Andrzej Olczak

The molecular basis of the coloration mechanism in lobster shell: β-Crustacyanin at 3.2-Å resolution

The binding of the carotenoid astaxanthin (AXT) in the protein multimacromolecular complex crustacyanin (CR) is responsible for the blue coloration of lobster shell. The structural basis of the bathochromic shift mechanism has long been elusive. A change in color occurs from the orange red of the unbound dilute AXT (λmax 472 nm in hexane), the well-known color of cooked lobster, to slate blue in the protein-bound live lobster state (λmax 632 nm in CR). Intriguingly, extracted CR becomes red on dehydration and on rehydration goes back to blue. Recently, the innovative use of softer x-rays and xenon derivatization yielded the three-dimensional structure of the A1 apoprotein subunit of CR, confirming it as a member of the lipocalin superfamily. That work provided the molecular replacement search model for a crystal form of the β-CR holo complex, that is an A1 with A3 subunit assembly including two bound AXT molecules. We have thereby determined the structure of the A3 molecule de novo. Lobster has clearly evolved an intricate structural mechanism for the coloration of its shell using AXT and a bathochromic shift. Blue/purple AXT proteins are ubiquitous among invertebrate marine animals, particularly the Crustacea. The three-dimensional structure of β-CR has identified the protein contacts and structural alterations needed for the AXT color regulation mechanism.

S-SWAT (softer single-wavelength anomalous technique): potential in high-throughput protein crystallography.

The drive for yet higher output of protein crystal structures sustains an interest in streamlining data-collection protocols. Highly organized automated robotics attend to one aspect. The data-collection elapsed time also depends on the number of wavelengths used, the data redundancy and the desired diffraction resolution limit. Synchrotron-radiation beamlines offer a centralized data-collection approach with considerable flexibility of choice of wavelength(s) with high beam intensity and fine collimation. The arrival of automated IPs (imaging plates) and CCDs (charge-coupled devices) with sample freezing has widened the scope for ease of data collection and achieving a high redundancy, the latter at the cost of elapsed time per sample. This paper reports a single-wavelength approach where a large optimized f signal involving the Xe L1-absorption edge is harnessed. Even with modest data redundancy (7), a high-quality electron-density map was obtained from that single data set when combined with phase improvement methods. Two phasing protocols are compared for the same data set. Resolution extension (to 1.4 A), in this test, was also done but via a second data set. In future, with a tilted detector geometry, full diffraction resolution will be collectable in one experiment on the one beamline. Moreover, careful minimization of beamline Be window thickness on the new Daresbury SRS Multipole Wiggler Beamline 10 will maximize the 2-3 A (softer) wavelength intensity performance specification. The work has application to the harnessing of iodine f optimized signals and enhanced sulfur f, as well as xenon f, in protein crystallography. This approach, softer-SWAT, should swat many protein structures when it comes online.

Structure of lobster apocrustacyanin A1 using softer X-rays

The molecular basis of the camouflage colouration of marine crustacea is often provided by carotenoproteins. The blue colour of the lobster carapace, for example, is intricately associated with a multimacromolecular 16-mer complex of protein subunits each with a bound astaxanthin molecule. The protein subunits of crustacyanin fall into two distinct subfamilies, CRTC and CRTA. Here, the crystal structure solution of the A(1) protein of the CRTC subfamily is reported. The problematic nature of the structure solution of the CRTC proteins (both C(1) and A(1)) warranted consideration and the development of new approaches. Three putative disulfides per protein subunit were likely to exist based on molecular-homology modelling against known lipocalin protein structures. With two such subunits per crystallographic asymmetric unit, this direct approach was still difficult as it involved detecting a weak signal from these sulfurs and suggested the use of softer X-rays, combined with high data multiplicity, as reported previously [Chayen et al. (2000), Acta Cryst. D56, 1064-1066]. This paper now describes the structure solution of CRTC in the form of the A(1) dimer based on use of softer X-rays (2 A wavelength). The structure solution involved a xenon derivative with an optimized xenon L(I) edge f signal and a native data set. The hand of the xenon SIROAS phases was determined by using the sulfur anomalous signal from a high-multiplicity native data set also recorded at 2 A wavelength. For refinement, a high-resolution data set was measured at short wavelength. All four data sets were collected at 100 K. The refined structure to 1.4 A resolution based on 60 276 reflections has an R factor of 17.7% and an R(free) of 22.9% (3137 reflections). The structure is that of a typical lipocalin, being closely related to insecticyanin, to bilin-binding protein and to retinol-binding protein. This A(1) monomer or dimer can now be used as a search motif in the structural studies of the oligomeric forms alpha- and beta-crustacyanins, which contain bound astaxanthin molecules.

Apocrustacyanin A1 from the lobster carotenoprotein α-crustacyanin: crystallization and initial X-ray analysis involving softer X-rays

The A1 subunit of the carotenoprotein alpha-crustacyanin, isolated from lobster carapace, has been crystallized using the vapour-diffusion method. The crystals, grown in solutions of ammonium sulfate containing methylpentanediol (MPD), diffracted to 2. 0 A. The crystals are stable to radiation. The space group of the crystals is P2(1)2(1)2(1). The unit-cell parameters are a = 41.9, b = 80.7, c = 110.8 A. Standard structure determination has been unsuccessful within this crustacyanin family. Instead, an approach based on the S atoms is being undertaken involving softer X-rays at the SRS, Daresbury.

Planarity of heteroaryldithiocarbazic acid derivatives showing tuberculostatic activity. II. Crystal structures of 3-[amino(pyrazin-2-yl)methylidene]-2-methylcarbazic acid esters

Four compounds showing moderate antituberculostatic activity have been studied to test the hypothesis that the planarity of the 2-[amino(pyrazin-2-yl)methylidene]dithiocarbazate fragment is crucial for activity. N-Anilinopyrazine-2-carboximidamide, C(11)H(11)N(5), D1, and diethyl 2,2-[({[amino(pyrazin-2-yl)methylidene]hydrazinylidene}methylidene)bis(sulfanediyl)]diacetate, C(14)H(19)N(5)O(4)S(2), B1, maintain planarity due to conjugation and attractive intramolecular hydrogen-bond contacts, while methyl 3-[amino(pyrazin-2-yl)methylidene]-2-methyldithiocarbazate, C(8)H(11)N(5)S(2), C1, and benzyl 3-[amino(pyrazin-2-yl)methylidene]-2-methyldithiocarbazate, C(14)H(15)N(5)S(2), C2, are not planar, due to methylation at one of the N atoms of the central N-N bond. The resulting twists of the two molecular halves (parts) of C1 and C2 are indicated by torsion angles of 116.5 (2) and -135.9 (2)°, respectively, compared with values of about 180° in the crystal structures of nonsubstituted compounds. As the methylated derivatives show similar activity against Mycobacterium tuberculosis to that of the nonsubstituted derivatives, maintaining planarity does not seem to be a prerequisite for activity.

Synthesis, structure, and tuberculostatic activity of dimethyl benzoylcarbonohydrazonodithioates

New dimethyl benzoylcarbonohydrazonodithioates were obtained by CS2 addition to arylcarboxylic acid hydrazides and methylation of the formed adduct. The new derivatives were tested for their activity against Mycobacterium tuberculosis. Some compounds exhibited high activity toward sensitive and resistant strains.Graphical abstract

Planarity of heteroaryldithiocarbazic acid derivatives showing tuberculostatic activity. III. Mono- and diesters of 3-(pyrazin-2-ylcarbonyl)dithiocarbazic acid.

Methyl 2-(pyrazin-2-ylcarbonyl)hydrazinecarbodithioate, C(7)H(8)N(4)OS(2), (E1), N-[bis(methylsulfanyl)methylidene]pyrazine-2-carbohydrazide, C(8)H(10)N(4)OS(2), (F1), N-[bis(methylsulfanyl)methylidene]-6-methoxypyrazine-2-carbohydrazide, C(9)H(12)N(4)O(2)S(2), (F2), and methyl 1-methyl-2-(pyrazin-2-ylcarbonyl)hydrazinecarbodithioate, C(8)H(10)N(4)OS(2), (G1), can be considered as derivatives of classical (thio)amide-type tuberculostatics, and all are moderately active against Mycobacterium tuberculosis. This study was undertaken in a search for relationships between activity and specific intramolecular interactions, especially conjugations and hydrogen-bond contacts, and the molecular structures were compared with respective amine analogues, also active against the pathogen. Despite the differences between the amine and carbonyl groups with opposite functions in the hydrogen bond, the two types of structure show a surprisingly similar planar geometry, mostly due to the conjugations aided by the bifurcated intramolecular hydrogen-bond contact between the N-H group of the central hydrazide group as donor and a pyrazine N atom and an S atom of the dithio function as acceptors. Planarity was suggested to be crucial for the tuberculostatic activity of these compounds. The N-methylated derivative (G1) showed a significant twist at the N-N bond [torsion angle = -121.9 (3)°] due to the methyl substitution, which precludes an intramolecular N-H···S contact and the planarity of the whole molecule. Nonetheless, the compound shows moderate tuberculostatic activity.

The role of wavelength and source in the search for sulfur-atom positions evaluated in two case studies: lysozyme at room temperature and cryo apocrustacyanin A1

Synchrotron radiation beamlines offer automated data collection with faster and larger detectors, a choice of wavelength(s), intense beams and fine collimation. An increasing output of protein crystal structures sustains an interest in streamlining data collection protocols. Thus, more and more investigators are looking into the use of the anomalous signal from sulfur to obtain initial phase information for medium-size proteins. This type of experiment ideally requires the use of synchrotron radiation, softer X-rays and cryocooling of the sample. Here the results are reported of an investigation into locating the weak, i.e. sulfur, anomalous scatterers in lysozyme using rotating anode or synchrotron radiation data recorded at room temperature. It was indeed possible to locate the sulfur atoms from a lysozyme crystal at room temperature. Accurate selection of images during scaling was needed where radiation damage effects were detected. Most interestingly, comparisons are provided of high-redundancy data sets recorded with synchrotron radiation at λ = 2.0 and 1.488u2005A, and with Cu Kα and Mo Kα radiation. Apocrustacyanin A1 was also investigated; from the results of a very high redundancy data collection using softer synchrotron X-rays and a cryo-cooled crystal, it was possible to find the sulfur atoms.

Planarity of heteroaryldithiocarbazic acid derivatives showing tuberculostatic activity. IV. Diesters of benzoylcarbonohydrazonodithioic acid.

Dimethyl (3,4-dichlorobenzoyl)carbonohydrazonodithioate, C(10)H(10)Cl(2)N(2)OS(2), (D1), dibenzyl (3,4-dichlorobenzoyl)carbonohydrazonodithioate, C(22)H(18)Cl(2)N(2)OS(2), (D2), dimethyl (3,4-dichlorobenzoyl)-1-methylcarbonohydrazonodithioate, C(11)H(12)Cl(2)N(2)OS(2), (D3), 3,4-dichloro-N-(1,3-dithiolan-2-ylidene)-N-methylbenzohydrazide, C(11)H(10)Cl(2)N(2)OS(2), (D4), were synthesized as potential tuberculostatics. Compound (D1) (with two molecules in the asymmetric unit) was the only one showing tuberculostatic activity of the same range as the common drugs isoniazid and pyrazinamide. The molecular structures of the studied compounds depend on the substitution at the N atom adjacent to the carbonyl group. In the case of the unsubstituted derivatives (D1) and (D2), their central frames are generally planar with a twist of the 3,4-dichlorophenyl ring by 30-40°. Until now, coplanarity of the aromatic ring with the (methylene)carbonohydrazone fragment has been considered a prerequisite for tuberculostatic activity. The N-methylated derivatives (D3) and (D4) show an additional twist along the N-C(=O) bond by 20-30° due to the spatial repulsion introduced by the methyl substituent.

Planarity of benzoylthiocarbazate tuberculostatics. III. Diesters of 3-(2-hydroxybenzoyl)dithiocarbazic acid

Searches for new tuberculostatic agents are important considering the occurrence of drug-resistant strains of Mycobacterium tuberculosis. The structures of three new potentially tuberculostatic compounds, namely isopropyl methyl (2-hydroxybenzoyl)carbonohydrazonodithioate, C12H16N2O2S2, (Z)-benzyl methyl (2-hydroxybenzoyl)carbonohydrazonodithioate, C16H16N2O2S2, and dibenzyl (2-hydroxybenzoyl)carbonohydrazonodithioate propan-2-ol monosolvate, C22H20N2O2S2·C3H8O, were determined by X-ray diffraction. The mutual orientation of the three main fragments of the compounds, namely an aromatic ring, a dithioester group and a hydrazide group, can influence the biological activity of the compounds. In all three of the structures studied, the C(=O)NH group is in the anti conformation. In addition, the presence of the hydroxy group in the ortho position of the aromatic ring in all three structures leads to the formation of an intramolecular hydrogen bond stabilizing the planarity of the molecules.