Diagnostic of precipitant for biomacromolecule crystallization by quasi-elastic light-scattering

The translational diffusion coefficient D25,w of hen egg-white lysozyme and concanavalin A from the jack bean is measured in various precipitating agent solutions as a function of salt and protein concentration using quasi-elastic light-scattering. With some precipitants, in undersaturated protein solutions, a protein or salt concentration dependence of the diffusion coefficient of the scatters is observed. It can be correlated with the inability of the protein to crystallize in this precipitant once the solution is supersaturated. These variations of D25,w are interpreted in terms of non-specific interactions and/or aggregation that prevent the protein from making appropriate contacts to form a crystal. With other precipitants known to lead to crystallization, no significant variation of the diffusion coefficient with increasing concentration was observed, indicating that under such conditions up to saturation the proteins remain essentially monodisperse. Application of this technique to find crystallization conditions of other proteins is discussed.     

Precipitants and additives for membrane crystallization of lysozyme

Membrane crystallization is a newly developed crystallization technique that has proven to be superior in producing good crystal forms under operating conditions that are not appropriate to perform the crystallization process by other traditional techniques. In this work, static membrane crystallization was carried out on lysozyme, with hollow-fiber microporous hydrophobic membranes. Numerous precipitant and additive types and concentrations were employed in the crystallization processes in order to select the most appropriate precipitant and additive types and to find their corresponding concentration levels that can yield the best crystal forms. The crystallization processes were analyzed in two ways: firstly, by evaluation of the transmembrane fluxes obtained by using different precipitants and additives; secondly, by utilization of the images and results obtained from the micrography and IR spectra in comparisons and evaluations of the crystals formed under all kinds of conditions. Moreover, the size distributions of the crystals yielded under several typical crystallization conditions were analyzed, and turbidity and induction time periods obtained during typical crystallization experiments were also measured. Amongst the numerous precipitants and additives tested, the most appropriate precipitant type and additive were chosen and their concentrations were optimized. Good lysozyme crystals were obtained using a certain precipitant and additive. The obtained results from this work further support the advantages of utilizing the membrane crystallization technique for macromolecule crystallizations.     

Effects of additives on surfactant phase behavior relevant to bacteriorhodopsin crystallization

The interactions leading to crystallization of the integral membrane protein bacteriorhodopsin solubilized in n-octyl-beta-D-glucoside were investigated. Osmotic second virial coefficients (B(22)) were measured by self-interaction chromatography using a wide range of additives and precipitants, including polyethylene glycol (PEG) and heptane-1,2,3-triol (HT). In all cases, attractive protein-detergent complex (PDC) interactions were observed near the surfactant cloud point temperature, and there is a correlation between the surfactant cloud point temperatures and PDC B(22) values. Light scattering, isothermal titration calorimetry, and tensiometry reveal that although the underlying reasons for the patterns of interaction may be different for various combinations of precipitants and additives, surfactant phase behavior plays an important role in promoting crystallization. In most cases, solution conditions that led to crystallization fell within a similar range of slightly negative B(22) values, suggesting that weakly attractive interactions are important as they are for soluble proteins. However, the sensitivity of the cloud point temperatures and resultant coexistence curves varied significantly as a function of precipitant type, which suggests that different types of forces are involved in driving phase separation depending on the precipitant used.     

沉淀剂类型对蛋白质晶体分子堆积的影响

以不对称单位只有一个分子的牛胰核糖核酸酶和T4溶菌酶晶体为材料,着重研究了无机盐、有机溶剂和PEG三类不同的沉淀剂对晶体分子堆积的影响,经研究发现两种蛋白质中用无机盐做沉淀剂的晶型几乎都含有面积较大的二次轴对称接触面和较少的相邻分子数,同时其含有的参与接触的非极性残基集中分布于二次轴对称接触面,而盐键则在二次轴对称接触面上分布稀少。用有机溶剂作沉淀剂的晶型却含有面积较小的非二次轴对称接触面和较多的相含分子数,而参与接触的非极性残基和直键在各个非二次轴对称接触面上随机分布,用PEG作沉淀剂的晶型其分子堆积特征总体上类似于用有机溶剂作沉淀剂的晶型,但个别晶型具有与用无机盐做沉淀剂的晶型相似的分子堆积特征,以上结果提示,用三类沉淀剂得到的不同的分子堆积特征可能与三类沉淀剂不同的诱导结晶机理密切相关。     

Crystal packing in six crystal forms of pancreatic ribonuclease.

We compare the molecular packing of bovine pancreatic ribonuclease A (RNase A) in six crystal forms, two grown with alcohol, three with high salt and one with polyethylene glycol as a precipitant. The six packings differ in the number of molecules in contact and in the extent of the contacts, which bury 1570 A2 to 2790 A2 of the RNase surface. Regions of the protein surface involved in the six packings cover almost the whole RNase molecule. The abundance of polar interactions, about one per 200 A2, is the same in all types of precipitants. All molecule-to-molecule contacts are different in the six crystal forms, except for the one that forms a RNase dimer. The dimer has a large interface covering 1800 A2 and eight to ten polar interactions. Its presence in the three salt-grown crystal forms suggests that it is an intermediate in salt induced crystallization. In contrast, the two alcohol-grown forms contain only small interfaces, implying a different mechanism of nucleation.     

Experimental determination of water equilibration rates in the hanging drop method of protein crystallization

The hanging drop method for protein crystallization consists of equilibrating a water droplet containing the protein and a precipitant against a reservoir solution at a higher precipitant concentration. The time for water equilibration--which affects the kinetics of crystallization--to reach 90% of completion is shown to vary between about 25 h and more than 25 days, depending on experimental conditions. Experiments were performed with three of the most widely used precipitants (ammonium sulfate, polyethylene glycol, 2-methyl-2,4-pentanediol), combining various parameters expected to affect the rate of water evaporation. The most dramatic effects were obtained when varying temperature and initial drop volume. A simple empirical equation for estimating the kinetics of water equilibration under given crystallization conditions is proposed.     

The binding of precipitant ions in the tetragonal crystals of hen egg white lysozyme

Abstract

The bonds between lysozyme molecules and precipitant ions in single crystals grown with chlorides of several metals are analysed on the basis of crystal structure data. Crystals of tetragonal hen egg lysozyme (HEWL) were grown with chlorides of several alkali and transition metals (LiCl, NaCl, KCl, NiCl₂ and CuCl₂) as precipitants and the three-dimensional structures were determined at 1.35?Å resolution by X-ray diffraction method. The positions of metal and chloride ions attached to the protein were located, divided into three groups and analysed. Some of them, in accordance with the recently proposed and experimentally confirmed crystal growth model, provide connections in protein dimers and octamers that are precursor clusters in the crystallization lysozyme solution. The first group, including Cu⁺², Ni⁺² and Na⁺¹ cations, binds specifically to the protein molecule. The second group consists of metal and chloride ions bound inside the dimers and octamers. The third group of ions can participate in connections between the octamers that are suggested as building units during the crystal growth. The arrangement of chloride and metal ions associated with lysozyme molecule at all stages of the crystallization solution formation and crystal growth is discussed.

Communicated by Ramaswamy H. Sarma     

Development of an alternative approach to protein crystallization

We are developing an alternate strategy for the crystallization of macromolecules that does not, like current methods, depend on the optimization of traditional variables such as pH and precipitant concentration, but is based on the hypothesis that many conventional small molecules might establish stabilizing, intermolecular, non covalent crosslinks in crystals, and thereby promote lattice formation. To test the hypothesis, we carried out preliminary experiments encompassing 18,240 crystallization trials using 81 different proteins, and 200 chemical compounds. Statistical analysis of the results demonstrated the validity of the idea. In addition, we conducted X-ray diffraction analyses of some of the crystals grown in the experiments. These clearly showed incorporation of conventional molecules into the protein crystal lattices, and further validated the underlying hypothesis. We are currently extending the investigations to include a broader and more diverse set of proteins, an expanded search of conventional and biologically active small molecules, and a wider range of precipitants. The strategy proposed here is essentially orthogonal to current approaches and has an objective of doubling the success rate of today.     

限制性酶切提高E.coli Hfq蛋白晶体结构分辨率

Hfq蛋白是一种促进sRNA结合互补RNA的细菌伴侣蛋白. Hfq蛋白可以抑制或上调目的蛋白的表达及促进mRNA的降解,从而调控细菌的生理功能使其适应周围胁迫环境.Hfq是同源六聚体蛋白质,在其中心部位有1个内环,每个单体上有1个极其保守的组氨酸分布在这个内环表面.根据Hfq这个特点,应用镍离子亲和柱直接从大肠杆菌中纯化Hfq,接着用凝胶过滤层析柱纯化,最终获得了纯度70%的Hfq蛋白.对该蛋白质进行结晶条件初筛前,应用胰凝乳蛋白酶对其进行限制性蛋白酶解,切除其C端无规则卷曲,便于Hfq结晶及提高晶体衍射率.初期得到的Hfq晶体是极小的针状晶体,通过优化结晶条件获得了较大的晶体.最终解析出了2个分辨率较高的Hfq晶体结构,其中1个晶体结构分辨率达到了1.63A.     

Optimization of crystals of an inhibitory antibody of urokinase plasminogen activator receptor (uPAR) with hydrogen peroxide and low protein concentration

Optimization of protein crystal formation is often a necessary step leading to diffraction-quality crystals to enable collection of a full X-ray data set. Typical protein crystal optimization involves screening different components, e.g., pH, precipitants, and additives of the precipitant solution. Here we present an example using an inhibitory antibody of urokinase plasminogen activator receptor (uPAR) where such procedures did not yield diffracting crystals. In contrast, it was the treatment of the protein with hydrogen peroxide incubation and the protein concentration reduction that were found to be key factors in obtaining diffracting crystals. Final crystals diffracted to 1.75 A, and belong to orthorhombic P2(1)2(1)2(1) space group with unit cell parameters a = 37.162 A, b = 84.474 A, c = 134.030 A, and contain one molecule of Fab fragment of anti-uro kinase receptor antibody in the asymmetric unit.     

The impact of crystallization conditions on structure‐based drug design: A case study on the methylene blue/acetylcholinesterase complex

Structure‐based drug design utilizes apoprotein or complex structures retrieved from the PDB. >57% of crystallographic PDB entries were obtained with polyethylene glycols (PEGs) as precipitant and/or as cryoprotectant, but <6% of these report presence of individual ethyleneglycol oligomers. We report a case in which ethyleneglycol oligomers' presence in a crystal structure markedly affected the bound ligand's position. Specifically, we compared the positions of methylene blue and decamethonium in acetylcholinesterase complexes obtained using isomorphous crystals precipitated with PEG200 or ammonium sulfate. The ligands' positions within the active‐site gorge in complexes obtained using PEG200 are influenced by presence of ethyleneglycol oligomers in both cases bound to W84 at the gorge's bottom, preventing interaction of the ligand's proximal quaternary group with its indole. Consequently, both ligands are ～3.0Å further up the gorge than in complexes obtained using crystals precipitated with ammonium sulfate, in which the quaternary groups make direct π‐cation interactions with the indole. These findings have implications for structure‐based drug design, since data for ligand‐protein complexes with polyethylene glycol as precipitant may not reflect the ligand's position in its absence, and could result in selecting incorrect drug discovery leads. Docking methylene blue into the structure obtained with PEG200, but omitting the ethyleneglycols, yields results agreeing poorly with the crystal structure; excellent agreement is obtained if they are included. Many proteins display features in which precipitants might lodge. It will be important to investigate presence of precipitants in published crystal structures, and whether it has resulted in misinterpreting electron density maps, adversely affecting drug design.     

PEG和盐对棕色固氮菌细菌铁蛋白和钼铁蛋白晶体生长的影响

棕色固氮菌（ＯＰ）体内的固氮酶钼铁（ＭｏＦｅ）蛋白和细菌铁蛋白均为重要的生物功能蛋白。前者为生物固氮的关键酶［１］,后者则可为生物代谢贮存丰富而又可溶的铁原子［２］。因而都得到了广泛而深入的研究。Ｋｉｍ［３］报道了ＭｏＦｅ蛋白衍射结果。赵宝光等［２］...     

Effect of PEG and Salt on Crystallization of Bacterioferritin and Nitrogenase MoFe Protein from Azotobacter vinelandii

MgCl2 was added to the supernatant of the first crystallization of MoFe protein to give a final concentration of 14.6 mmol/L, followed by centrifugation. The treated supematant solution and MoFe protein could be crystallized by using method of siting drop with PEG 6000 and MgC12 as a precipitant and salt, respectively. The larger crystal from the supermatant was observed when the final concentration of PEG and MgCl2 was 4.5% and 15.6 mmol/L, respectively; but small crystal was observed when the concentration was 0 and 23.8 mmol/L, respectively. The larger crystal in brown rectangular prism of MoFe protein was also obtained using the same crystallization method when the final concentration of PEG and MgCI2 was 7.44% and 338.0 mmol/L, respectively. It suggests that the two protein crystals seem to be different, the former being bacterioferritin and the later as nitrogenase MoFe protein.     

Dependence of Protein Crystal Stability on Residue Charge States and Ion Content of Crystal Solvent

Protein crystallization is frequently induced by the addition of various precipitants, which directly affect protein solubility. In addition to organic cosolvents and long-chain polymers, salts belong to the most widely used precipitants in protein crystallography. However, despite such widespread usage, their mode of action at the atomistic level is still largely unknown. Here, we perform extensive molecular dynamics simulations of the villin headpiece crystal unit cell to examine its stability at different concentrations of sodium sulfate. We show that the inclusion of ions in crystal solvent at high concentration can prevent large rearrangements of the asymmetric units and a loss of symmetry of the unit cell without significantly affecting protein dynamics. Of importance, a similar result can be achieved by neutralizing several specific charged residues suggesting that they may play an active role in crystal destabilization due to unfavorable electrostatic interactions. Our results provide a microscopic picture behind salt-induced stabilization of a protein crystal and further suggest that adequate modeling of realistic crystallization conditions may be necessary for successful molecular dynamics simulations of protein crystals.     

The crystallization of outer membrane proteins from Escherichia coli. Studies on lamB and ompA gene products

The outer membrane protein LambB from Escherichia coli has been crystallized from detergent-containing solutions. Several different crystal habits can be obtained under the same ionic and precipitant conditions by altering the detergent head group composition of the protein-detergent mixed micelle or by adding polar organic compounds. Two crystal forms have been partially characterized as P1 and C2221, the former diffracting to beyond 4 A resolution and the latter to 6 A. The detergents used were beta-octyl glucoside, octyl tetraoxyethylene, and octyl polyoxyethylene (polydisperse) either alone or as mixtures. In some experiments, the addition of small nonionic amphiphiles having n-butyl alkyl tails significantly influenced crystallization. The experiments suggest that the detergent region of the mixed micelle plays a critical role in crystal formation. Using the methods developed here for LamB and also for matrix porin (Garavito, R. M., Jenkins, J. A., Jansonius, J. N., Karlsson, R., and Rosenbusch, J. P. (1983) J. Mol. Biol. 164, 313-327), an additional protein from the outer membrane, OmpA, has been obtained as a microcrystalline preparation.     

Optimization of protein precipitation based upon effectiveness of protein removal and ionization effect in liquid chromatography-tandem mass spectrometry

Four categories of protein precipitation techniques (organic solvent, acid, salt and metal ion) were tested in plasma using spectrophotometry to assess protein removal efficiency across a range of volumes, species and lots. Acetonitrile, trichloroacetic acid (TCA) and zinc sulfate were found to be optimal at removing protein in their categories (>96, 92 and 91% protein precipitation efficiency at a 2:1 ratio of precipitant to plasma, respectively). A post-column infusion LC-MS/MS system was used to assess ionization effect of a protein-bound drug caused by the endogenous components remaining after using various protein precipitants. The extent of ionization effect varied with mobile phase (-20 to 93%), protein precipitant (0.3-86%), but only slightly with species (86-93%). The optimal bioanalytical methodologies for removal of plasma proteins and minimal ionization effect for the probe molecule in positive ion turboionspray LC-MS/MS involve the use of TCA for precipitation with mobile phases consisting of either pure organic solvents (methanol:water or acetonitrile:water) or precipitation with all of the mass spectrometer compatible precipitants evaluated with a methanol:aqueous 0.1% formic acid mobile phase.     

Crystallization and preliminary characterization of mitogillin, a ribosomal ribonuclease from Aspergillus restrictus.

Mitogillin is a ribonuclease secreted by the fungus Aspergillus restrictus. The substrate for mitogillin is a short, universally conserved, sequence in ribosomal RNA. Cleavage of this sequence inactivates protein synthesis. Mitogillin was crystallized by a two-chamber vapor/liquid diffusion method using ethanol as the precipitant. This method has wider potential in the use of volatile organic solvents as precipitants. Crystals of mitogillin diffract X-rays to lattice d-spacings of at least 1.6 A, and belong to the monoclinic space group P2(1), with a = 50.4 A, b = 82.4 A, c = 38.2 A and beta = 99.8 degrees.     

Protein crystallization by design: chymotrypsinogen without precipitants

Protein crystals are usually obtained by an empirical approach based on extensive screening to identify suitable crystallization conditions. In contrast, we have used a systematic predictive procedure to produce data-quality crystals of bovine chymotrypsinogen A and used them to obtain a refined X-ray structure to 3 A resolution. Measurements of the osmotic second virial coefficient of chymotrypsinogen solutions were used to identify suitable solvent conditions, following which crystals were grown for approximately 30 hours by ultracentrifugal crystallization, without the use of any precipitants. Existing structures of chymotrypsinogen were obtained in solutions including 10-30 % ethanol, whereas simple buffered NaCl solutions were used here. The protein crystallized in the tetragonal space group P4(1)2(1)2, with one molecule per asymmetric unit. The quality of the refined map was very high throughout, with the main-chain atoms of all but four residues clearly defined and with nearly all side-chains also defined. Although only minor differences are seen compared to the structures previously reported, they indicate the possibility of structural changes due to the crystallization conditions used in those studies. Our results show that more systematic crystallization of proteins is possible, and that the procedure can expand the range of conditions under which crystals can be grown successfully and can make new crystal forms available.     

Phase behavior of an intact monoclonal antibody

Understanding protein phase behavior is important for purification, storage, and stable formulation of protein drugs in the biopharmaceutical industry. Glycoproteins, such as monoclonal antibodies (MAbs) are the most abundant biopharmaceuticals and probably the most difficult to crystallize among water-soluble proteins. This study explores the possibility of correlating osmotic second virial coefficient (B(22)) with the phase behavior of an intact MAb, which has so far proved impossible to crystallize. The phase diagram of the MAb is presented as a function of the concentration of different classes of precipitants, i.e., NaCl, (NH4)2SO4, and polyethylene glycol. All these precipitants show a similar behavior of decreasing solubility with increasing precipitant concentration. B(22) values were also measured as a function of the concentration of the different precipitants by self-interaction chromatography and correlated with the phase diagrams. Correlating phase diagrams with B(22) data provides useful information not only for a fundamental understanding of the phase behavior of MAbs, but also for understanding the reason why certain proteins are extremely difficult to crystallize. The scaling of the phase diagram in B(22) units also supports the existence of a universal phase diagram of a complex glycoprotein when it is recast in a protein interaction parameter.