Madeleine Riès-Kautt

High-resolution structure (1.33 A) of a HEW lysozyme tetragonal crystal grown in the APCF apparatus. Data and structural comparison with a crystal grown under microgravity from SpaceHab-01 mission.

Crystals of tetragonal hen egg-white lysozyme were grown using Advanced Protein Crystallization Facility (APCF) apparatus under a microgravity environment (SpaceHab-01 mission) and ground control conditions. Crystals were grown from NaCl as a crystallizing agent at pH 4.3. The X-ray diffraction patterns of the best diffracting ground- and space-grown crystals were recorded using synchrotron radiation and an image plate on the W32 beamline at LURE. Both ground- and space-grown crystals showed nearly equivalent maximum resolution of 1.3-1.4 A. Refinements were carried out with the program X-PLOR with final R values of 18.45 and 18.27% for structures from ground- and space- grown crystals, respectively. The two structures are nearly identical with the root-mean-square difference on all protein atoms being 0.13 A. Some residues of the two refined structures show multiple alternative conformations. Two ions were localized into the electron-density maps of the two structures: one chloride ion at the interface between two symmetry-related molecules and one sodium ion stabilizing the loop Ser60-Leu75. The sodium ion is surrounded by six ligands which form a bipyramid around it at distances of 2.2-2.6 A.

No salting-in of lysozyme chloride observed at low ionic strength over a large range of pH.

Solubility of lysozyme chloride was determined in the absence of added salt and in the presence of 0.05-1.2 M NaCl, starting from isoionic lysozyme, which was then brought to pH values from 9 to 3 by addition of HCl. The main observation is the absence of a salting-in region whatever the pH studied. This is explained by a predominant electrostatic screening of the positively charged protein and/or by adsorption of chloride ions by the protein. The solubility increases with the protein net charge at low ionic strength, but the reverse is observed at high ionic strength. The solubility of lysozyme chloride seems to become independent of ionic strength at pH approximately 9.5, which is interpreted as a shift of the isoionic pH (10.8) to an isoelectric pH due to chloride binding. The crystallization at very low ionic strength, where lysozyme crystallizes at supersaturation values as low as 1.1, amplifies the effect of pH on protein solubility. Understanding the effect of the net charge and of ionic strength on protein-protein interactions is valuable not only for protein crystal growth but more generally also for the formation of protein-protein or protein-ligand complexes.

[3] Inferences drawn from physicochemical studies of crystallogenesis and precrystalline state

Publisher Summary This chapter focuses on those aspects of the physicochemical state of a macromolecule that are amenable to physicochemical studies, including its interactions with solvent components and with other identical macromolecules. The chapter presents experimental data on solubility and small-angle X-ray scattering (SAXS) measurements. Deviations of experimental data from theory are discussed in the chapter. Crystallogenesis explains the way different parameters affect each step of the crystallization process—that is, the solubility, nucleation, growth, and stability of crystals. Some parameters may affect only one step, while others, such as pH or temperature, can intervene at different levels. The same parameter may affect one stage differently from another, and different parameters may have synergistic effects. New crystallization methods can be conceived, based on binding ions or small molecules to a protein, thus changing the physicochemical properties of the protein. As buffers are weak acids or bases that bind to charged groups of the opposite charge of the protein, they can be used as crystallizing agents.

Structural effects of monovalent anions on polymorphic lysozyme crystals.

Understanding direct salt effects on protein crystal polymorphism is addressed by comparing different crystal forms (triclinic, monoclinic, tetragonal and orthorhombic) for hen, turkey, bob white quail and human lysozymes. Four new structures of hen egg-white lysozyme are reported: crystals grown in the presence of NapTS diffracted to 1.85 A, of NaI to 1.6 A, of NaNO(3) to 1.45 A and of KSCN to 1.63 A. These new structures are compared with previously published structures in order to draw a mapping of the surface of different lysozymes interacting with monovalent anions, such as nitrate, chloride, iodide, bromide and thiocyanate. An analysis of the structural sites of these anions in the various lysozyme structures is presented. This study shows common anion sites whatever the crystal form and the chemical nature of anions, while others seem specific to a given geometry and a particular charge environment induced by the crystal packing.

Strong and specific effects of cations on lysozyme chloride solubility.

The influence of salt nature and concentration on tetragonal lysozyme chloride crystal solubility is presented for a set of mono-, di- and trivalent cations (Cs(+), Rb(+), Mn(2+), Co(2+) and Yb(3+)). The results show that cations have as strong an effect on protein solubility as anions and that they present their own particular effects as co-ions. Indeed, after decreasing at low ionic strength, lysozyme solubility increases with high concentration of polyvalent cations, probably due to co-ion binding and therefore the concomitant increase of the net charge of the protein-salt complex. These new results are discussed in order to progress in the understanding of the crystallisation process at the atomic level.

Importance of the nature of anions in lysozyme crystallisation correlated with protein net charge variation

Hofmeister anion series are studied to examine the coupled influence of pH and ionic strength on the solubility of previously desalted lysozyme. Solubility curves are measured at pH 4.3 and 8.3 and 18 degrees C for nitrate, para-toluenesulfonate, citrate, sulphate, phosphate, and acetate. Extreme low ionic strength is explored, confirming the decrease of lysozyme solubility while increasing the protein net charge and the ionic strength. The classification of specific salt effects takes into account the valence of the anions with respect to the protein net charge.

Crystallogenesis studies in microgravity with the Advanced Protein Crystallization Facility on SpaceHab-01

Abstract The Advanced Protein Crystallization Facility (APCF), a new protein crystallization device developed by ESA for the IML-2 Mission in 1994, was tested in its maiden flight on STS-57 Mission in SpaceHab-01 with a physico-chemical experiment on lysozyme crystallization. In pre-flight ground experiments, prior to the Shuttle Mission, the protocol for lysozyme crystallization with NaCl was based on its solubility diagram at 18°C and pH 4.5. Crystallization was conducted under microgravity in 25 APCF reactors using vapor diffusion, dialysis, and free liquid interface diffusion, with control on earth in 25 identical reactors. Identical supersaturation values were tested by the three crystallization techniques. Values of supersaturation derived from ground experiments allowed for conditions that yielded crystals in microgravity. The average number and size of crystals from the flight experiment and the earth control showed no significant difference; however many crystals were not free floating and grew on the walls of some of the protein chambers. The dialysis technique proved to be suitable, since no additional nucleation was generated by the membrane. Protein concentration measurements indicated that 13 days after activation of the experiment as much as 70–90% of the protein in supersaturated state had already crystallized. Data indicated differences in the crystallization behavior depending upon the crystallization set-up. Images of the protein chamber of 6 reactors, recorded during the flight, allowed us to evaluate the early stage of crystallization, to verify that recovered crystals had actually grown under microgravity conditions, and showed motions of crystals during the Mission. Using synchrotron radiation, resolution and rocking curve measurements of ground and space lysozyme crystals grown in APCF reactors showed no significant differences, although the values are much better than previously recorded diffraction limits and mosaicity data obtained with tetragonal lysozyme crystals grown in other set-ups and under different conditions. All controls foreseen throughout the microgravity experiment proved to be essential for the interpretation of the flight data, as concerning the effect of microgravity.

The decameric structure of bovine pancreatic trypsin inhibitor (BPTI) crystallized from thiocyanate at 2.7 A resolution.

The structure of a monoclinic form of bovine pancreatic trypsin inhibitor (BPTI) crystallized from a thiocyanate solution has been determined and refined at 2.7 A resolution. The space group is P21 with a = 71.56, b = 73.83, c = 64.47 A, beta = 93.9 degrees and Z = 20. The ten independent molecules were located by a multi-body molecular-replacement search as developed in the AMoRe program, starting from a single monomer model (PDB code: 6PTI). The molecular arrangement of the subunits is a decamer resulting from the combination of two orthogonal fivefold and twofold non-crystallographic axes. This builds a globular micelle-like particle which minimizes hydrophobic interactions with the solvent. The refinement was conducted with non-crystallographic symmetry constraints up to a final residual of R = 0.20 (Rfree= 0.26). The root-mean-square deviations from ideal geometry were 0.015 A and 1.6 degrees on bond distances and bond angles, respectively. Several sites for thiocyanate ions were analyzed.

Solubility diagram analysis and the relative effectiveness of different ions on protein crystal growth

The solubility of a protein can be defined as the concentration of the soluble protein in equilibrium with the solid phase under given conditions of pH, temperature, buffer, and/or various additives. A solubility curve represents the dependence of this saturating concentration on a single parameter, which could be the concentration of a crystallizing or precipitating agent. For accurate determinations, the solubility should be determined by both crystallization and redissolution of crystals, and it should be demonstrated that in the two methods the protein concentration converges asymptotically to the same value. In cases where fairly large amounts of protein are readily available, solubility diagram analysis provides a means of quantitatively characterizing the effect of one parameter, and hence of interpreting its influence in a meaningful way. Our analysis of lysozyme (p I = 11.1) solubility curves in presence of various salts, at pH 4.5 and 18°C, indicates that solubility is affected more by anions than by cations and that the effectiveness of the anions is in an order roughly the reverse of that of the Hofmeister series. Under these conditions, thiocyanate, a chaotropic anion at high concentration, is found to be a very effective crystallizing agent at low ( m ) concentrations. Two other monomeric proteins (erabutoxin and bovine pancreatic trypsin inhibitor) of high isoelectric point crystallized also at pH 4.5 and 18°C, confirming the much higher effectiveness of KSCN compared to NaCl. The width of the crystallization zone between the solubility curve and the curve above which only precipitation occurs also varies considerably with different anions. These observations may have useful implications in screening and optimizing crystallization conditions.

Probing the quality of crystals of biological macromolecules using maximum resolution of diffraction data, Bragg reflection profiles and X-ray topographs

Abstract The quality of a macromolecular crystal can be probed by combining maximum resolution of diffraction data, Bragg reflection profiles and X-ray topographs. Concerning maximum resolution, critical parameters are the overall rms displacement of atoms, which includes both dynamic and static disorder components, and the dynamic range of integrated intensities that can be achieved for given sample and experimental conditions. Bragg reflection profiles and topographs recorded with a quasi-plane wave of X-rays give a wealth of information on the domain structure and macroscopic disorder.