43 results found
Govada L, Chayen NE, 2023, Crystallisation and characterisation of muscle proteins: a mini-review, Journal of Muscle Research and Cell Motility, Vol: 44, Pages: 209-215, ISSN: 0142-4319
The techniques of X-ray protein crystallography, NMR and high-resolution cryo-electron microscopy have all been used to determine the high-resolution structure of proteins. The most-commonly used method, however, remains X-ray crystallography but it does rely heavily on the production of suitable crystals. Indeed, the production of diffraction quality crystals remains the rate-limiting step for most protein systems. This mini-review highlights the crystallisation trials that used existing and newly developed crystallisation methods on two muscle protein targets - the actin binding domain (ABD) of α-actinin and the C0-C1 domain of human cardiac myosin binding protein C (cMyBP-C). Furthermore, using heterogenous nucleating agents the crystallisation of the C1 domain of cMyBP-C was successfully achieved in house along with preliminary actin binding studies using electron microscopy and co-sedimentation assays .
<jats:p>The techniques of X-ray protein crystallography, NMR and high-resolution cryo-electron microscopy have all been used to determine the high-resolution structure of proteins. The most-commonly used method, however, remains X-ray diffraction but it does rely heavily on the production of suitable crystals. Indeed, the production of diffraction quality crystals remains the rate-limiting step for most protein systems. Crystallisation trials, initiated by John Squire with the assistance of the authors, used existing and newly developed crystallisation methods for two different muscle proteins - the actin binding domain (ABD) of α-actinin and the C0-C1 domain of human cardiac myosin binding protein C (cMyBP-C). Diffracting crystals of α-actinin ABD, with a resolution of 1.9Å, however, did not prove to be in a form suitable for diffraction methods. In contrast, when the C0-C1 domain of cMyBP-C underwent proteolysis during crystallogenesis very satisfactory crystals of the C1 domain were obtained. These employed a modified hanging drop vapour diffusion method and diffracted to high resolution (1.5Å). This has led to the determination of the structure of the C1 domain of cMyBP-C at atomic resolution and has thereby provided new insights into its function. </jats:p>
Govada L, Rubio N, Saridakis E, et al., 2022, Graphene-based nucleants for protein crystallization, Advanced Functional Materials, Vol: 32, ISSN: 1616-301X
Protein crystallization remains a major bottleneck for the determination of high resolution structures. Nucleants can accelerate the process but should ideally be compatible with high throughput robotic screening. Polyethylene glycol grafted (PEGylated) graphenes can be stabilized in water providing dispensable, nucleant systems. Two graphitic feedstocks are exfoliated and functionalized with PEG using a non-destructive, scalable, chemical reduction method, delivering good water dispersibility (80 and 750 µg mL−1 for large and small layers, respectively). The wide utility of these nucleants has been established across five proteins and three different screens, each of 96 conditions, demonstrating greater effectiveness of the dispersed PEGylated graphenes. Smaller numbers of larger, more crystalline flakes consistently act as better protein nucleants. The delivered nucleant concentration is optimized (0.1 mg mL−1 in the condition), and the performance benchmarked against existing state of the art, molecularly imprinted polymer nucleants. Strikingly, graphene nucleants are effective even when decreasing both the nucleant and protein concentration to unusually low concentrations. The set-up to scale-up nucleant production to liter volumes can provide sufficient material for wide implementation. Together with the optimized crystallization conditions, the results are a step forward toward practical synthesis of a readily accessible “universal” nucleant.
Nanev CN, Saridakis E, Govada L, et al., 2022, Protein crystals nucleated and grown by means of porous materials display improved X-ray diffraction quality, International Journal of Molecular Sciences, Vol: 23, Pages: 1-13, ISSN: 1422-0067
Well-diffracting protein crystals are indispensable for X-ray diffraction analysis, which is still the most powerful method for structure-function studies of biomolecules. A promising approach to growing such crystals is the use of porous nucleation-inducing materials. However, while protein crystal nucleation in pores has been thoroughly considered, little attention has been paid to the subsequent growth of crystals. Although the nucleation stage is decisive, it is the subsequent growth of crystals outside the pore that determines their diffraction quality. The molecular-scale mechanism of growth of protein crystals in and outside pores is theoretically considered. Due to the low degree of metastability, the crystals that emerge from the pores grow slowly, which is a prerequisite for better diffraction. This expectation has been corroborated by experiments carried out with several types of porous material, such as bioglass (“Naomi’s Nucleant”), buckypaper, porous gold and porous silicon. Protein crystals grown with the aid of bioglass and buckypaper yield significantly better diffraction quality compared with crystals grown conventionally. In all cases, visually superior crystals are usually obtained. Our theoretical conclusion is that heterogeneous nucleation of a crystal outside the pore is an exceptional case. Rather, the protein crystals nucleating inside the pores continue growing outside them.
Govada L, Saridakis E, Kassen SC, et al., 2021, X-ray crystallographic studies of RoAb13 bound to PIYDIN, a part of the N-terminal domain of C-C chemokine receptor 5, IUCrJ, Vol: 8, Pages: 678-683, ISSN: 2052-2525
C-C chemokine receptor 5 (CCR5) is a major co-receptor molecule used by HIV-1 to enter cells. This led to the hypothesis that stimulating an antibody response would block HIV with minimal toxicity. Here, X-ray crystallographic studies of the anti-CCR5 antibody RoAb13 together with two peptides were undertaken: one peptide is a 31-residue peptide containing the PIYDIN sequence and the other is the PIDYIN peptide alone, where PIYDIN is part of the N-terminal region of CCR5 previously shown to be important for HIV entry. In the presence of the longer peptide (the complete N-terminal domain), difference electron density was observed at a site within a hypervariable CDR3 binding region. In the presence of the shorter core peptide PIYDIN, difference electron density is again observed at this CDR3 site, confirming consistent binding for both peptides. This may be useful in the design of a new biomimetic to stimulate an antibody response to CCR5 in order to block HIV infection.
Nanev C, Govada L, Chayen N, 2021, Theoretical and experimental investigation on protein crystal nucleation in pores and crevices, IUCrJ, Vol: 8, Pages: 270-280, ISSN: 2052-2525
The nucleation ability of pores is explained using the equilibration between the cohesive energy maintaining the integrity of a crystalline cluster and the destructive energy tending to tear it up. It is shown that to get 3D crystals it is vital to have 2D crystals nucleating in the pores first. By filling the pore orifice, the 2D crystal nuclei are more stable because their peripheries are protected from the destructive action of water molecules. Furthermore, the periphery of the 2D crystal is additionally stabilized as a result of its cohesion with the pore wall. The understanding provided by this study combining theory and experiment will facilitate the design of new nucleants.
Gillis RB, Solomon HV, Govada L, et al., 2021, Analysis of insulin glulisine at the molecular level by X-ray crystallography and biophysical techniques, Scientific Reports, Vol: 11, ISSN: 2045-2322
This study concerns glulisine, a rapid-acting insulin analogue that plays a fundamental role in diabetes management. We have applied a combination of methods namely X-ray crystallography, and biophysical characterisation to provide a detailed insight into the structure and function of glulisine. X-ray data provided structural information to a resolution of 1.26 Å. Crystals belonged to the H3 space group with hexagonal (centred trigonal) cell dimensions a = b = 82.44 and c = 33.65 Å with two molecules in the asymmetric unit. A unique position of D21Glu, not present in other fast-acting analogues, pointing inwards rather than to the outside surface was observed. This reduces interactions with neighbouring molecules thereby increasing preference of the dimer form. Sedimentation velocity/equilibrium studies revealed a trinary system of dimers and hexamers/dihexamers in dynamic equilibrium. This new information may lead to better understanding of the pharmacokinetic and pharmacodynamic behaviour of glulisine which might aid in improving formulation regarding its fast-acting role and reducing side effects of this drug.
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.
Levenstein MA, Anduix-Canto C, Kim YY, et al., 2019, Droplet microfluidics XRD identifies effective nucleating agents for calcium carbonate, Advanced Functional Materials, Vol: 29, 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.
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
Govada L, Chayen N, 2019, Choosing the method of crystallization to obtain optimal results, Crystals, Vol: 9, ISSN: 2073-4352
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.
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.
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.
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
Khurshid S, Saridakis E, Govada L, et al., 2014, Porous nucleating agents for protein crystallization, Nature Protocols, Vol: 9, Pages: 1621-1633, ISSN: 1750-2799
Solving the structure of proteins is pivotal to achieving success in rational drug design and in other biotechnological endeavors. The most powerful method for determining the structure of proteins is X-ray crystallography, which relies on the availability of high-quality crystals. However, obtaining such crystals is a major hurdle. Nucleation is the crucial prerequisite step, which requires overcoming an energy barrier. The presence in a protein solution of a nucleant, a solid or a semiliquid substance that facilitates overcoming that barrier allows crystals to grow under ideal conditions, paving the way for the formation of high-quality crystals. The use of nucleants provides a unique means for optimizing the diffraction quality of crystals, as well as for discovering new crystallization conditions. We present a protocol for controlling the nucleation of protein crystals that is applicable to a wide variety of nucleation-inducing substances. Setting up crystallization trials using these nucleating agents takes an additional few seconds compared with conventional setup, and it can accelerate crystallization, which typically takes several days to months.
Lansky S, Alalouf O, Solomon V, et al., 2014, Crystallization and preliminary crystallographic analysis of Axe2, an acetylxylan esterase from <i>Geobacillus stearothermophilus</i>. (vol 69, pg 430, 2013), ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS, Vol: 70, Pages: 685-685
Lansky S, Alalouf O, Solomon HV, et al., 2014, A unique octameric structure of Axe2, an intracellular acetyl-xylooligosaccharide esterase from <i>Geobacillus stearothermophilus</i>, ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY, Vol: 70, Pages: 261-278, ISSN: 1399-0047
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
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
Solomon HV, Tabachnikov O, Feinberg H, et al., 2013, Crystallization and preliminary crystallographic analysis of GanB, a GH42 intracellular β-galactosidase from <i>Geobacillus stearothermophilus</i>, ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS, Vol: 69, Pages: 1114-1119
Lansky S, Alalouf O, Solomon V, et al., 2013, Crystallization and preliminary crystallographic analysis of Axe2, an acetylxylan esterase from <i>Geobacillus stearothermophilus</i>, 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
Reddy SM, Phan QT, El-Sharif H, et al., 2012, Protein Crystallization and Biosensor Applications of Hydrogel-based Molecularly Imprinted Polymers (HydroMIPs), Biomacromolecules, Vol: 13, Pages: 3959-3965
Saridakis E, Khurshid S, Govada L, et al., 2011, Protein crystallization facilitated by molecularly imprinted polymers (vol 108, pg 11081, 2011), PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 108, Pages: 18566-18566, ISSN: 0027-8424
Boudjemline A, Saridakis E, Swann MJ, et al., 2011, Use of Dual Polarization Interferometry as a Diagnostic Tool for Protein Crystallization, ANALYTICAL CHEMISTRY, Vol: 83, Pages: 7881-7887, ISSN: 0003-2700
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