Crystallisation and characterisation of muscle proteins: a mini-review.

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 .

Growing Crystals for X-ray Free-Electron Laser Structural Studies of Biomolecules and Their Complexes.

Currently, X-ray crystallography, which typically uses synchrotron sources, remains the dominant method for structural determination of proteins and other biomolecules. However, small protein crystals do not provide sufficiently high-resolution diffraction patterns and suffer radiation damage; therefore, conventional X-ray crystallography needs larger protein crystals. The burgeoning method of serial crystallography using X-ray free-electron lasers (XFELs) avoids these challenges: it affords excellent structural data from weakly diffracting objects, including tiny crystals. An XFEL is implemented by irradiating microjets of suspensions of microcrystals with very intense X-ray beams. However, while the method for creating microcrystalline microjets is well established, little attention is given to the growth of high-quality nano/microcrystals suitable for XFEL experiments. In this study, in order to assist the growth of such crystals, we calculate the mean crystal size and the time needed to grow crystals to the desired size in batch crystallization (the predominant method for preparing the required microcrystalline slurries); this time is reckoned theoretically both for microcrystals and for crystals larger than the upper limit of the Gibbs-Thomson effect. The impact of the omnipresent impurities on the growth of microcrystals is also considered quantitatively. Experiments, performed with the model protein lysozyme, support the theoretical predictions.

Protein Crystals Nucleated and Grown by Means of Porous Materials Display Improved X-ray Diffraction Quality.

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.

Automating the application of smart materials for protein crystallization.

The fabrication and validation of the first semi-liquid nonprotein nucleating agent to be administered automatically to crystallization trials is reported. This research builds upon prior demonstration of the suitability of molecularly imprinted polymers (MIPs; known as `smart materials') for inducing protein crystal growth. Modified MIPs of altered texture suitable for high-throughput trials are demonstrated to improve crystal quality and to increase the probability of success when screening for suitable crystallization conditions. The application of these materials is simple, time-efficient and will provide a potent tool for structural biologists embarking on crystallization trials.

Three-dimensional structure of the human breast cancer resistance protein (BCRP/ABCG2) in an inward-facing conformation.

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.

A unique octameric structure of Axe2, an intracellular acetyl-xylooligosaccharide esterase from Geobacillus stearothermophilus.

Geobacillus stearothermophilus T6 is a thermophilic, Gram-positive soil bacterium that possesses an extensive and highly regulated hemicellulolytic system, allowing the bacterium to efficiently degrade high-molecular-weight polysaccharides such as xylan, arabinan and galactan. As part of the xylan-degradation system, the bacterium uses a number of side-chain-cleaving enzymes, one of which is Axe2, a 219-amino-acid intracellular serine acetylxylan esterase that removes acetyl side groups from xylooligosaccharides. Bioinformatic analyses suggest that Axe2 belongs to the lipase GDSL family and represents a new family of carbohydrate esterases. In the current study, the detailed three-dimensional structure of Axe2 is reported, as determined by X-ray crystallography. The structure of the selenomethionine derivative Axe2-Se was initially determined by single-wavelength anomalous diffraction techniques at 1.70 Šresolution and was used for the structure determination of wild-type Axe2 (Axe2-WT) and the catalytic mutant Axe2-S15A at 1.85 and 1.90 Šresolution, respectively. These structures demonstrate that the three-dimensional structure of the Axe2 monomer generally corresponds to the SGNH hydrolase fold, consisting of five central parallel ß-sheets flanked by two layers of helices (eight α-helices and five 310-helices). The catalytic triad residues, Ser15, His194 and Asp191, are lined up along a substrate channel situated on the concave surface of the monomer. Interestingly, the Axe2 monomers are assembled as a `doughnut-shaped' homo-octamer, presenting a unique quaternary structure built of two staggered tetrameric rings. The eight active sites are organized in four closely situated pairs, which face the relatively wide internal cavity. The biological relevance of this octameric structure is supported by independent results obtained from gel-filtration, TEM and SAXS experiments. These data and their comparison to the structural data of related hydrolases are used for a more general discussion focusing on the structure-function relationships of enzymes of this category.

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.

Structural basis for paxillin binding and focal adhesion targeting of ß-parvin.

ß-Parvin is a cytoplasmic adaptor protein that localizes to focal adhesions where it interacts with integrin-linked kinase and is involved in linking integrin receptors to the cytoskeleton. It has been reported that despite high sequence similarity to α-parvin, ß-parvin does not bind paxillin, suggesting distinct interactions and cellular functions for these two closely related parvins. Here, we reveal that ß-parvin binds directly and specifically to leucine-aspartic acid repeat (LD) motifs in paxillin via its C-terminal calponin homology (CH2) domain. We present the co-crystal structure of ß-parvin CH2 domain in complex with paxillin LD1 motif to 2.9 Å resolution and find that the interaction is similar to that previously observed between α-parvin and paxillin LD1. We also present crystal structures of unbound ß-parvin CH2 domain at 2.1 Å and 2.0 Å resolution that show significant conformational flexibility in the N-terminal α-helix, suggesting an induced fit upon paxillin binding. We find that ß-parvin has specificity for the LD1, LD2, and LD4 motifs of paxillin, with K(D) values determined to 27, 42, and 73 µM, respectively, by surface plasmon resonance. Furthermore, we show that proper localization of ß-parvin to focal adhesions requires both the paxillin and integrin-linked kinase binding sites and that paxillin is important for early targeting of ß-parvin. These studies provide the first molecular details of ß-parvin binding to paxillin and help define the requirements for ß-parvin localization to focal adhesions.

Crystallization and preliminary crystallographic analysis of GanB, a GH42 intracellular ß-galactosidase from Geobacillus stearothermophilus.

Geobacillus stearothermophilus T-6 is a Gram-positive thermophilic soil bacterium that contains a multi-enzyme system for the utilization of plant cell-wall polysaccharides, including xylan, arabinan and galactan. The bacterium uses a number of endo-acting extracellular enzymes that break down the high-molecular-weight polysaccharides into decorated oligosaccharides. These oligosaccharides enter the cell and are further hydrolyzed into sugar monomers by a set of intracellular glycoside hydrolases. One of these intracellular degrading enzymes is GanB, a glycoside hydrolase family 42 ß-galactosidase capable of hydrolyzing short ß-1,4-galactosaccharides to galactose. GanB and related enzymes therefore play an important part in the hemicellulolytic utilization system of many microorganisms which use plant biomass for growth. The interest in the biochemical characterization and structural analysis of these enzymes stems from their potential biotechnological applications. GanB from G. stearothermophilus T-6 has recently been cloned, overexpressed, purified, biochemically characterized and crystallized in our laboratory as part of its complete structure-function study. The best crystals obtained for this enzyme belong to the primitive orthorhombic space group P212121, with average crystallographic unit-cell parameters of a=71.84, b=181.35, c=196.57 Å. Full diffraction data sets to 2.45 and 2.50 Šresolution have been collected for both the wild-type enzyme and its E323A nucleophile catalytic mutant, respectively, as measured from flash-cooled crystals at 100 K using synchrotron radiation. These data are currently being used for the full three-dimensional crystal structure determination of GanB.

Crystallization and preliminary crystallographic analysis of Axe2, an acetylxylan esterase from Geobacillus stearothermophilus.

Acetylxylan esterases are part of the hemi-cellulolytic system of many microorganisms which utilize plant biomass for growth. Xylans, which are polymeric sugars that constitute a significant part of the plant biomass, are usually substituted with acetyl side groups attached at position 2 or 3 of the xylose backbone units. Acetylxylan esterases hydrolyse the ester linkages of the xylan acetyl groups and thus improve the ability of main-chain hydrolysing enzymes to break down the sugar backbone units. As such, these enzymes play an important part in the hemi-cellulolytic utilization system of many microorganisms that use plant biomass for growth. Interest in the biochemical characterization and structural analysis of these enzymes stems from their numerous potential biotechnological applications. An acetylxylan esterase (Axe2) of this type from Geobacillus stearothermophilus T-6 has recently been cloned, overexpressed, purified, biochemically characterized and crystallized. One of the crystal forms obtained (RB1) belonged to the tetragonal space group I422, with unit-cell parameters a = b = 110.2, c = 213.1 Å. A full diffraction data set was collected to 1.85 Å resolution from flash-cooled crystals of the wild-type enzyme at 100 K using synchrotron radiation. A selenomethionine derivative of Axe2 has also been prepared and crystallized for single-wavelength anomalous diffraction experiments. The crystals of the selenomethionine-derivatized Axe2 appeared to be isomorphous to those of the wild-type enzyme and enabled the measurement of a full 1.85 Å resolution diffraction data set at the selenium absorption edge and a full 1.70 Å resolution data set at a remote wavelength. These data are currently being used for three-dimensional structure determination of the Axe2 protein.

Protein crystallization and biosensor applications of hydrogel-based molecularly imprinted polymers.

We have characterized the imprinting capability of a family of acrylamide polymer-based molecularly imprinted polymers (MIPs) for bovine hemoglobin (BHb) and trypsin (Tryp) using spectrophotometric and quartz crystal microbalance (QCM) sensor techniques. Bulk gel characterization on acrylamide (AA), N-hydroxymethylacrylamide (NHMA), and N-isopropylacrylamide (NiPAM) gave varied selectivities when compared with nonimprinted polymers. We have also harnessed the ability of the MIPs to facilitate protein crystallization as a means of evaluating their selectivity for cognate and noncognate proteins. Crystallization trials indicated improved crystal formation in the order NiPAM

The CXCL16 A181V mutation selectively inhibits monocyte adhesion to CXCR6 but is not associated with human coronary heart disease.

OBJECTIVE: The chemokine CXCL16 serves as a scavenger receptor for oxidized low-density lipoprotein and as an adhesion molecule and chemoattractant for cells expressing the receptor CXCR6. A commonly occurring CXCL16 allele has been described containing 2 nonsynonymous single-nucleotide polymorphisms in complete linkage disequilibrium, although the effects on CXCL16 function are unknown. Here, we examined the effect of the single-nucleotide polymorphisms on CXCL16 function and assessed the association of the mutant allele with coronary heart disease (CHD). METHODS AND RESULTS: Both wild-type and mutant T123V181-CXCL16 were readily expressed in vitro and were similarly functional in assays of oxidized low-density lipoprotein scavenging and chemotaxis. However, unlike wild-type CXCL16, T123V181-CXCL16 was unable to promote adhesion of CXCR6(+) cells. Findings were confirmed ex vivo, with monocytes from donors homozygous for the T123V181 allele unable to facilitate adhesion of CXCR6 transfectants. In the London Life Sciences Prospective Population cohort (n = 2797), we found that the T123V181 allele was not associated with protection or susceptibility to CHD (adjusted odds ratio, 1.01; 95% CI, 0.95 to 1.10; P = 0.74). CONCLUSIONS: CXCL16-mediated cell adhesion plays at best a modest role in CHD, and the scavenging and chemotactic properties of the chemokine are more likely to be more important in disease pathogenesis.

Two independent histidines, one in human prolactin and one in its receptor, are critical for pH-dependent receptor recognition and activation.

Human prolactin (hPRL), a member of the family of hematopoietic cytokines, functions as both an endocrine hormone and autocrine/paracrine growth factor. We have previously demonstrated that recognition of the hPRL·receptor depends strongly on solution acidity over the physiologic range from pH 6 to pH 8. The hPRL·receptor binding interface contains four histidines whose protonation is hypothesized to regulate pH-dependent receptor recognition. Here, we systematically dissect its molecular origin by characterizing the consequences of His to Ala mutations on pH-dependent receptor binding kinetics, site-specific histidine protonation, and high resolution structures of the intermolecular interface. Thermodynamic modeling of the pH dependence to receptor binding affinity reveals large changes in site-specific protonation constants for a majority of interface histidines upon complexation. Removal of individual His imidazoles reduces these perturbations in protonation constants, which is most likely explained by the introduction of solvent-filled, buried cavities in the crystallographic structures without inducing significant conformational rearrangements.

Use of dual polarization interferometry as a diagnostic tool for protein crystallization.

The use of dual polarization interferometry (DPI) as a tool for probing the different possible outcomes of protein crystallization experiments is described. DPI is a surface analytical technique used for the characterization of structure and interactions of molecular layers on an optical waveguide surface for a wide range of applications, including protein-protein interactions and conformational changes. The application of this technique provides a "signature" of crystallization events, thus predicting if there will be protein crystal formation, amorphous precipitate, or clear solution. The technique was demonstrated on a number of model proteins, and it also produced meaningful results in the case of two problematic target proteins. DPI in conjunction with a dialysis setup, allows changes in the protein solution above the waveguide surface to be monitored simultaneously with continuous control of its precipitant content. DPI has the potential to be used as a powerful method for discovering crystallization conditions, for obtaining information on the crystallization process, and as an aid in crystal optimization. It has also provided what is, to the best of our knowledge, the most direct observation to date of salting-in behavior in a protein-salt solution.

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.

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.

X-ray crystallographic studies of RoAb13 bound to PIYDIN, a part of the N-terminal domain of C-C chemokine receptor 5.

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.

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.

The structure-function relationship of oncogenic LMTK3.

Elucidating signaling driven by lemur tyrosine kinase 3 (LMTK3) could help drug development. Here, we solve the crystal structure of LMTK3 kinase domain to 2.1Å resolution, determine its consensus motif and phosphoproteome, unveiling in vitro and in vivo LMTK3 substrates. Via high-throughput homogeneous time-resolved fluorescence screen coupled with biochemical, cellular, and biophysical assays, we identify a potent LMTK3 small-molecule inhibitor (C28). Functional and mechanistic studies reveal LMTK3 is a heat shock protein 90 (HSP90) client protein, requiring HSP90 for folding and stability, while C28 promotes proteasome-mediated degradation of LMTK3. Pharmacologic inhibition of LMTK3 decreases proliferation of cancer cell lines in the NCI-60 panel, with a concomitant increase in apoptosis in breast cancer cells, recapitulating effects of LMTK3 gene silencing. Furthermore, LMTK3 inhibition reduces growth of xenograft and transgenic breast cancer mouse models without displaying systemic toxicity at effective doses. Our data reinforce LMTK3 as a druggable target for cancer therapy.

Towards a 'universal' nucleant for protein crystallization.

The expression 'crystal clear' stems from the science of crystallography, which determines the detailed atomic structures of materials by exposing crystals to X-rays. Protein structures are pivotal to the success of rational drug design and other biotechnology applications; however, obtaining high quality crystals poses a major problem to progress. Nucleation is the first step that determines the entire crystallization process, thus control of crystal nucleation would tackle the problem at its conception. A search for a 'universal' nucleant, a substance that can induce nucleation of any protein, is therefore on-going. We report the advances that have been made in this area, highlighting the success of especially engineered as well as naturally structured surfaces.