171 results found
Li Y, Govada L, Solomon HV, et al., 2019, Analysis of glulisine crystallisation utilising phase diagrams and nucleants, Crystals, Vol: 9, ISSN: 2073-4352
Glulisine is a US Food and Drug Administration (FDA) approved insulin analogue, used for controlling hyperglycaemia in patients with diabetes mellitus (DM). It is fast acting which better approximates physiological insulin secretion, improving patient outcome. Crystallisation of Glulisine was analysed by its crystallisation phase diagram and nucleation-inducing materials. Both the hanging drop vapour diffusion and microbatch-under-oil methods were used and compared. We have shown that the same protein can have different solubility behaviours depending on the nature of the salt in the precipitating agent. In the case of Glulisine with magnesium formate, lowering the precipitant concentration drove the system further into supersaturation resulting in the formation of crystals and precipitation. This was the opposite effect to the usual scenario where raising the precipitant concentration leads to supersaturation. Glulisine with sodium potassium tartrate tetrahydrate (NaKT) followed the expected trend of forming crystals or precipitate at higher concentrations and clear drops at lower concentrations of the precipitant. The outcomes of crystallisation using the different crystallisation methods is also described. Glulisine was successfully crystallised and the crystals diffracted up to a resolution limit of 1.4 Å.
Birtley JR, Alomary M, Zanini E, et al., 2019, Inactivating mutations and X-ray crystal structure of the tumor suppressor OPCML reveal cancer-associated functions, Nature Communications, Vol: 10, ISSN: 2041-1723
OPCML, a tumor suppressor gene, is frequently silenced epigenetically in ovarian and other cancers. Here we report, by analysis of databases of tumor sequences, the observation of OPCML somatic missense mutations from various tumor types and the impact of these mutations on OPCML function, by solving the X-ray crystal structure of this glycoprotein to 2.65 Å resolution. OPCML consists of an extended arrangement of three immunoglobulin-like domains and homodimerizes via a network of contacts between membrane-distal domains. We report the generation of a panel of OPCML variants with representative clinical mutations and demonstrate clear phenotypic effects in vitro and in vivo including changes to anchorage-independent growth, interaction with activated cognate receptor tyrosine kinases, cellular migration, invasion in vitro and tumor growth in vivo. Our results suggest that clinically occurring somatic missense mutations in OPCML have the potential to contribute to tumorigenesis in a variety of cancers.
Nanev C, Saridakis E, Govada L, et al., 2019, Hydrophobic Interface-assisted protein crystallization: theory and experiment, ACS Applied Materials and Interfaces, Vol: 11, Pages: 12931-12940, ISSN: 1944-8244
Macromolecular crystallization is crucially important to a large number of scientific fields, including structural biology, drug design, formulation and delivery, the manufacture of biomaterials, and the preparation of foodstuffs. The purpose of this study is to facilitate control of crystallization, by investigating hydrophobic interface-assisted protein crystallization both theoretically and experimentally. The application of hydrophobic liquids as nucleation promoters or suppressors has rarely been investigated, and provides an underused avenue to explore in protein crystallization. Theoretically, crystal nucleation is regarded as a two-step process, the first step being a local increase in protein concentration due to its adsorption on the hydrophobic surface. Subsequently, the protein is ordered in a crystal lattice. The energetic aspect of crystal nucleation on water/hydrophobic substance interfaces is approached by calculating the balance between the cohesive energy maintaining integrity of the 2D-crystal nucleus and the sum of destructive energies tending to tear up the crystal. This is achieved by comparing the number of bonds shared by the units forming the crystal and the number of unshared (dangling) bonds on the crystal surface pointing toward the solution. The same approach is extended to 3D protein crystal nucleation at water/hydrophobic liquid interfaces. Experimentally, we studied protein crystalliza-tion over oils and other hydrophobic liquids (paraffin oil, FC-70 Fluorinert fluorinated oil, and three chlorinated hydrocarbons). Crystallization of α-lactalbumin and lysozyme are compared, and additional information is acquired by studying α-crustacyanin, trypsin, an insulin analogue and protein Lpg2936. Depending on the protein type, concentration, and the interface aging time, the proteins exhibit different crystallization propensities depending on the hydrophobic liquid used. Some hydrophobic liquids provoke an increase in the effective
Levenstein MA, Anduix-Canto C, Kim YY, et al., 2019, Droplet microfluidics XRD identifies effective nucleating agents for calcium carbonate, Advanced Functional Materials, ISSN: 1616-301X
The ability to control crystallization reactions is required in a vast range of processes including the production of functional inorganic materials and pharmaceuticals and the prevention of scale. However, it is currently limited by a lack of understanding of the mechanisms underlying crystal nucleation and growth. To address this challenge, it is necessary to carry out crystallization reactions in well-defined environments, and ideally to perform in situ measurements. Here, a versatile microfluidic synchrotron-based technique is presented to meet these demands. Droplet microfluidic-coupled X-ray diffraction (DMC-XRD) enables the collection of time-resolved, serial diffraction patterns from a stream of flowing droplets containing growing crystals. The droplets offer reproducible reaction environments, and radiation damage is effectively eliminated by the short residence time of each droplet in the beam. DMC-XRD is then used to identify effective particulate nucleating agents for calcium carbonate and to study their influence on the crystallization pathway. Bioactive glasses and a model material for mineral dust are shown to significantly lower the induction time, highlighting the importance of both surface chemistry and topography on the nucleating efficiency of a surface. This technology is also extremely versatile, and could be used to study dynamic reactions with a wide range of synchrotron-based techniques.
Govada L, Chayen N, 2019, Choosing the Method of Crystallization to Obtain Optimal Results, Crystals, Vol: 9, Pages: 106-106
<jats:p>Anyone who has ever attempted to crystallise a protein or other biological macromolecule has encountered at least one, if not all of the following scenarios: No crystals at all, tiny low quality crystals; phase separation; amorphous precipitate and the most frustrating; large, beautiful crystals that do not diffract at all. In this paper we review a number of simple ways to overcome such problems, which have worked well in our hands and in other laboratories. It brings together information that has been dispersed in various publications and lectures over the years and includes further information that has not been previously published.</jats:p>
Khurshid S, Govada L, Wills G, et al., 2018, Chlamydia protein Pgp3 studied at high resolution in a new crystal form, IUCrJ, Vol: 5, Pages: 439-448, ISSN: 2052-2525
The protein Pgp3 is implicated in the sexually transmitted disease chlamydia and comprises an extended complex arrangement of a C terminal domain (CTD) and an N terminal domain (NTD), each linked by a triple helix coiled coil (THCC). We report the X-ray crystal structure of Pgp3 from a LGV1 strain at the highest X-ray diffraction resolution obtained to date for the full protein. The protein was crystallised using a high KBr salt concentration, which resulted in a new crystal form with relatively low solvent content diffracting to a resolution of 1.98 Å. We describe the 3D structure of this new crystal form, compare it with other crystal forms, describe the KBr salt binding sites and the relevance to chlamydia isolates from around the globe. The crystal packing is apparently driven by the CTDs. Since the three fold axes of the THCC and NTD are not collinear with a CTD’s three fold axis this naturally leads to a disorder in the THCC and the portion of the NTD not directly interacting with the CTD via crystal packing. The key avenue to resolve these oddities of the crystal structure analysis was a complete new analysis in space group P1 and determining the space group as P212121. This space group assignment was the one originally determined from the diffraction pattern but perhaps complicated by a translational non crystallographic symmetry. We found this crystal structure of a three domain multi macromolecular complex, with two misaligned three fold axes, a unique challenge, something not encountered before. A specific intermolecular interaction, possibly of functional significance in receptor binding in chlamydia, we suggest might allow design of a new chemotherapeutic agent against chlamydia.
Adams GG, Meal A, Morgan PS, et al., 2018, Characterisation of insulin analogues therapeutically available to patients., PLoS ONE, Vol: 13, ISSN: 1932-6203
The structure and function of clinical dosage insulin and its analogues were assessed. This included 'native insulins' (human recombinant, bovine, porcine), 'fast-acting analogues' (aspart, glulisine, lispro) and 'slow-acting analogues' (glargine, detemir, degludec). Analytical ultracentrifugation, both sedimentation velocity and equilibrium experiments, were employed to yield distributions of both molar mass and sedimentation coefficient of all nine insulins. Size exclusion chromatography, coupled to multi-angle light scattering, was also used to explore the function of these analogues. On ultracentrifugation analysis, the insulins under investigation were found to be in numerous conformational states, however the majority of insulins were present in a primarily hexameric conformation. This was true for all native insulins and two fast-acting analogues. However, glargine was present as a dimer, detemir was a multi-hexameric system, degludec was a dodecamer (di-hexamer) and glulisine was present as a dimer-hexamer-dihexamer system. However, size-exclusion chromatography showed that the two hexameric fast-acting analogues (aspart and lispro) dissociated into monomers and dimers due to the lack of zinc in the mobile phase. This comprehensive study is the first time all nine insulins have been characterised in this way, the first time that insulin detemir have been studied using analytical ultracentrifugation and the first time that insulins aspart and glulisine have been studied using sedimentation equilibrium. The structure and function of these clinically administered insulins is of critical importance and this research adds novel data to an otherwise complex functional physiological protein.
Adams GG, Alzahrani Q, Jiwani SI, et al., 2017, Glargine and degludec: Solution behaviour of higher dose synthetic insulins., Scientific Reports, Vol: 7, ISSN: 2045-2322
Single, double and triple doses of the synthetic insulins glargine and degludec currently used in patient therapy are characterised using macromolecular hydrodynamic techniques (dynamic light scattering and analytical ultracentrifugation) in an attempt to provide the basis for improved personalised insulin profiling in patients with diabetes. Using dynamic light scattering and sedimentation velocity in the analytical ultracentrifuge glargine was shown to be primarily dimeric under solvent conditions used in current formulations whereas degludec behaved as a dihexamer with evidence of further association of the hexamers ("multi-hexamerisation"). Further analysis by sedimentation equilibrium showed that degludec exhibited reversible interaction between mono- and-di-hexamer forms. Unlike glargine, degludec showed strong thermodynamic non-ideality, but this was suppressed by the addition of salt. With such large injectable doses of synthetic insulins remaining in the physiological system for extended periods of time, in some case 24-40 hours, double and triple dose insulins may impact adversely on personalised insulin profiling in patients with diabetes.
Nanev CN, Saridakis E, Chayen N, 2017, Protein crystal nucleation in pores, Scientific Reports, Vol: 7, ISSN: 2045-2322
The most powerful method for protein structure determination is X-ray crystallography which relies on the availability of high quality crystals. Obtaining protein crystals is a major bottleneck, and inducing their nucleation is of crucial importance in this field. An effective method to form crystals is to introduce nucleation-inducing heterologous materials into the crystallization solution. Porous materials are exceptionally effective at inducing nucleation. It is shown here that a combined diffusion-adsorption effect can increase protein concentration inside pores, which enables crystal nucleation even under conditions where heterogeneous nucleation on flat surfaces is absent. Provided the pore is sufficiently narrow, protein molecules approach its walls and adsorb more frequently than they can escape. The decrease in the nucleation energy barrier is calculated, exhibiting its quantitative dependence on the confinement space and the energy of interaction with the pore walls. These results provide a detailed explanation of the effectiveness of porous materials for nucleation of protein crystals, and will be useful for optimal design of such materials.
Govada L, Saridakis E, Kassen S, et al., 2017, Smart materials for increasing the success of protein crystallization, Publisher: INT UNION CRYSTALLOGRAPHY, Pages: C1138-C1138, ISSN: 2053-2733
Govada L, Saridakis E, Khurshid S, et al., 2017, Enhancing the success of crystallization: strategies and techniques, Publisher: INT UNION CRYSTALLOGRAPHY, Pages: C1082-C1082, ISSN: 2053-2733
Leese HS, Govada L, Saridakis E, et al., 2016, Reductively PEGylated carbon nanomaterials andtheir use to nucleate 3D protein crystals:a comparison of dimensionality, Chemical Science, Vol: 7, Pages: 2916-2923, ISSN: 2041-6539
A range of carbon nanomaterials, with varying dimensionality, were dispersed by a non-damaging and versatile chemical reduction route, and subsequently grafted by reaction with methoxy polyethylene glycol (mPEG) monobromides. The use of carbon nanomaterials with different geometries provides both a systematic comparison of surface modification chemistry and the opportunity to study factors affecting specific applications. Multi-walled carbon nanotubes, single-walled carbon nanotubes, graphite nanoplatelets, exfoliated few layer graphite and carbon black were functionalized with mPEG-Br, yielding grafting ratios relative to the nanocarbon framework between ca. 7 and 135 wt%; the products were characterised by Raman spectroscopy, TGA-MS, and electron microscopy. The functionalized materials were tested as nucleants by subjecting them to rigorous protein crystallization studies. Sparsely functionalized flat sheet geometries proved exceptionally effective at inducing crystallization of six proteins. This new class of nucleant, based on PEG grafted graphene-related materials, can be widely applied to promote the growth of 3D crystals suitable for X-ray crystallography. The association of the protein ferritin with functionalized exfoliated few layer graphite was directly visualized by transmission electron microscopy, illustrating the formation of ordered clusters of protein molecules critical to successful nucleation.
Chayen N, shaffer, govada, et al., 2016, Exploring Carbon Nanomaterial Diversity for Nucleation of Protein Crystals, Scientific Reports, Vol: 6, ISSN: 2045-2322
Controlling crystal nucleation is a crucial step in obtaining high quality protein crystals for structure determination by X-ray crystallography. Carbon nanomaterials (CNMs) including carbon nanotubes, graphene oxide, and carbon black provide a range of surface topographies, porosities and length scales; functionalisation with two different approaches, gas phase radical grafting and liquid phase reductive grafting, provide routes to a range of oligomer functionalised products. These grafted materials, combined with a range of controls, were used in a large-scale assessment of the effectiveness for protein crystal nucleation of 20 different carbon nanomaterials on five proteins. This study has allowed a direct comparison of the key characteristics of carbon-based nucleants: appropriate surface chemistry, porosity and/or roughness are required. The most effective solid system tested in this study, carbon black nanoparticles functionalised with poly(ethylene glycol) methyl ether of mean molecular weight 5000, provides a novel highly effective nucleant, that was able to induce crystal nucleation of four out of the five proteins tested at metastable conditions.
Chayen NE, 2016, How to ... enhance the success of protein crystallization, Publisher: INT UNION CRYSTALLOGRAPHY, Pages: S173-S173, ISSN: 2053-2733
Rosenberg MF, Bikadi Z, Hazai E, et al., 2015, Three-dimensional structure of the human breast cancer resistance protein (BCRP/ABCG2) in an inward-facing conformation, Acta Crystallographica Section D - Biological Crystallography, Vol: 71, Pages: 1725-1735, ISSN: 0907-4449
ABCG2 is an efflux drug transporter that plays an important role in drug resistance and drug disposition. In this study, the first three-dimensional structure of human full-length ABCG2 analysed by electron crystallography from two-dimensional crystals in the absence of nucleotides and transported substrates is reported at 2 nm resolution. In this state, ABCG2 forms a symmetric homodimer with a noncrystallographic twofold axis perpendicular to the two-dimensional crystal plane, as confirmed by subtomogram averaging. This configuration suggests an inward-facing configuration similar to murine ABCB1, with the nucleotide-binding domains (NBDs) widely separated from each other. In the three-dimensional map, densities representing the long cytoplasmic extensions from the transmembrane domains that connect the NBDs are clearly visible. The structural data have allowed the atomic model of ABCG2 to be refined, in which the two arms of the V-shaped ABCG2 homodimeric complex are in a more closed and narrower conformation. The structural data and the refined model of ABCG2 are compatible with the biochemical analysis of the previously published mutagenesis studies, providing novel insight into the structure and function of the transporter.Keywords: ABCG2; BCRP; ABC transporter; ATP-binding cassette transporter; cryo-electron microscopy; three-dimensional structure from two-dimensional crystals.
Chain B, Arnold J, Akthar S, et al., 2015, A Linear Epitope in the N-Terminal Domain of CCR5 and Its Interaction with Antibody., PLOS One, Vol: 10, ISSN: 1932-6203
The CCR5 receptor plays a role in several key physiological and pathological processes and is an important therapeutic target. Inhibition of the CCR5 axis by passive or active immunisation offers one very selective strategy for intervention. In this study we define a new linear epitope within the extracellular domain of CCR5 recognised by two independently produced monoclonal antibodies. A short peptide encoding the linear epitope can induce antibodies which recognise the intact receptor when administered colinear with a tetanus toxoid helper T cell epitope. The monoclonal antibody RoAb 13 is shown to bind to both cells and peptide with moderate to high affinity (6x10^8 and 1.2x107 M-1 respectively), and binding to the peptide is enhanced by sulfation of tyrosines at positions 10 and 14. RoAb13, which has previously been shown to block HIV infection, also blocks migration of monocytes in response to CCR5 binding chemokines and to inflammatory macrophage conditioned medium. A Fab fragment of RoAb13 has been crystallised and a structure of the antibody is reported to 2.1 angstrom resolution.
Khurshid S, Govada L, El-Sharif HF, et al., 2015, Automating the application of smart materials for protein crystallization, ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY, Vol: 71, Pages: 534-540, ISSN: 2059-7983
Chayen NE, 2015, Successful crystal formation - the journey from idea to fruition, Publisher: INT UNION CRYSTALLOGRAPHY, Pages: S47-S47, ISSN: 2053-2733
Khurshid S, Saridakis E, Govada L, et al., 2014, Porous nucleating agents for protein crystallization, NATURE PROTOCOLS, Vol: 9, Pages: 1621-1633, ISSN: 1754-2189
Lansky S, Alalouf O, Solomon V, et al., 2014, Crystallization and preliminary crystallographic analysis of Axe2, an acetylxylan esterase from Geobacillus stearothermophilus. (vol 69, pg 430, 2013), ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS, Vol: 70, Pages: 685-685
Chayen NE, Shaffer M, Leese H, et al., 2014, NUCLEANT FOR MACROMOLECULE CRYSTALLISATION.
Lansky S, Alalouf O, Solomon HV, et al., 2014, A unique octameric structure of Axe2, an intracellular acetyl-xylooligosaccharide esterase from Geobacillus stearothermophilus, ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY, Vol: 70, Pages: 261-278, ISSN: 1399-0047
Govada L, Saridakis E, Kassen S, et al., 2014, Smart materials for increasing the success of protein crystallization, Publisher: INT UNION CRYSTALLOGRAPHY, Pages: C1138-C1138, ISSN: 2053-2733
Govada L, Saridakis E, Khurshid S, et al., 2014, Enhancing the success of crystallization: strategies and techniques, Publisher: INT UNION CRYSTALLOGRAPHY, Pages: C1082-C1082, ISSN: 2053-2733
Chayen N, Reddy SM, 2013, Novel Protein Crystallization Technique, GB2470744
Solomon HV, Tabachnikov O, Feinberg H, et al., 2013, Crystallization and preliminary crystallographic analysis of GanB, a GH42 intracellular beta-galactosidase from Geobacillus stearothermophilus, ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS, Vol: 69, Pages: 1114-1119
Saridakis E, Chayen NE, 2013, Imprinted polymers assisting protein crystallization, TRENDS IN BIOTECHNOLOGY, Vol: 31, Pages: 515-520, ISSN: 0167-7799
Lansky S, Alalouf O, Solomon V, et al., 2013, Crystallization and preliminary crystallographic analysis of Axe2, an acetylxylan esterase from Geobacillus stearothermophilus, ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS, Vol: 69, Pages: 430-434
Kertis F, Khurshid S, Okman O, et al., 2012, Heterogeneous nucleation of protein crystals using nanoporous gold nucleants, JOURNAL OF MATERIALS CHEMISTRY, Vol: 22, Pages: 21928-21934, ISSN: 0959-9428
Glassford SE, Govada L, Chayen NE, et al., 2012, Micro ATR FTIR imaging of hanging drop protein crystallisation, VIBRATIONAL SPECTROSCOPY, Vol: 63, Pages: 492-498, ISSN: 0924-2031
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