bacillus stearothermophilus and mutant protease (M1; Gly144 replaced by Ala144) with enhanced thermostability. When casein was used as a substrate, M1 showed 1.5-times higher specific activity than that of WT. In contrast, the specific activities of M1 for soluble reduced lysozyme and insulin B chain were lower than those of WT by 17.2 and 13.2%, respectively. After digestion of the insulin A chain by these enzymes, the peptide products were purified and the N-terminal amino acid sequences were determined. WT enzyme cleaved insulin A chain at three sites, whereas no digestion was observed with M1. Using Z-Gly-Leu-NH2 as a substrate, the kinetic parameters were determined. The Km values are nearly equal for both enzymes, whereas the kcat of M1 (240 min-1) was much smaller compared to the WT (830 min-1). The data indicate that the mutation (addition of a methyl group) exerts an effect by changing both the catalytic velocity and thermostability.

Selective decrease in low-Mr HMG proteins HMG I and HMG Y during differentiation of mouse teratocarcinoma cells.

We have studied the presence of high-mobility-group (HMG) chromatin proteins in undifferentiated F9 mouse teratocarcinoma cells and F9 cells, which were induced to differentiate by treatment with retinoic acid and dibutyryl-cAMP for 5 days. Acetic acid/urea-polyacrylamide gel electrophoresis and reversed-phase HPLC revealed that the induced F9 cells contained 77 and 62% less HMG I and HMG Y, respectively, than their untreated counterparts. The relative amounts of two other low-Mr HMG proteins HMG 14 and HMG 17 remained essentially unchanged and only a minor decrease was observed in the content of one of the high-Mr HMG proteins, HMG 2. The identity of the low-Mr HMG proteins was verified by amino acid analysis or partial sequencing. These results suggest that HMG I and HMG Y are HMG proteins specific for undifferentiated cells.

The identification of large peptide fragments produced from proteins of known sequence: a computerized approach using amino acid composition indexes and its application to thermolysin.

An approach to identify fragments produced from proteins of known sequence, based on their amino acid composition, is described. A BASIC computer program (SEARCH) was used to quantitate the degree of relatedness between an experimentally determined amino acid composition and theoretical test peptide compositions calculated from a protein of known amino acid sequence. This computerized approach provides a rapid and objective identification of autolytic peptide fragments produced from thermolysin. Three different types composition indexes were compared with respect to their value versus the number of sequence differences between experimental and test compositions. The difference index was found to show a linear relationship and the lowest level of variability in this regard. On the basis of this comparison, we conclude that the difference index is the most reliable indicator of peptide fragment identity.

The primary structure of Bacillus cereus neutral proteinase and comparison with thermolysin and Bacillus subtilis neutral proteinase.

The complete amino-acid sequence of a neutral proteinase, produced by Bacillus cereus, was determined by protein sequencing. The neutral proteinase consists of 317 amino-acid residues. The primary structure is 70% homologous to thermolysin, a thermostable neutral proteinase and 45% homologous to Bacillus subtilis neutral proteinase. The zinc-binding site and the hydrophobic pocket of the active site are highly similar in all three proteinases. B. cereus neutral proteinase which is 20 degrees C less thermostable (60 degrees C) than thermolysin (80 degrees C) shows only minor differences in calcium binding sites and salt bridges compared to thermolysin (known from its X-ray diffraction analysis), whereas B. subtilis neutral proteinase (50 degrees C) differs considerably. Therefore it was assumed that the difference in thermostability between B. cereus neutral proteinase and thermolysin is not caused by different metal binding properties, or differences in the active site, but by changes within the rest of the molecule. Calculation of secondary structure potentials according to Chou & Fasman, hydrophobicity and bulkiness of the different structural elements and preferred cold----hot amino-acid residue exchanges indicated, that the thermostability of thermolysin compared to B. cereus neutral proteinase is caused by small effects contributed by numerous amino-acid exchanges distributed over the whole molecule, resulting in increased hydrophobicity of beta-pleated sheet and higher bulkiness of alpha-helical regions.

A modification for increasing the sensitivity of the casein-agar plate assay: a simple semiquantitative assay for thermophilic and mesophilic proteases.

A casein-agar plate assay was used for the quantitative determination of both mesophilic and thermophilic proteases. Because many proteases are thermostable, assay at higher temperatures is possible. The sensitivity of the plate assay increased with temperature, the optimum assay temperature depending on the thermostability of the enzyme (e.g. Thermus protease, 75 degrees C; thermolysin, 65 degrees C; trypsin, 65 degrees C; alpha-chymotrypsin, 45 degrees C). A positive correlation was observed between incubation temperature and the density of the para-casein precipitate, increasing the accuracy of diameter measurement. Using this modified method, thermostable proteases could be assayed at levels well below the limits of detection of other methods (e.g. 40 pg of thermolysin and 300 pg of trypsin detectable at 65 degrees C, a 16-fold increase in the sensitivity for trypsin compared with a conventional plate assay (Fossum, K. (1970) Acta Pathol. Microbiol. Scand. Sect. B 78, 350-361)). The sensitivity of the plate assay could be further increased by the inclusion of some detergents and chaotropic agents in the gel.

Circular dichroism comparative studies of two bacterial collagenases and thermolysin.

The recently isolated and purified collagenase produced by Achromobacter iophagus, the collagenase from Clostridium histolyticum, and thermolysin, three enzymes having common properties, were studied by circular dichroism. From the spectra of the aqueous solutions obtained in the peptide region, the fraction of alpha helix, beta sheet and aperiodic segments in the three proteins could be estimated. Good similarity was found between Achromobacter collagenase and thermolysin, which both contain a high fraction of alpha helix. Side-chain contributions were analyzed in the aromatic region of thespectra: effects of pH and of organic solvents were observed, showing the strong influence of surroundings on the stabilization of the proteins.

Fluorescence properties of neutral protease from Bacillus subtilis.

The fluorescence properties of neutral protease from B. subtilis have been investigated under a variety of conditions and the results compared with those previously reported for the homologous metalloendopeptidase thermolysin (Fontana et al., 1977). In the pH range 5-9 neutral protease displayed a quite unusual fluorescence emission spectrum with a maximum near 320 nm, when excitation was at 295 nm. At this wavelength the protein fluorescence is due to tryptophan residues only, which, considering their blue-shift emission, appear rather buried in the hydrophobic protein interior. Specific removal of the functional zinc ion from the enzyme with tetraethylenepentamine does not lead to alteration of the microenvironment around tryptophan residues. On the other hand, removal of both zinc and calcium with ethylenediaminetetraacetic acid brings these residues in full contact with the aqueous solvent medium. Fluorescence quenching measurements were also used to determine the exposure of tryptophan residues in the native enzyme as well as in the presence of chelating agents and protein denaturants. Melting profile experiments carried out by monitoring the fluorescence intensity at 320 nm indicated a cooperative transition at 60-70 degrees. Temperature effects were also determined under conditions perturbed with respect to pH, guanidine hydrochloride and chelating agents. The results reveal differences in the fluorescence properties of the tryptophanyl residues of B. subtilis neutral protease relative to those of thermolysin, which are interpretable considering the location of these residues in the sequences of the two homologous proteins.

[Biospecific chromatography of Bacillus subtilis metalloendoproteinase and detection of multiple forms of the enzyme]. / Biosletsificheskaia khromatografiia metalloendoproteinazy Bacillus subtilis i obnaruzhenie mnozhestvennykh form fermenta

Metalloendoproteinase was isolated from protosubtilin, i.e. a mixture of enzymes produced by Bacillus subtilis, during adsorption on activated carbon and a subsequent biospecific chromatography on DNP--hexamethylene diamine--Sepharose 4B and gramicidin S--Sepharose 4B. The molecular weight of the enzyme estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was found to be 36.000. The preparation isolated contained four molecular forms of the enzyme, each splitting DNP-Gly-Gly-Val-Arg. It is shown that thermolysine, purified by biospecific chromatography, consists of three molecular forms. The amino acid composition of metalloendoproteinase was established.

Thermal stability of homologous neutral metalloendopeptidases in thermophilic and mesophilic bacteria: structural considerations.

Thermolysin and neutral protease A are neutral metalloendopeptidases having similar specificity, molecular weight, metal content, and amino acid composition. Thermolysin, derived from the thermophilic organism Bacillus thermoproteolyticus, is heat inactivated at about 84 degrees whereas neutral protease A, derived from the mesophilic organism Bacillus subtilis, is inactivated at about 59 degrees. Structural analyses reveal that the two enzymes are homologous. Of the 326 residues of neutral protease A, 171 have been placed in sequence and 49% of these have been found in identical loci in thermolysin. These include many of the residues corresponding to the active site of thermolysin. The sensitivity of both enzymes to thermal inactivation is dependent upon the presence of calcium and neutral protease appears to bind less calcium than thermolysin. Structural data indicate that many of the ligands associated with calcium sites 1 and 2 (double site of thermolysin) are present in neutral protease and that calcium site 4 cannot exist in neutral protease. The structural homology and functional analogy of these two proteins support the concept that they have similar conformations. The known structure of thermolysin is used as a model to discuss structural differences which might be related to thermal stability.

Evidence of homologous relationship between thermolysin and neutral protease A of Bacillus subtilis.

A comparison of the partial amino-acid sequence of neutral protease A from Bacillus subtilis with the structure of thermolysin (EC 3.4.24.4) from Bacillus thermoproteolyticus reveals that these two proteins are homologous. Of 171 residues placed in neutral protease (54% of the sequence), 83 residues (49%) occur in identical positions in thermolysin, and include nine of the 13 residues previously identified as components of the active site of thermolysin. This similarity provides support for the hypothesis that the two enzymes have similar three-dimensional structures and a common mechanism of action. Since these enzymes differ markedly in their resistance to heat inactivation, a comparison of their structures may eventually provide a chemical basis for explaining the differences in their thermal stability.