Substrate binding induces a cooperative conformational change in the 12S subunit of transcarboxylase: Raman crystallographic evidence

The 12S subunit of transcarboxylase is a 338 000 Da hexamer that transfers carboxlylate from methylmalonyl-CoA (MM-CoA) to biotin; in turn, the biotin transfers the carboxylate to pyruvate on another subunit, the 5S. Here, Raman difference microscopy is used to study the binding of substrate and product, and their analogues, to single crystals of 12S. A single crystal is the medium of choice because it provides Raman data of unprecedented quality. Crystalline ligand-protein complexes were formed by cocrystallization or by the soaking in/soaking out method. Raman difference spectra were obtained by subtracting the spectrum of the apo crystal from that of a crystal with the substrate or product bound. Raman difference spectra from crystals with the substrate bound are dominated by bands from the protein's amide bonds and aromatic side chain residues. In contrast, Raman difference spectra involving the product, propionyl-CoA, are dominated by modes from the ligand. These results show that substrate binding triggers a conformational change in 12S, whereas product binding does not. The conformational change involves an increase in the amount of alpha-helix since markers for this secondary structure are prominent in the difference spectra of the substrate complex. The number of MM-CoA ligands bound per 12S hexamer can be gauged from the intensity of the MM-CoA Raman features and the fact that the protein concentration in the crystals is known from X-ray crystallographic data. Most crystal samples had six MM-CoAs per hexamer although a few, from different soaking experiments, contained only 1-2. However, both sets of crystals showed the same degree of protein conformational change, indicating that the change induced by the substrate is cooperative. This effect allowed us to record the Raman spectrum of bound MM-CoA without interference from protein modes; the Raman spectrum of a 12S crystal containing 2 MM-CoA ligands per hexamer was subtracted from the Raman spectrum of a 12S crystal containing six MM-CoA ligands per hexamer. The conformational change is reversible and can be controlled by soaking out or soaking in the ligand, using either concentrated ammonium sulfate solutions or the solution used in the crystallization trials. Malonyl-CoA also binds to 12S crystals and brings about conformational changes identical to those seen for MM-CoA; in addition, butyryl-CoA binds and behaves in a manner similar to propionyl-CoA. These data implicate the -COO- group on MM-CoA (that is transferred to biotin in the reaction on the intact enzyme) as the agent bringing about the cooperative conformational change in 12S.     

Comparison of protein structure in crystals and in solution by laser raman scattering. I. Lysozyme

The laser Raman-scattering technique was employed to examine the question of whether the structure of a globular protein is the same in crystals as in solution. Lysozyme was selected as a model system for this study. In the amide I and amide III regions we found a good agreement between the Raman spectra of lysozyme chloride crystals (in 100% relative humidity) and lysozyme solution (at pH 4.50), indicating that the main-chain conformation is the same between two phases. However, small but definite spectral differences were observed near 464, 622, 644, 934, 960, 978, 1032, 1129, and 1196 cm^?1. Some of these spectral differences may be interpreted in terms of side-chain conformational changes. Additionally, we present Raman spectrum of lysozyme in the lyophilized form and compare it to those of crystals and solution. It was concluded that lyophilization caused conformational changes appreciably, both in the main chain and side chain.     

Laser-diffusion measurements on δ-endotoxin crystals from Bacillus thuringiensis var. kurstaki HD1

Laser light scattering has been employed to investigate changes in the hydrodynamic properties of δ-endotoxin crystals of Bacillus thuringiensis var. kurstaki HD1. Crystals are polydisperse. Estimates of the mean hydrodynamic diameter agree with those obtained by light microscopy. Sodium dodecyl sulphate (SDS) at a final concentration of 1% results in swelling of the toxin crystals. Dithiothreitol-induced solubilization of the swollen crystals indicates the importance of disulphide bridges to the maintenance of the assembled crystal.     

An X-ray study on the interaction between indole ring and pyridine coenzymes: Crystal structure of 1-methyl-3-carbamoylpyridinium: Indole-3-acetic acid (1:1) monohydrate charge-transfer complex

The crystal and molecular structures of the title complex has been determined by X-ray diffraction methods, as a model for tryptophan residues in protein-pyridine coenzyme interactions. The structure was solved by direct methods and was refined by standard methods (final R = 0.073). The light-green crystals consist of alternate layers of indole-3-acetic acid and 1-methyl-3-carbamoylpyridinium molecules piled up to the c-direction, and are stabilized by the crystal water participating in hydrogen bonds in the a- and b-directions. The parallel stackings and interplanar spacing distances between indole and pyridinium rings strongly suggest a II–II¹ charge transfer from the indole ring to the lowest unoccupied orbital of the pyridinium ring in the ground state. Furthermore, this crystal structure provides evidence that quaternization of the N1 position enhances electron-acceptor properties of pyridine. On the other hand, the proton magnetic resonance spectra suggest that the stacking mode between both rings in solution is very similar to the one observed in the crystal structure.     

Raman structural markers of tryptophan and histidine side chains in proteins

The Raman spectrum of a protein contains a wealth of information on the structure and interaction of the protein. To extract the structural information from the Raman spectrum, it is necessary to identify and interpret the marker bands that reflect the structure and interaction in the protein. Recently, new Raman structural markers have been proposed for the tryptophan and histidine side chains by examining the spectra-structure correlations of model compounds. Raman structural markers are now available for the conformation, hydrogen bonding, hydrophobic interaction, and cation-pi interaction of the indole ring of Trp. For His, protonation, tautomerism, and metal coordination of the imidazole ring can be studied by using Raman markers. The high-resolution X-ray crystal structures of proteins provide the basis for testing and modifying the Raman structural markers of Trp and His. The structures derived from Raman spectra are generally consistent with the X-ray crystal structures, giving support for the applicability of most Raman structural makers. Possible modifications and limitations to some marker bands are also discussed.     

A laser Raman study of lysozyme denaturation

The Raman spectrum of chemically denatured lysozyme was studied. The denaturants studied included dimethyl sulfoxide, LiBr, guanidine · HCl, sodium dodecyl sulfate, and urea. Previous studies have shown that the amide I and amide III regions of the Raman spectrum are sensitive to the nature of the hydrogen bond involving the amide group. The intensity of the amide III band at 1260 cm^?1 (assigned to strongly hydrogen-bonded α-helix structure) relative to the intensity of the amide III band near 1240 cm^?1 (assigned to less strongly hydrogen-bonded groups) is used as a parameter for comparison with other physical parameters used to assess denaturation. The correlation between this Raman parameter and denaturation as evidenced by enzyme activity and viscosity measurements is good, leading to the conclusion that the amide III Raman spectrum is useful for assessing the degree of denaturation. The Raman spectrum clearly depends on the type of denaturant employed, suggesting that there is not one unique denatured state for lysozyme. The data, as interpreted, place constraints on the possible models for lysozyme denaturation. One of these is that the simple two-state model does not seem consistent with the observed Raman spectral changes.     

Raman studies of the conformation of the basic pancreatic trypsin inhibitor.

The vibrational Raman spectra of the basic pancreatic trypsin inhibitor in aqueous solution, as lyophilized powder and in a single crystal and presented. The thermal stability of this protein is demonstrated by the fact that minor alterations in the spectrum, mainly in the amide III band near 1260 cm-1, occur in the solution spectrum only at temperatures above 75 degrees C. No significant spectral changes appear when the pH value of the solution is varied in the range from 1.5 to 8.7. The distinct differences of the powder spectrum compared to that of the solution, show that lyophilization causes appreciable conformational changes both in the main-chain and in the side-chains. A difference in main chain conformation of the basic pancreatic trypsin inhibitor in single crystal and in solution is suggested by different amide III frequencies.     

Isolation and Characterization of Coproporphyrin Produced by Four Subspecies of Bacillus thuringiensis

It was found by using spectrophotometric, spectrofluorometric, and high-pressure liquid chromatography that four subspecies of Bacillus thuringiensis produce coproporphyrin. The porphyrin isomer was identified as coproporphyrin I for B. thuringiensis subsp. kurstaki (HD1). The porphyrin was isolated both from spores and from a variety of spent growth media. The quantity of porphyrin released by each Bacillus subspecies differed. The rank order of porphyrin production follows: B. thuringiensis subsp. kurstaki HD1 > B. thuringiensis subsp. thuringiensis HD27 > B. thuringiensis subsp. thuringiensis HD41 > B. thuringiensis subsp. darmstadiensis HD199.     

Constructing Bacillus thuringiensis strain that co-expresses Cry2Aa and chitinase

A triple recombineering technique was used with plasmid pHT315 to produce pHTEC, a construct carrying chitinase and cry2Aa genes from Bacillus thuringiensis subsp. kurstaki 4.0718. Transformation of wild-type B. thuringiensis strain HD73 and the acrystalliferous strain Cry-B with pHTEC resulted in the recovery of recombinant strains that expressed Cry2Aa as cubic crystals in the cell pellet and soluble chitinase protein. The toxicity of HD73 (pHTEC) against Helicoverpa armigera larvae increased sevenfold when compared with HD73 (pHT315) harboring pHT315 vector. The triple recombineering protocol was optimized by comparing recombination efficacy mediated by RecE/RecT and Redα/Redβ and by using single-strand DNA as substrate.     

Identification of Bacillus thuringiensis subsp. kurstaki Strain HD1-Like Bacteria from Environmental and Human Samples after Aerial Spraying of Victoria, British Columbia, Canada, with Foray 48B

Aerial applications of Foray 48B, which contains Bacillus thuringiensis strain HD1, were carried out on 9 to 10 May, 19 to 21 May, and 8 to 9 June 1999 to control European gypsy moth (Lymantria dispar) populations in Victoria, British Columbia, Canada. A major assessment of the health impact of B. thuringiensis subsp. kurstaki was conducted by the Office of the Medical Health Officer of the Capital Health Region during this period. Environmental (air and water) and human (nasal swab) samples, collected before and after aerial applications of Foray 48B, both in the spray zone and outside of the spray zone, were analyzed for the presence of strain HD1-like bacteria. Random amplified polymorphic DNA analysis, cry gene-specific PCR, and dot blot DNA hybridization techniques were used to screen over 11,000 isolates of bacteria. We identified bacteria with genetic patterns consistent with those of B. thuringiensis subsp. kurstaki HD1 in 9,102 of 10,659 (85.4%) isolates obtained from the air samples, 13 of 440 (2.9%) isolates obtained from the water samples, and 131 of 171 (76.6%) isolates from the nasal swab samples. These analyses suggest that B. thuringiensis subsp. kurstaki HD1-like bacteria were present both in the environment and in the human population of Victoria prior to aerial applications of Foray 48B. The presence of B. thuringiensis subsp. kurstaki HD1-like bacteria in human nasal passages increased significantly after the application of Foray 48B, both inside and outside the spray zone.     

Conformational states of the bacteriophage P22 capsid subunit in relation to self-assembly

The formation of closed icosahedral capsids from a single species of coat protein subunit requires that the subunits assume different conformations at different lattice positions. In the double-stranded DNA bacteriophage P22, formation of correctly dimensioned capsids is mediated by interaction between coat protein subunits and scaffolding protein. Raman spectroscopy has been employed to compare the conformations of coat protein subunits which have been polymerized to form capsids in the presence and absence of the of scaffolding protein display a Raman spectrum characterized by a broad amide I band centered at 1665 cm-1 with a discernible shoulder near 1653 cm-1, and a broad amide III profile centered at 1238 cm-1 but asymmetrically skewed to higher frequency. These spectral features indicate that the protein conformation in procapsid shells is rich in beta-sheet secondary structure but contains also a significant distribution of alpha-helix. When biologically active, purified subunits assemble in the absence of scaffolding protein, they form polydisperse multimers lacking the proper dimensions of procapsid closed shells. We designate these multimers as "associated subunits" (AS). The Raman spectrum of associated subunits indicates a narrower distribution of secondary structure. The associated subunits are characterized by a sharper and more intense Raman amide I band at 1666 cm-1, with no prominent amide I shoulder of lower frequency. An analogous narrowing of the Raman amide III profile is also observed for AS particles, with an accompanying shift of the amide III band center to 1235 cm-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spore Coat Protein Synergizes Bacillus thuringiensis Crystal Toxicity for the Indianmeal Moth (Plodia interpunctella)

Spores from Bacillus thuringiensis serovars kurstaki and entomocidus synergized crystal protein toxicity for larvae of the Indianmeal moth (Plodia interpunctella). Preparations of spore-crystal mixtures of either serovar were more toxic for the larvae than either purified spores or crystals alone (based on dry weight). Spores lost 53% of their toxicity for the Indianmeal moth after 2 h of UV-irradiation, but remained partially toxic (28%) even after 4 h of irradiation. Spore coat protein was toxic for the Indianmeal moth and was synergistic with B. thuringiensis serovar kurstaki HD-1 crystal protein. Enhanced toxicity of the combined spore-crystal preparation was attributed to a combination of crystal and spore coat protein, and included the effects of spore germination and resulting septicemia in the larval hemolymph. Ultraviolet irradiation of spores reduced the toxicity from septicemia but not the synergism caused by spore coat protein. The potencies of spore-crystal preparations must be carefully evaluated on the basis of contributions from all three factors. Received: 15 September 1997/Accepted: 21 October 1997     

Environments and conformations of tryptophan side chains of gramicidin A in phospholipid bilayers studied by Raman spectroscopy.

Raman spectroscopy has been used to investigate the hydrophobic interaction of the indole ring with the environments, the water accessibility to the N1H site, and the conformation about the C beta-C3 bond for the four tryptophan side chains of gramicidin A incorporated into phospholipid bilayers. Most of the tryptophan side chains of the head-to-head helical dimer transmembrane channel are strongly interacting with the lipid hydrocarbon chains, and the hydrophobic interactions for the rest increase with increasing hydrocarbon chain length of the lipid. One tryptophan side chain (probably Trp-15) is accessible to water molecules, another (Trp-9) is deeply buried in the bilayer and inaccessible, and the accessibilities of the remaining two (Trp-11 and Trp-13) depend on the bilayer thickness. The torsional angle about the C beta-C3 bond is found to be +/- 90 degrees for all the tryptophans irrespective of the membrane thickness. Binding of the sodium cation to the channel does not change the torsional angles but decreases the water accessibilities of two tryptophans (Trp-11 and Trp-13) considerably. In conjunction with a slight spectral change in the amide III region, it is suggested that the sodium binding causes a partial change in the main-chain conformation around Trp-11 and Trp-13, which results in the movements of these side chains toward the bilayer center. Two models consistent with the present Raman data are proposed for the tryptophan orientation in the dominant channel structure.     

Dissolution and Degradation of Bacillus thuringiensis δ-Endotoxin by Gut Juice Protease of the Silkworm Bombyx mori

The dissolution and degradation of †-endotoxin (crystal) of Bacillus thuringiensis subsp. kurstaki strain HD-1 were investigated. Crystals were dissolved in 0.1 M phosphate-carbonate-NaOH buffer at pH > 12. Swelling of crystals occurred in the buffer between pH 10 and 11, and crystals dissolved in the same buffer supplemented with gut juice protease of the silkworm Bombyx mori. The proteolytic dissolution of crystals occurred after a time lag of several minutes in 0.1 M carbonate-NaOH buffer, pH 10.2. The time lag was not observed when crystals were suspended in the buffer for 30 min before the addition of protease. After the dissolution of the crystals and further degradation of the solubilized protein, the appearance of a toxic protein with a molecular weight of 59,000, designated P-59, was observed. Lower-molecular-weight peptides (less than 40,000) showed no toxicity to the silkworm larvae on feeding. Digestion of the 120,000-dalton subunit of the crystal by gut juice protease also produced P-59. These observations suggest the occurrence of a similar process in vivo, i.e., the swelling of crystals due to the alkalinity of gut juice and the production of P-59, dependent on the hydrolysis of swollen crystals by gut juice protease.     

Immunological properties of entomocidal protein of Bacillus thuringiensis and its insecticidal activity

The immunological properties of the proteinaceous component of the parasporal crystal (δ-endotoxin) of Bacillus thuringiensis var. kurstaki were analyzed by rocket immunoelectrophoresis. Two antisera, one against the k-l-type crystal containing two components, and the other against the k-73-type crystal containing one component, were made in rabbits. The antigens consisting of purified and dissociated crystals were run in electrophoresis with these two antisera. The ratio between the two peak heights of precipitin lines, which were formed by the dissociated crystal of one B. thuringiensis isolate in two antisera, was compared with the ratios of other isolates under identical conditions. The difference in the ratio reflected a difference in the structure of the crystal component and correlated closely with the insecticidal activity spectrum. This method can be used to evaluate a newly isolated B. thuringiensis, and it can further differentiate the isolates which have been classified as one serotype.     

Tyrosine and tryptophan modification monitored by ultraviolet resonance Raman spectroscopy

Nitration of tyrosine with tetranitromethane shifts the tyrosine absorption spectrum and abolishes its 200 nm-excited resonance Raman spectrum. There is no detectable resonance Raman contribution from either reactants or products. Likewise, modification of tryptophan with 2-hydroxy-5-nitrobenzyl bromide (HNBB) shifts its absorption spectrum and abolishes its 218 nm-excited resonance Raman spectrum. In this case resonance Raman bands due to HNBB are seen, but are readily distinguishable from the tryptophan spectrum, can be computer-subtracted. When stellacyanin was treated with tetranitromethane the UV resonance Raman spectrum was greatly attenuated; quantitation of the 850 cm-1 tyrosine band intensity gave a value of 4.3 tyrosines modified out of the seven present in stellacyanin, in good agreement with an estimate of 4.7 from the absorption spectrum. For cytochrome c, the resonance Raman spectrum indicates that two out of the four tyrosines are modified by tetranitromethane treatment, consistent with the crystal structure, which shows two buried tyrosines and two at the protein surface. Treatment of stellacyanin with HNBB gave a reduction in the tryptophan spectrum, excited at 218 nm, consistent with one of the three tryptophans being modified. These modification procedures should be useful in distinguishing spectra of buried tyrosine and tryptophan residues from those at the surface.     

Following ligand binding and ligand reactions in proteins via Raman crystallography

Raman crystallography permits the monitoring of chemical events in single-protein crystals in real time. Using a Raman microscope, it is possible to obtain protein Raman spectroscopic data of unprecedented quality and stability. The latter features allow us to obtain the Raman spectrum for small molecules soaking into crystals under normal (nonresonance) Raman conditions. Thus, via an approach utilizing Raman difference spectroscopy, we can quantitate the amount of ligand in the crystal, determine the chemistry of inhibitor-protein interactions, and follow chemical reactions in the active site on the time scale of minutes. While providing unique chemical insights, these data also provide an invaluable guide for determining the conditions for flash-freezing crystals for X-ray crystallographic analysis. In addition, the Raman difference spectra often contain contributions from protein modes due to protein conformational changes occurring upon ligand binding. These features allow us to probe events ranging from small cooperative conformational changes to massive and unexpected secondary structure changes in the crystal. An experimental advantage of Raman crystallography is that the data can be collected from crystals in situ, in sitting or hanging drops, under the conditions used to grow the crystals.     

Host Range of an Insecticidal Crystal Protein of Bacillus thuringiensis subsp. kurstaki Produced in Escherichia coli

A crylA(c)-like gene of Bacillus thuringiensis subsp. kurstaki strain HD1 was over-expressed in Escherichia coli from a multicopy plasmid. Biological toxicity tests conducted on the larvae of three lepidopteran insects showed that the host range of transgenic E. coli HB 101 (pRT 200) was a subset of the host range of B. thuringiensis kurstaki HD1. Both were toxic to the larvae of Helicoverpa armigera (Gram pod borer) and Bombyx mori (Silkworm). However. though the sporecrystal formulation of HDl was toxic to the larvae of Phthorimaea operculella (Potato tuber moth). the transgenic E. coli was not. Product of St toxin gene other than crylA(c) present In HD1 may be responsible for Its toxicity to the larvae of P. opercuiella.     

Raman spectra of chemically modified lysozyme. Oxindole derivatives.

The Raman spectra of oxidation products of lysozyme have been investigated. The protein was oxidized by N-bromosuccinimide and dimethyl sulfoxide/HCl. Depending on the experimental conditions one to six tryptophan residues are oxidized to oxindole. The most prominent difference between the spectra of lysozyme and its oxindole derivatives is the strong band at 1017 cm^?1 which displaces the tryptophan peak at 1010 cm^?1. Other tryptophan bands are also weakened corresponding to the number of the tryptophan side chains destroyed. Shifts are observed in the amide I and in the amide III regions sensitive to conformational changes. These shifts indicate conformational differences in the higher oxidized species and in the native enzyme, although the amide III maxima overlap with a strong oxindole band. Similar effects are observed in the range of the C-C stretching vibrations of the peptide backbone. If more than one tryptophan side chain is oxidized changes have also been found in the S-S stretching range. The evaluation of this effect is difficult because of the strong oxindole vibration appearing in this region. In species oxidized by great excess of N-bromosuccinimide the tyrosine vibrations can no longer be detected, indicating the modification of this amino acid too.     

Probing inhibitors binding to human urokinase crystals by Raman microscopy: implications for compound screening

Inhibition of urokinase activity represents a promising target for antimetastatic therapy for several types of tumor. The present study sets out to investigate the potential of Raman spectroscopy for defining the molecular details of inhibitor binding to this enzyme, with emphasis on single crystal studies. It is demonstrated that high quality Raman spectra from a series of five inhibitors bound individually to the active site of human urokinase can be obtained in situ from urokinase single crystals in hanging drops by using a Raman microscope. After recording the spectrum of the free crystal, a solution of inhibitor containing an amidine functional group on a naphthalene ring was added, and the spectrum of the crystal-inhibitor complex was obtained. The resulting difference Raman spectrum contained only vibrational modes due to bound inhibitor, originating from the protonated group, i.e., the amidinium moiety, as well as naphthalene ring modes and features from other functionalities that made up each inhibitor. The identification of the amidinium modes was placed on a quantitative basis by experimental and theoretical work on naphthamidine compounds. For the protonated group, -C-(NH2)(2)(+), the symmetric stretch occurs near 1520 cm(-1), and a less intense antisymmetric mode appears in the Raman spectra near 1680 cm(-1). The presence of vibrational modes near 1520 cm(-1) in each of the Raman difference spectra of the five complexes examined unambiguously identifies the protonated form of the amidinium group in the active site. Several advantages were found for single crystal experiments over solution studies of inhibitor-enzyme complexes, and these are discussed. The use of single crystals permits competitive binding experiments that cannot be undertaken in solution in any kind of homogeneous assay format. The Raman difference spectrum for a single crystal that had been exposed to equimolar amounts of all five inhibitors in the hanging drop showed only the Raman signature of the compound with the lowest K(i). These findings suggest that the Raman approach may offer a route in the screening of compounds in drug design applications as well as an adjunct to crystallographic analysis.