Protein Crystal Lattice Research Articles

Insights into the solubility of D-crystallin from multiscale atomistic simulations.

The molecular basis underlying the rich phase behavior of globular proteins remains poorly understood. We use atomistic multiscale molecular simulations to model the solution-state conformational dynamics and interprotein interactions of D-crystallin and its P23T-R36S mutant, which drastically limits the protein solubility, at both infinite dilution and at a concentration where the mutant fluid phase and crystalline phase coexist. We find that while the mutant conserves the protein fold, changes to the surface exposure of residues in the neighborhood of residue-36 enhance protein-protein interactions and develop specific protein-protein contacts found in the protein crystal lattice.

Peculiarities of Protein Crystal Nucleation and Growth

This paper reviews investigations on protein crystallization. It aims to present a comprehensive rather than complete account of recent studies and efforts to elucidate the most intimate mechanisms of protein crystal nucleation. It is emphasized that both physical and biochemical factors are at play during this process. Recently-discovered molecular scale pathways for protein crystal nucleation are considered first. The bond selection during protein crystal lattice formation, which is a typical biochemically-conditioned peculiarity of the crystallization process, is revisited. Novel approaches allow us to quantitatively describe some protein crystallization cases. Additional light is shed on the protein crystal nucleation in pores and crevices by employing the so-called EBDE method (equilibration between crystal bond and destructive energies). Also, protein crystal nucleation in solution flow is considered.

Response to comment on 'Protein crystal lattices are dynamic assemblies: the role of conformational entropy in the protein condensed phase'.

A response is given to Nespolo's comment [IUCrJ (2018), 5, https://doi.org/10.1107/S2052252518006267] about the usage of the the term 'crystal lattice' in Dimova & Devedjiev [IUCrJ (2018), 5, 130-140].

Comment on the article 'Protein crystal lattices are dynamic assemblies: the role of conformational entropy in the protein condensed phase'.

A comment is given on Dimova & Devedjiev [IUCrJ (2018), 5, 130-140].

Protein crystal lattices are dynamic assemblies: the role of conformational entropy in the protein condensed phase.

Until recently, the occurrence of conformational entropy in protein crystal contacts was considered to be a very unlikely event. A study based on the most accurately refined protein structures demonstrated that side-chain conformational entropy and static disorder might be common in protein crystal lattices. The present investigation uses structures refined using ensemble refinement to show that although paradoxical, conformational entropy is likely to be the major factor in the emergence and integrity of the protein condensed phase. This study reveals that the role of shape entropy and local entropic forces expands beyond the onset of crystallization. For the first time, the complete pattern of intermolecular interactions by protein atoms in crystal lattices is presented, which shows that van der Waals interactions dominate in crystal formation.

Growth of Ultrastable Protein–Silica Composite Crystals

Protein crystals were obtained in a wide range of silica gel concentrations, 2.0–22.0% (v/v), using the counter-diffusion technique. The protein crystal lattice incorporates silica fibers during their growth, making the crystal appear optically translucent while maintaining the diffraction quality. The effect of the silica fibers on the nucleation and growth morphology is discussed, and the amount of incorporated silica matrix is quantified. The practical implications of the presence of a high hygroscope phase on the crystal properties are discusse, and the improvement of the mechanical properties and stability of the crystals is shown. These reinforced protein crystals, able to include large amounts of silica, open a new range of possibilities for the characterization of protein crystals and the application in the biotechnological industry.

Equilibrium forms of protein crystals

Models for equilibrium forms of protein crystals were suggested using classical methods, such as Stranski–Kaischew's mean-separation-work method and the Gibbs–Curie–Wulff law. Although absolute bonding energies in protein crystal lattices are unknown, their relative strength was estimated from the type of lattice contacts observed in the protein x-ray structures. Equilibrium forms of ferritin crystals deduced on this basis were compared with experimental observations of Yau and Vekilov.

Brittleness of protein crystals

AbstractA simple technique for studying the brittleness of small crystals is reported. The limits of fracture toughness of tetragonal hen‐egg white lysozyme crystals, oriented with their c‐axis normally to the substrate, were measured. The strong mechanics anisotropy of those crystals was confirmed. The role of the water present in the protein crystal lattice was re‐considered in seek for a more holistic understanding of the process, the idea being that the intra‐crystalline solution sustains the globular protein molecules in their native configuration. Also it is argued that this water may contribute for holding together the huge bio‐molecules in the crystal lattice (that is to act as additional “glue” in the crystal). The hypothesis is that dynamic chains of H‐bonds in the intra‐crystalline water are likely to be prolonged to connect protein‐to‐water‐to‐protein.

Time Resolved Crystallographic Analysis of Cooperative Ligand Binding and Ligand Migration in a Dimeric Hemoglobin

Invertebrate hemoglobins, which range in size from dimers to assemblies of hundreds of subunits, offer excellent models for the investigation of allosteric protein function. A recurring theme among cooperative invertebrate hemoglobins is a subunit pairing involving the heme-embedding E and F helices, despite a lack of sequence conservation in these helices. This is quite different from the assembly of vertebrate hemoglobins and suggests that such a pairing is amenable for regulating oxygen delivery. We have carried out extensive time-resolved crystallographic analysis on the simplest of these hemoglobins, the cooperative homodimeric hemoglobin (HbI) from the blood clam Scapharca inaequivalvis. Limited ligand-linked subunit rotation permits the full allosteric transition to be followed in crystals of HbI. These studies have been combined with mutant and functional studies along with conventional crystallographic analysis to provide a comprehensive understanding of the communication between subunits and the movement of ligands through the protein. Investigation of ligand migration through this protein suggests, surprisingly, that ligands primarily escape through a distal histidine gate, despite its involvement in the subunit interface. As a result, ligand escape may require transient subunit movement, which is inhibited in the protein crystal lattice. These findings emphasize the central role of dynamics in protein function. Our studies are also directed towards understanding cooperative protein function in larger invertebrate hemoglobin assemblies. Preliminary time-resolved crystallogaphic analysis on the tetrameric Scapharca HbII suggests similar structural transitions as HbI and offers the possibility of investigating how changes in one subunit directly impact a neighboring subunit in the heterodimeric halves of HbII. In addition, we will discuss new insights that may provide unifying themes for the basis of cooperativity among diverse invertebrate hemoglobins that form EF dimeric pairing.

Characterizing a monotopic membrane enzyme. Biochemical, enzymatic and crystallization studies on Aquifex aeolicus sulfide:quinone oxidoreductase

Monotopic membrane proteins are membrane proteins that interact with only one leaflet of the lipid bilayer and do not possess transmembrane spanning segments. They are endowed with important physiological functions but until now only few of them have been studied. Here we present a detailed biochemical, enzymatic and crystallographic characterization of the monotopic membrane protein sulfide:quinone oxidoreductase. Sulfide:quinone oxidoreductase is a ubiquitous enzyme involved in sulfide detoxification, in sulfide-dependent respiration and photosynthesis, and in heavy metal tolerance. It may also play a crucial role in mammals, including humans, because sulfide acts as a neurotransmitter in these organisms. We isolated and purified sulfide:quinone oxidoreductase from the native membranes of the hyperthermophilic bacterium Aquifex aeolicus. We studied the pure and solubilized enzyme by denaturing and non-denaturing polyacrylamide electrophoresis, size-exclusion chromatography, cross-linking, analytical ultracentrifugation, visible and ultraviolet spectroscopy, mass spectrometry and electron microscopy. Additionally, we report the characterization of its enzymatic activity before and after crystallization. Finally, we discuss the crystallization of sulfide:quinone oxidoreductase in respect to its membrane topology and we propose a classification of monotopic membrane protein crystal lattices. Our data support and complement an earlier description of the three-dimensional structure of A. aeolicus sulfide:quinone oxidoreductase (M. Marcia, U. Ermler, G. Peng, H. Michel, Proc Natl Acad Sci USA, 106 (2009) 9625-9630) and may serve as a reference for further studies on monotopic membrane proteins.

Progress in the Development of an Alternative Approach to Macromolecular 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, noncovalent cross-links in crystals, and thereby promote lattice formation. Earlier experiments provided encouraging results that suggested further research was warranted (Larson, S. B.; Day, J. S.; Cudney, R.; McPherson, A. A novel strategy for the crystallization of proteins: X-ray diffraction validation. Acta Crystallogr., D: Biol. Crystallogr. 2007, 63, 310−318. McPherson, A.; Cudney, B. Searching for silver bullets: an alternative strategy for crystallizing macromolecules. J. Struct. Biol. 2006, 156, 387−406). Here we report additional, large-scale crystallization screening experiments that lend further support, though they suggest that additional mechanisms may play a positive role as well. As before, we accompanied the crystallization experiments with X-ray diffraction analyses of some of the crystals grown. A number of these showed incorporation of conventional molecules into protein crystal lattices, and further validated the underlying hypothesis. The strategy we are pursuing is essentially orthogonal to current approaches and has an objective of doubling the success rate of today.

Crystal packing of plant-typeL-asparaginase fromEscherichia coli

Plant-type L-asparaginases hydrolyze the side-chain amide bond of L-asparagine or its beta-peptides. They belong to the N-terminal nucleophile (Ntn) hydrolases and are synthesized as inactive precursor molecules. Activation occurs via the autoproteolytic release of two subunits, alpha and beta, the latter of which carries the nucleophile at its N-terminus. Crystallographic studies of plant-type asparaginases have focused on an Escherichia coli homologue (EcAIII), which has been crystallized in several crystal forms. Although they all belong to the same P2 1 2 1 2 1 space group with similar unit-cell parameters, they display different crystal-packing arrangements and thus should be classified as separate polymorphs. This variability stems mainly from different positions of the EcAIII molecules within the unit cell, although they also exhibit slight differences in orientation. The intermolecular interactions that trigger different crystal lattice formation are mediated by ions, which represent the most variable component of the crystallization conditions. This behaviour confirms recent observations that small molecules might promote protein crystal lattice formation.

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.

'Crystal lattice engineering,' an approach to engineer protein crystal contacts by creating intermolecular symmetry: crystallization and structure determination of a mutant human RNase 1 with a hydrophobic interface of leucines.

A protein crystal lattice consists of surface contact regions, where the interactions of specific groups play a key role in stabilizing the regular arrangement of the protein molecules. In an attempt to control protein incorporation in a crystal lattice, a leucine zipper-like hydrophobic interface (comprising four leucine residues) was introduced into a helical region (helix 2) of the human pancreatic ribonuclease 1 (RNase 1) that was predicted to form a suitable crystallization interface. Although crystallization of wild-type RNase 1 has not yet been reported, the RNase 1 mutant having four leucines (4L-RNase 1) was successfully crystallized under several different conditions. The crystal structures were subsequently determined by X-ray crystallography by molecular replacement using the structure of bovine RNase A. The overall structure of 4L-RNase 1 is quite similar to that of the bovine RNase A, and the introduced leucine residues formed the designed crystal interface. To characterize the role of the introduced leucine residues in crystallization of RNase 1 further, the number of leucines was reduced to three or two (3L- and 2L-RNase 1, respectively). Both mutants crystallized and a similar hydrophobic interface as in 4L-RNase 1 was observed. A related approach to engineer crystal contacts at helix 3 of RNase 1 (N4L-RNase 1) was also evaluated. N4L-RNase 1 also successfully crystallized and formed the expected hydrophobic packing interface. These results suggest that appropriate introduction of a leucine zipper-like hydrophobic interface can promote intermolecular symmetry for more efficient protein crystallization in crystal lattice engineering efforts.

Reinforced protein crystals

Large tetragonal hen egg white lysozyme single crystals (up to 16 mm 3) can be obtained by the counter-diffusion method, using high concentration silica gels. The protein crystal lattice is able to incorporate large amounts of silica while still maintaining its short-range crystallographic order. The crystal morphology is controlled by the concentration of the silica gel, which can reduce surface energy anisotropy to such an extent that spherical single crystals can be obtained as growth forms. The mechanical properties and the stability of the crystals against dehydration are improved by the incorporated hydrophilic silica polymeric network. This makes it possible to record full diffraction data sets with a resolution better than 1.5 Å from crystals glued to glass fibers. Such reinforcement of the crystals facilitates their handling at ambient conditions and opens new possibilities for the measurement of physical properties of large biological macromolecules as well as for their technological applications.

Topochemical Catalysis Achieved by Structure-based Ligand Design

Recently, a cyclic peptide ligand, cyclo-Ac-[CHPQG-PPC]-NH2, that binds to streptavidin with high affinity was discovered by screening phage libraries. From the streptavidin-bound crystal structures of cyclo-Ac-[CHPQGPPC]-NH2 and of a related but more weakly binding linear ligand, FSHPQNT, we designed linear thiol-containing streptavidin binding ligands, FCH-PQNT-NH2 and Ac-CHPQNT-NH2, which are dimerized catalytically by the streptavidin crystal lattice of space group I222, as demonstrated by high performance liquid chromatography and mass spectrometry. The catalytic dimerization relies on presentation of the ligand thiols toward one another in the lattice. The streptavidin crystal lattice-mediated catalysis achieved by structure-based design is the first example of catalysis of a chemical reaction by a protein crystal lattice. The spontaneous and crystal catalyzed rates of disulfide formation were determined by high performance liquid chromatography at pH 3.1, 4.0, 5.0, and 6.0. The ratio of the catalyzed to uncatalyzed rate was maximal at pH 3.1 (kcat/kuncat = 3.8), diminishing to 1.2 at pH 6.0. The crystal structures of the streptavidin-bound dimerized peptide ligands, FCHPQNT-NH2 dimer at 1.95 A and Ac-CHPQNT-NH2 dimer at 1.80 A, are described and compared with the structures of streptavidin-bound FSHPQNT monomer and cyclo-Ac-[CHPQGPPC]-NH2 dimer.

Topochemistry for preparing ligands that dimerize receptors

Background: The cyclic, disulfide-containing peptide cyclo-Ac-[Cys-His-Pro-Gln-Gly-Pro-Pro-Cys]-NH 2, binds to streptavidin with high affinity. In streptavidin-peptide cocrystals of space group 1222, cyclic peptide monomers are bound on adjacent streptavidin tetramers related by a crystallographic two-fold symmetry axis. We set out to determine whether the disulfide bonds of the peptide, presented close to one another in the crystal, could undergo disulfide interchange to form a dimer. Results: When juxtaposed, the disulfides of neighboring peptides undergo disulfide interchange, breaking and forming covalent disulfide bonds, to produce a peptide dimer adopting the symmetry of the crystal. This is the first example of a chemical transformation mediated by a protein crystal lattice. The structure of the streptavidin-bound monomer, and that of the dimer that was eventually produced from it in the crystal, were both determined from the same single crystal studied at different times. The two-fold symmetric peptide dimer was independently synthesized and shown to form crystals of dimerized streptavidin. Conclusions: We have shown that formation of a covalently linked peptide dimer can be mediated by a protein crystal lattice. The dimer thus produced dimerizes its target, streptavidin, suggesting that solid-state (or topochemical) reactions of this kind may be broadly useful for the preparation of ligands that can dimerize other protein targets.

Electron cryomicroscopy of frozen-hydrated biological specimens: analysis of freezing artifacts by x-ray cryocrystallography

Freezing artifacts have been evaluated by X-ray cryocrystallography on pellets of two-dimensional membrane protein crystals: purple membrane and maltoporin. The comparison of the X-ray patterns recorded when the specimens are maintained at room temperature to those obtained when the specimens are maintained at about -160°C shows that (i) membrane proteins have a positive thermal dilatation coefficient: the protein crystal lattice shrinks upon cooling; (ii) the asymmetric unit of crystal containing water is changed upon freezing; the relative intensities of the diffraction rings of such crystals are different after freezing. From these results, it can be postulated that freezing may lead to partial dehydration of biological objects. Electron cryomicroscopy visualizes objects which are structurally influenced by the cooling procedure. However, our microscopy study on maltoporin crystals shows that freezing artifacts are negligible in comparison to artifacts associated with conventional techniques such as negative staining.

Active-site-directed inactivators of the Zn2+-containing D-alanyl-D-alanine-cleaving carboxypeptidase of Streptomyces albus G.

Several types of active-site-directed inactivators (inhibitors) of the Zn2+-containing D-alanyl-D-alanine-cleaving carboxypeptidase were tested. (i) Among the heavy-atom-containing compounds examined, K2Pt(C2O4)2 inactivates the enzyme with a second-order rate constant of about 6 X 10(-2)M-1 X S-1 and has only one binding site located close to the Zn2+ cofactor within the enzyme active site. (ii) Several compounds possessing both a C-terminal carboxylate function and, at the other end of the molecule, a thiol, hydroxamate or carboxylate function were also examined. 3-Mercaptopropionate (racemic) and 3-mercaptoisobutyrate (L-isomer) inhibit the enzyme competitively with a Ki value of 5 X 10 X 10(-9)M. (iii) Classical beta-lactam compounds have a very weak inhibitory potency. Depending on the structure of the compounds, enzyme inhibition may be competitive (and binding occurs to the active site) or non-competitive (and binding causes disruption of the protein crystal lattice). (iv) 6-beta-Iodopenicillanate inactivates the enzyme in a complex way. At high beta-lactam concentrations, the pseudo-first-order rate constant of enzyme inactivation has a limit value of 7 X 10(-4)S-1 X 6-beta-Iodopenicillanate binds to the active site just in front of the Zn2+ cofactor and superimposes histidine-190, suggesting that permanent enzyme inactivation is by reaction with this latter residue.