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"Chayen, Naomi E"
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Protein crystallization: from purified protein to diffraction-quality crystal
Determining the structure of biological macromolecules by X-ray crystallography involves a series of steps: selection of the target molecule; cloning, expression, purification and crystallization; collection of diffraction data and determination of atomic positions. However, even when pure soluble protein is available, producing high-quality crystals remains a major bottleneck in structure determination. Here we present a guide for the non-expert to screen for appropriate crystallization conditions and optimize diffraction-quality crystal growth.
Journal Article
Porous nucleating agents for protein crystallization
A Protocol for obtaining high-quality protein crystals that involves addition to the protein solution of solid or semi-liquid nucleants that provide the experimenter the means to control crystal growth.
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.
Journal Article
Structural basis of topoisomerase targeting by delafloxacin
Delafloxacin is a potent anionic fluoroquinolone approved for the treatment of respiratory infections that acts by trapping the DNA cleavage complexes of bacterial topoisomerase IV and gyrase. Its N-1-pyridinyl-, C-7-azetidinyl- and C-8-chlorine substituents confer enhanced antibiotic activity against bacteria resistant to other fluoroquinolones, but its mode of action is unclear. Here we present the X-ray crystal structures of a delafloxacin-DNA cleavage complex obtained by co-crystallization with
Streptococcus pneumoniae
topo IV using a graphene nucleant and solved at 2.0 and 2.4 Å resolution. The two Mg
2+
-chelated delafloxacin molecules intercalated at the DNA cleavage site are bound in an unusual conformation involving interacting out-of-plane N-1-aromatic- and C-8-chlorine- substituents. The unprecedented resolution allows comprehensive imaging of water-metal ion links integrating enzyme and DNA through drug-bound and active-site Mg
2+
ions plus the discovery of enzyme-bound K
+
ions. Our studies on delafloxacin action suggest that intrinsic target affinity contributes to its activity against quinolone-resistant bacteria.
Here, the authors show that delafloxacin, a respiratory antibacterial fluoroquinolone, binds the Streptococcus pneumoniae topoisomerase IV-DNA cleavage complex in a distinct tilted-ring conformation involving multiple Mg
2 +
, K
+
and water links. Intrinsic target affinity likely contributes to activity against quinolone-resistant bacteria.
Journal Article
Experiment and Theory for Heterogeneous Nucleation of Protein Crystals in a Porous Medium
The determination of high-resolution structures of proteins requires crystals of suitable quality. Because of the new impetus given to structural biology by structural genomics/proteomics, the problem of crystallizing proteins is becoming increasingly acute. There is therefore an urgent requirement for the development of new efficient methods to aid crystal growth. Nucleation is the crucial step that determines the entire crystallization process. Hence, the holy grail is to design a \"universal nucleant,\" a substrate that induces the nucleation of crystals of any protein. We report a theory for nucleation on disordered porous media and its experimental testing and validation using a mesoporous bioactive gelglass. This material induced the crystallization of the largest number of proteins ever crystallized using a single nucleant. The combination of the model and the experimental results opens up the scope for the rational design of nucleants, leading to alternative means of controlling crystallization.
Journal Article
Protein crystallization facilitated by molecularly imprinted polymers
We present a previously undescribed initiative and its application, namely the design of molecularly imprinted polymers (MIPs) for producing protein crystals that are essential for determining high-resolution 3D structures of proteins. MIPs, also referred to as \"smart materials,\" are made to contain cavities capable of rebinding protein; thus the fingerprint of the protein created on the polymer allows it to serve as an ideal template for crystal formation. We have shown that six different MIPs induced crystallization of nine proteins, yielding crystals in conditions that do not give crystals otherwise. The incorporation of MIPs in screening experiments gave rise to crystalline hits in 8-10% of the trials for three target proteins. These hits would have been missed using other known nucleants. MIPs also facilitated the formation of large single crystals at metastable conditions for seven proteins. Moreover, the presence of MIPs has led to faster formation of crystals in all cases where crystals would appear eventually and to major improvement in diffraction in some cases. The MIPs were effective for their cognate proteins and also for other proteins, with size compatibility being a likely criterion for efficacy. Atomic force microscopy (AFM) measurements demonstrated specific affinity between the MIP cavities and a protein-functionalized AFM tip, corroborating our hypothesis that due to the recognition of proteins by the cavities, MIPs can act as nucleation-inducing substrates (nucleants) by harnessing the proteins themselves as templates.
Journal Article
Theoretical and experimental investigation of protein crystal nucleation in pores and crevices
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.
Journal Article
Analysis of insulin glulisine at the molecular level by X-ray crystallography and biophysical techniques
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.
Journal Article
Choosing the Method of Crystallization to Obtain Optimal Results
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.
Journal Article
Inactivating mutations and X-ray crystal structure of the tumor suppressor OPCML reveal cancer-associated functions
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.
OPCML is a tumour suppressor gene that is epigenetically silenced in ovarian cancer and is somatically mutated in various cancers. Here, the authors solve the X-ray crystal structure of OPCML and model clinically relevant mutations that could contribute to tumorigenesis.
Journal Article