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PubMed2004

Adsorption of fluorescein by protein crystals.

Adsorption characteristics of native and cross-linked lysozyme crystals were examined using fluorescein as model adsorbate. The adsorption isotherms exhibited Langmuir or linear behavior. The affinity constant (b1) and the adsorption capacity (Qsat) for fluorescein were found to depend on the type and concentration of co-solute present in the solution. The dynamics of adsorption isotherm transition from Langmuir to linear showed that affinity of lysozyme for solutes increases in the order 2-(cyclohexylamino)ethanesulphonic acid (CHES), 4-morpholinepropanesulphonic acid (MOPS), acetate, fluorescein. Furthermore, the crystal morphology, the degree of cross-linking of the crystals, and, in particular, solution pH were identified as factors determining fluorescein adsorption by the lysozyme crystals. These factors seem to affect crystal capacity for the solute more than affinity for the solute. Adsorption of fluorescein by cross-linked tetragonal lysozyme crystals was exponentially dependent on the lysozyme net charge calculated from the final solution pH. The 3-5-fold increase in the fluorescein adsorption as a result of cross-linking is presumably due to the increasing hydrophobicity of the lysozyme crystal.

Adsorption↗
PubMed2004

New simulation model of multicomponent crystal growth and inhibition.

We review a novel computational model for the study of crystal structures both on their own and in conjunction with inhibitor molecules. The model advances existing Monte Carlo (MC) simulation techniques by extending them from modeling 3D crystal surface patches to modeling entire 3D crystals, and by including the use of "complex" multicomponent molecules within the simulations. These advances makes it possible to incorporate the 3D shape and non-uniform surface properties of inhibitors into simulations, and to study what effect these inhibitor properties have on the growth of whole crystals containing up to tens of millions of molecules. The application of this extended MC model to the study of antifreeze proteins (AFPs) and their effects on ice formation is reported, including the success of the technique in achieving AFP-induced ice-growth inhibition with concurrent changes to ice morphology that mimic experimental results. Simulations of ice-growth inhibition suggest that the degree of inhibition afforded by an AFP is a function of its ice-binding position relative to the underlying anisotropic growth pattern of ice. This extended MC technique is applicable to other crystal and crystal-inhibitor systems, including more complex crystal systems such as clathrates.

Animals↗
PubMed2005

Effect of flavored beverage crystals on salivary cortisol enzyme-immunoreactive assay measurements.

We examined the effects of using flavored drink crystals as a salivary stimulant on salivary cortisol values. The effects of both amount and method of flavored crystal administration on cortisol values were evaluated using a high sensitivity enzyme immunoassay (EIA) kit to measure cortisol. Repeated saliva sampling, one sample without the drink crystal stimulant, followed immediately by a second sample using the drink crystal stimulant, allowed direct analysis of the effect of the stimulant on cortisol values. Repeated sampling, with the stimulant used when obtaining both the first and second samples, allowed analysis of the repeatability of cortisol measurements using the drink crystals. The results suggest that the use of drink mix crystals as an oral stimulant causes a small increase in measured cortisol values, as well as an increase in the variability of these values. The results also suggest that the effect of drink crystals is sufficiently regular so that it will not distort either within or between subject comparisons as long as there is consistency in use or non-use of the stimulant.

Analysis of Variance↗
PubMed2002

Effect of sodium content on the crystallization behavior of fluoramphibole glass-ceramics.

The purpose of this study was to evaluate the effect of sodium content on the crystallization behavior of glass-ceramics in the system SiO(2)-MgO-CaO-Na(2)O-K(2)O-F. Five glass compositions were prepared with increasing sodium content. The compositions were melted at 1400 degrees C for 2 h and cast into 60 x 12-mm ingots. Differential thermal analyses were performed on the glasses up to 1400 degrees C. Discs (1.5 x 12 mm) were cut from the ingots, nucleated in the temperature range 600-650 degrees C for 1-2 h, and crystallized in the temperature range 900-1000 degrees C for 0.5-4 h. The crystalline phases were analyzed by x-ray diffraction. The microstructure was investigated by scanning electron microscopy. Differential thermal analyses showed that the crystallization exotherm occurred in the temperature range 600-750 degrees C. There was a linear relationship between the amount of sodium and the transformation temperatures. X-ray diffraction revealed the presence of mica and diopside as major crystalline phases in the sodium-free composition. Mica, diopside, and fluorrichterite were present in all other glass-ceramics. The microstructure of the sodium-free glass-ceramic was characterized by the presence of hexagonal mica crystals and prismatic diopside crystals. All other compositions exhibited needle-shaped fluorrichterite crystals (2-5 microm in length) in addition to mica and diopside crystals.

Aluminum Silicates↗
PubMed1982

Parameters for crystal growth of ribosomal subunits.

A systematic analysis of the parameters that control the crystal growth of the large subunit of ribosomes from B stearothermophilus has been carried out. Several parameters have been identified and classified according to their significance. It was found that only biologically active particles can crystallize and that the critical period for the crystallization process is the first few days, during which changes in the volume and content of the crystallization drop are of importance for both nucleation and crystal growth. Consequently, an experimental procedure for fine control of these variables has been developed. As a result of these studies, the reproducibility of crystal formation was increased, and larger and more stable crystals have been obtained.

Bacillus↗
PubMed2005

Isothermal crystallization kinetics of lidocaine in supersaturated lidocaine/polyacrylate pressure sensitive adhesive systems.

Isothermal crystallization of lidocaine (LC) in supersaturated LC/Duro-Tak 87-2287 (DT2287) polyacrylate pressure sensitive adhesive (PSA) systems has been studied by differential scanning calorimetry (DSC). It was found that crystallization of LC in supersaturated LC/DT2287 systems was governed by the nucleation process, which in turn was dependent on temperature and composition of the systems. A critical temperature T(crit) was found at approximately 26 degrees C, above which the crystallization of LC in LC/DT2287 systems becomes slow. The lack of dependence of T(crit) on the composition of the mixtures indicates that the presence of the PSA affected the kinetics (diffusion) rather than the thermodynamics of the nucleation process. A critical degree of saturation S(crit) of approximately 4 was also found, above which the nucleation rate sharply increases. Kinetic analysis based on the classical theory of nucleation indicates that nucleation of LC in the PSA medium is a diffusion-controlled process. The activation energy of crystallization had a two-phase dependence on temperature suggesting that the mechanism of crystallization may change at the transition temperatures. As the weight fraction of LC increased in the systems, the activation energy of crystallization, DeltaG(c), was minimal at approximately 15 degrees C, indicating that the nucleation of LC in the LC/DT2287 systems is at its fastest rate around this temperature. These fundamental analyses of nucleation and crystallization mechanisms are of practical significance in the design of supersaturated drug delivery systems.

Acrylic Resins↗
PubMed1993

Changes in interactions in complexes of hirudin derivatives and human alpha-thrombin due to different crystal forms.

The three-dimensional structures of D-Phe-Pro-Arg-chloromethyl ketone-inhibited thrombin in complex with Tyr-63-sulfated hirudin (ternary complex) and of thrombin in complex with the bifunctional inhibitor D-Phe-Pro-Arg-Pro-(Gly)4-hirudin (CGP 50,856, binary complex) have been determined by X-ray crystallography in crystal forms different from those described by Skrzypczak-Jankun et al. (Skrzypczak-Jankun, E., Carperos, V.E., Ravichandran, K.G., & Tulinsky, A., 1991, J. Mol. Biol. 221, 1379-1393). In both complexes, the interactions of the C-terminal hirudin segments of the inhibitors binding to the fibrinogen-binding exosite of thrombin are clearly established, including residues 60-64, which are disordered in the earlier crystal form. The interactions of the sulfate group of Tyr-63 in the ternary complex structure explain why natural sulfated hirudin binds with a 10-fold lower K(i) than the desulfated recombinant material. In this new crystal form, the autolysis loop of thrombin (residues 146-150), which is disordered in the earlier crystal form, is ordered due to crystal contacts. Interactions between the C-terminal fragment of hirudin and thrombin are not influenced by crystal contacts in this new crystal form, in contrast to the earlier form. In the bifunctional inhibitor-thrombin complex, the peptide bond between Arg-Pro (P1-P1') seems to be cleaved.

Amino Acid Sequence↗
PubMed1994

Conservation of solvent-binding sites in 10 crystal forms of T4 lysozyme.

Solvent-binding sites were compared in 10 different crystal forms of phage T4 lysozyme that were refined using data from 2.6 A to 1.7 A resolution. The sample included 18 crystallographically independent lysozyme molecules. Despite different crystallization conditions, variable crystal contacts, changes due to mutation, and varying attention to solvent during crystallographic refinement, 62% of the 20 most frequently occupied sites were conserved. Allowing for potential steric interference from neighboring molecules in the crystal lattice, this fraction increased to 79% of the sites. There was, however, no solvent-binding site that was occupied in all 18 lysozyme molecules. A buried double site was occupied in 17 instances and 2 other internal sites were occupied 15 times. Apart from these buried sites, the most frequently occupied sites were often at the amino-termini of alpha-helices. Solvent molecules at the most conserved sites tended to have crystallographic thermal factors lower than average, but atoms with low B-factors were not restricted to these sites. Although superficial inspection may suggest that only 50-60% (or less) of solvent-binding sites are conserved in different crystal forms of a protein, it appears that many sites appear to be empty either because of steric interference or because the apparent occupancy of a given site can vary from crystal to crystal. The X-ray method of identifying sites is somewhat subjective and tends to result in specification only of those solvent molecules that are well ordered and bound with high occupancy, even though there is clear evidence for solvent bound at many additional sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophage T4↗
PubMed1996

Crystallization and preliminary X-ray diffraction studies of the Lb proteinase from foot-and-mouth disease virus.

Different crystal forms of the C23A mutant from the leader proteinase of foot-and-mouth disease virus were obtained by the hanging drop vapor diffusion technique, using MgCl2 and PEG 6000 as precipitants. Well-developed crystals, with cubic morphology growing to approximately 1.0 mm3 in size, presented a large unit cell parameter of 274.5 A and diffracted to, at most, 5 A resolution. A second type of crystal had a tetragonal appearance and these were obtained in droplets soaked in a silica gel matrix. These crystals, with an approximate size of 0.3 X 0.3 X 0.7 mm3, diffracted to approximately 4.0 A resolution, but presented a strong anisotropic mosaicity around the longest crystal axis. Crystals with a needlelike morphology and reaching sizes of about 0.2 X 0.3 X 1.2 mm3 diffracted beyond 3.5 A resolution and were stable to X-ray radiation for approximately one day when using a conventional source at room temperature. These crystals are orthorhombic with space group I222 (or I2(1)2(1)2(1)) and unit cell dimensions a = 65.9 A, b = 104.3 A, and c = 124.0 A, and appear well suited for high-resolution studies. Density packing considerations are consistent with the presence of two molecules in the asymmetric unit and a solvent content of approximately 54%.

Aphthovirus↗
PubMed1996

Conformational flexibility and crystallization of tandemly linked type III modules of human fibronectin.

Fibronectin is a large cell adhesion molecule that is composed of several functional domains. The cell-binding domain that binds to cell surface integrins consists of repeated homologous type III modules. In this study, recombinant fragments from the cell-binding domain of human fibronectin that participate in a newly characterized fibronectin-fibronectin interaction with FNIII1 were crystallized. In each case, the crystals had more than one fibronectin fragment in the asymmetric unit. Crystals of FNIII10-11 grew in the space group C2 with a = 117.1 A, b = 38.6 A, c = 80.6 A, beta = 97.2 degrees, and two molecules in the asymmetric unit. These crystals diffracted to 2.5 A resolution. Fragment FNIII8-11 and a shorter fragment, FNIII8-10, crystallized in hexagonal space groups with large unit cells and two to four molecules per asymmetric unit. Even very large crystals of these fragments did not diffract beyond 4 A. The crystal packing for this collection of fibronectin fragments suggests conformational flexibility between linked type III modules. The functional relevance of this flexibility for elongated versus compact models of the cell-binding domain of fibronectin is discussed.

Ammonium Sulfate↗
PubMed1997

Allosteric effectors do not alter the oxygen affinity of hemoglobin crystals.

In solution, the oxygen affinity of hemoglobin in the T quaternary structure is decreased in the presence of allosteric effectors such as protons and organic phosphates. To explain these effects, as well as the absence of the Bohr effect and the lower oxygen affinity of T-state hemoglobin in the crystal compared to solution, Rivetti C et al. (1993a, Biochemistry 32:2888-2906) suggested that there are high- and low-affinity subunit conformations of T, associated with broken and unbroken intersubunit salt bridges. In this model, the crystal of T-state hemoglobin has the lowest possible oxygen affinity because the salt bridges remain intact upon oxygenation. Binding of allosteric effectors in the crystal should therefore not influence the oxygen affinity. To test this hypothesis, we used polarized absorption spectroscopy to measure oxygen binding curves of single crystals of hemoglobin in the T quaternary structure in the presence of the "strong" allosteric effectors, inositol hexaphosphate and bezafibrate. In solution, these effectors reduce the oxygen affinity of the T state by 10-30-fold. We find no change in affinity (< 10%) of the crystal. The crystal binding curve, moreover, is noncooperative, which is consistent with the essential feature of the two-state allosteric model of Monod J, Wyman J, and Changeux JP (1965, J Mol Biol 12:88-118) that cooperative binding requires a change in quaternary structure. Noncooperative binding by the crystal is not caused by cooperative interactions being masked by fortuitous compensation from a difference in the affinity of the alpha and beta subunits. This was shown by calculating the separate alpha and beta subunit binding curves from the two sets of polarized optical spectra using geometric factors from the X-ray structures of deoxygenated and fully oxygenated T-state molecules determined by Paoli M et al. (1996, J Mol Biol 256:775-792).

Allosteric Regulation↗
PubMed1997

Protein-protein crystal-packing contacts.

Protein-protein contacts in monomeric protein crystal structures have been analyzed and compared to the physiological protein-protein contacts in oligomerization. A number of features differentiate the crystal-packing contacts from the natural contacts occurring in multimeric proteins. The area of the protein surface patches involved in packing contacts is generally smaller and its amino acid composition is indistinguishable from that of the protein surface accessible to the solvent. The fraction of protein surface in crystal contacts is very variable and independent of the number of packing contacts. The thermal motion at the crystal packing interface and that of the protein core, even for large packing interfaces, though the tendency is to be closer to that of the core. These results suggest that protein crystallization depends on random protein-protein interactions, which have little in common with physiological protein-protein recognition processes, and that the possibility of engineering macromolecular crystallization to improve crystal quality could be widened.

Crystallization↗
PubMed1998

Contaminant inclusion into protein crystals analyzed by electrospray mass spectrometry and X-ray crystallography.

The inclusion of protein contaminants into crystals of turkey egg white lysozyme (TEWL) was investigated by electrospray mass spectrometry of the dissolved crystals. The results show that significant amounts of the structurally related contaminant hen egg white lysozyme (HEWL) are included in the crystals of TEWL. The structurally unrelated contaminant RNAse A, on the other hand, is not included. The X-ray diffraction data statistics of a hybrid TEWL/HEWL crystal and an uncontaminated TEWL crystal were of similar quality. This indicates that, even though the crystals contain much higher levels of the contaminant than one would have expected after a recrystallization experiment, they are still suitable for X-ray diffraction experiments. However, attempts to detect the presence of the contaminant in the crystal by crystallographic structure refinement did not yield conclusive results.

Animals↗
PubMed2002

Towards atomic resolution with crystals grown in gel: the case of thaumatin seen at room temperature.

One reason for introducing a gel in the crystallization medium of proteins is its ability to reduce convection in solution. This can lead to better nucleation and growth conditions, and to crystals having enhanced diffraction properties. We report here the X-ray characterization at room temperature of high-quality crystals of the intensely sweet thaumatin prepared in a sodium tartrate solution gelified with 0.15% (m/v) agarose. Using a synchrotron radiation, these crystals diffracted to a previously unachieved resolution. A diffraction dataset was collected from four crystals at a resolution of 1.2 A with a R(sym) of 3.6% and a completeness of 99%. Refinement was carried out to a final crystallographic R-factor of 12.0%. The quality of the electron density map allowed for the observation of fine structural details in the protein and its solvation shell. Crystallization in gel might be used more generally to improve the quality of macromolecular crystals. Advantages provided by the gelified medium in the frame of structural studies are emphasized.

Crystallization↗
PubMed1991

Crystallization of yeast orotidine 5'-monophosphate decarboxylase complexed with 1-(5'-phospho-beta-D-ribofuranosyl) barbituric acid.

Using an incomplete factorial experimental design, we have identified conditions for crystallization of yeast orotidine 5'-monophosphate decarboxylase (ODCase) in an unliganded state and complexed separately to two inhibitors: 6-azauridine 5'-monophosphate (aza-UMP) and 1-(5'-phospho-beta-D-ribofuranosyl) barbituric acid (BMP). Crystals of X-ray diffraction quality have been obtained of yeast ODCase complexed with BMP, a putative transition state analog inhibitor (Ki = 8.8 x 10(-12) M). ODCase:BMP complex crystals with a hexagonal rod habit were grown from a solution initially containing 12 mg/ml ODCase (205 microM dimer) plus 450 microM BMP by microdialysis at 4 degrees C against a mother liquor which consisted of 0.1 M Na-PIPES-acetate (pH 6.4), 37.5 microM BMP, 5 mM mercaptoethanol, 1% polyethylene glycol 400, and 2.3 M ammonium sulfate. Crystals were analyzed using precession photography and were assigned to trigonal space group R32 with unit cell dimensions a = b = 115 A, c = 385 A. The crystal density is 1.245 g/cm3 indicating the presence of two ODCase: BMP complex dimers (118 kDa each) per asymmetric unit with a packing density of 2.08 A3/Da and 41% solvent content. The morphological habit of crystals of the ODCase:BMP complex changed when the initial ammonium sulfate concentration was increased in 0.05 M steps from 2.3 to 2.45 M. All of these crystals diffracted to at least 3.0 A resolution over a period of several weeks at room temperature and are isomorphous.

Ammonium Sulfate↗
PubMed1995

Crystallization and preliminary X-ray diffraction studies of leishmanolysin, the major surface metalloproteinase from Leishmania major.

The membrane-bound GPI-anchored zinc metalloproteinase leishmanolysin purified from Leishmania major promastigotes has been crystallized in its mature form. Two crystal forms of leishmanolysin have been grown by the vapor diffusion method using 2-methyl-2,4-pentanediol as the precipitant. Macroseeding techniques were employed to produce large single crystals. Protein microheterogeneity in molecular size and charge was incorporated into both crystal forms. The tetragonal crystal form belongs to the space group P4(1)2(1)2 or the enantiomorph P4(3)2(1)2, has unit cell parameters of a = b = 63.6 A, c = 251.4 A, and contains one molecule per asymmetric unit. The second crystal form is monoclinic, space group C2, with unit cell dimensions a = 107.2 A, b = 90.6 A, c = 70.6 A, beta = 110.6 degrees, and also contains one molecule per asymmetric unit. Both crystal forms diffract X-rays beyond 2.6 A resolution and are suitable for X-ray analysis. Native diffraction data sets have been collected and the structure determination of leishmanolysin using a combination of the isomorphous replacement and the molecular replacement methods is in progress.

Animals↗
PubMed1994

Crystallization and preliminary crystallographic analysis of two endo-beta-N-acetylglucosaminidases, endo H and endo F1.

Endo H and F1 are endoglycosidases that cleave the oligosaccharide moiety of asparagine-linked glycoproteins by hydrolysis of the glycosidic bond in the N,N'-diacetylchitobiose core. The two enzymes are specific for high-mannose oligosaccharides. Here, we report the crystallization and preliminary crystallographic analysis of Endo H and Endo F1. Crystals were grown by hanging drop vapor diffusion methods. Both proteins crystallize from crystallization buffers containing polyethyleneglycol 8000 and zinc acetate as precipitating agents in cacodylate buffer. The crystals of Endo H belong to the tetragonal space group P4(1)2(1)2 (or P4(3)2(1)2) with cell dimensions: a = 85.22 A, c = 89.41 A. The crystals of Endo F1 belong to the hexagonal space group P6(1) (or P6(5)) with cell dimensions: a = 70.61 A, c = 100.32 A. Crystals of both proteins diffract to at least 1.8 A resolution.

Carbohydrate Sequence↗
PubMed1994

Five viral peptide-HLA-A2 co-crystals. Simultaneous space group determination and X-ray data collection.

We prepared and crystallized five complexes of the human histocompatibility molecule HLA-A2 with peptides derived from human immunodeficiency virus type 1, human T lymphotropic virus type 1, influenza A virus and hepatitis B virus proteins. Each HLA-A2 complex was refolded in vitro from insoluble proteins produced in bacteria; to crystallize, two of the complexes required seeding with microcrystals of another complex. Maintained at -160 degrees C, single co-crystals of each of the five peptide-HLA-A2 complexes yielded complete X-ray diffraction data sets to a resolution of approximately 2.5 A. After a sufficient number of diffraction peaks were acquired during data collection, the direct analysis of integrated intensities established the point group of the co-crystal, thus allowing an efficient data collection strategy to be designed. The subsequent examination of systematic absences revealed that the five peptide-HLA-A2 co-crystals formed in space groups P1, P2(1), or P2(1)2(1)2(1). Molecular replacement structure solutions yielded unambiguous protein electron density maps, thus confirming the space group determinations. The system of obtaining HLA-A2 co-crystal structures described here is applicable to other crystallographic problems where structures of several related molecules from uncharacterized single crystals are required.

Amino Acid Sequence↗