1181 Citations
Many bacteria kill related bacteria by secretion of bacteriocins In Escherichia coli the colicin M protein kills E coli after uptake into the periplasm Self-protection from destruction is provided by the co-expressed immunity protein The colicin M immunity protein Cmi was cloned overexpressed and purified to homogeneity The correct fold of purified Cmi was analyzed by activity tests and circular-dichroism spectroscopy Crystallization trials yielded crystals one of which diffracted to a resolution of in the orthorhombic space group C The crystal packing with unit-cell parameters a b c indicated the presence of one monomer in the asymmetric unit with a ... More
Many bacteria kill related bacteria by secretion of bacteriocins. In Escherichia coli, the colicin M protein kills E. coli after uptake into the periplasm. Self-protection from destruction is provided by the co-expressed immunity protein. The colicin M immunity protein (Cmi) was cloned, overexpressed and purified to homogeneity. The correct fold of purified Cmi was analyzed by activity tests and circular-dichroism spectroscopy. Crystallization trials yielded crystals, one of which diffracted to a resolution of 1.9 � in the orthorhombic space group C2221. The crystal packing, with unit-cell parameters a = 66.02, b = 83.47, c = 38.30 �, indicated the presence of one monomer in the asymmetric unit with a solvent content of 53%. Less
HAMP domains mediate signal transduction in over enzyme-coupled receptors represented in all kingdoms of life The HAMP domain of the putative archaeal receptor Af has a parallel dimeric four-helical coiled coil structure but with unusual core packing related to canonical packing by concerted axial rotation of the helices This has led to the gearbox model for signal transduction whereby the alternate packing modes correspond to signaling states Here we present structures of a series of Af HAMP variants We show that substitution of a conserved small side chain within the domain core A for larger residues induces a gradual transition ... More
HAMP domains mediate signal transduction in over 7500 enzyme-coupled receptors represented in all kingdoms of life. The HAMP domain of the putative archaeal receptor Af1503 has a parallel, dimeric, four-helical coiled coil structure, but with unusual core packing, related to canonical packing by concerted axial rotation of the helices. This has led to the gearbox model for signal transduction, whereby the alternate packing modes correspond to signaling states. Here we present structures of a series of Af1503 HAMP variants. We show that substitution of a conserved small side chain within the domain core (A291) for larger residues induces a gradual transition in packing mode, involving both changes in helix rotation and bundle shape, which are most prominent at the C-terminal, output end of the domain. These are correlated with activity and ligand response in vitro and in vivo by incorporating Af1503 HAMP into mycobacterial adenylyl cyclase assay systems. Less
Gramicidin is an apolar pentadecapeptide antibiotic consisting of alternating D-and L-amino acids It functions in part by creating pores in membranes of susceptible cells rendering them leaky to monovalent cations The peptide should be able to traverse the host membrane either as a double stranded intertwined double helix DSDH or as a head-to-head single stranded helix HHSH Current structure models are based on macromolecular X-ray crystallography MX and nuclear magnetic resonance NMR However the HHSH form has only been observed by NMR The shape and size of the different gramicidin conformations differ We speculated therefore that reconstituting it into a ... More
Gramicidin is an apolar pentadecapeptide antibiotic consisting of alternating D-and L-amino acids. It functions, in part, by creating pores in membranes of susceptible cells rendering them leaky to monovalent cations. The peptide should be able to traverse the host membrane either as a double stranded, intertwined double helix (DSDH) or as a head-to-head single stranded helix (HHSH). Current structure models are based on macromolecular X-ray crystallography (MX) and nuclear magnetic resonance (NMR). However, the HHSH form has only been observed by NMR. The shape and size of the different gramicidin conformations differ. We speculated therefore that reconstituting it into a lipidic mesophase with bilayers of different microstructures would preferentially stabilize one form over the other. By using such mesophases for in meso crystallogenesis the expectation was that at least one would generate crystals of gramicidin in the HHSH form for structure determination by MX. This was tested using commercial and in-house synthesised lipids that support in meso crystallogenesis. Lipid acyl chain lengths were varied from 14 to 18 carbons to provide mesophases with a range of bilayer thicknesses. Unexpectedly, all lipids produced high quality, structure-grade crystals with gramicidin only in the DSDH conformation. Less
The Toll interleukin- receptor TIR domain is a protein protein interaction domain that is found in both animal and plant immune receptors In animal Toll-like receptor signalling both homotypic TIR-domain interactions between two receptor molecules and heterotypic interactions between receptors and TIR-domain-containing adaptors are required for initiation of an innate immune response The TIR domains in cytoplasmic nucleotide-binding leucine-rich repeat NB-LRR plant disease-resistance proteins are not as well characterized but recent studies have suggested a role in defence signalling In this study the crystallization X-ray diffraction analysis and preliminary structure determination of the TIR domain from the flax resistance protein ... More
The Toll/interleukin-1 receptor (TIR) domain is a protein�protein interaction domain that is found in both animal and plant immune receptors. In animal Toll-like receptor signalling, both homotypic TIR-domain interactions between two receptor molecules and heterotypic interactions between receptors and TIR-domain-containing adaptors are required for initiation of an innate immune response. The TIR domains in cytoplasmic nucleotide-binding/leucine-rich repeat (NB-LRR) plant disease-resistance proteins are not as well characterized, but recent studies have suggested a role in defence signalling. In this study, the crystallization, X-ray diffraction analysis and preliminary structure determination of the TIR domain from the flax resistance protein L6 (L6TIR) are reported. Plate-like crystals of L6TIR were obtained using PEG 200 as a precipitant and diffracted X-rays to 2.3 � resolution. Pseudo-translation complicated the initial assignment of the crystal symmetry, which was ultimately found to correspond to space group P21212 with two molecules per asymmetric unit. The structure of L6TIR was solved by molecular replacement using the structure of the TIR-domain-containing protein AT1G72930 from Arabidopsis as a template. Less
G protein-coupled receptors GPCRs constitute a highly diverse and ubiquitous family of integral membrane proteins transmitting signals inside the cells in response to an assortment of disparate extracellular stimuli Their strategic location on the cell surface and their involvement in crucial cellular and physiological processes turn these receptors into highly important pharmaceutical targets Recent technological developments aimed at stabilization and crystallization of these receptors have led to significant breakthroughs in GPCR structure determination efforts One of the successful approaches involved receptor stabilization with the help of a fusion partner combined with crystallization in lipidic cubic phase LCP The success of ... More
G protein-coupled receptors (GPCRs) constitute a highly diverse and ubiquitous family of integral membrane proteins, transmitting signals inside the cells in response to an assortment of disparate extracellular stimuli. Their strategic location on the cell surface and their involvement in crucial cellular and physiological processes turn these receptors into highly important pharmaceutical targets. Recent technological developments aimed at stabilization and crystallization of these receptors have led to significant breakthroughs in GPCR structure determination efforts. One of the successful approaches involved receptor stabilization with the help of a fusion partner combined with crystallization in lipidic cubic phase (LCP). The success of using LCP matrix for crystallization is generally attributed to the creation of a more native, membrane-like stabilizing environment for GPCRs just prior to nucleation and to the formation of type I crystal lattices, thus, generating highly ordered and strongly diffracting crystals. Here we describe protocols for reconstituting purified GPCRs in LCP, performing pre-crystallization assays, setting up crystallization trials in manual mode, detecting crystallization hits, optimizing crystallization conditions, harvesting, and collecting crystallographic data The protocols provide a sensible framework for approaching crystallization of stabilized GPCRs in LCP, however, as in any crystallization experiment extensive screening and optimization of crystallization conditions as well as optimization of protein construct and purification steps are required. The process remains risky and these protocols do not necessarily guarantee success. Less
Colicin M Cma is specifically imported into the periplasm of Escherichia coli and kills the cells Killing depends on the periplasmic peptidyl prolyl cis-trans isomerase chaperone FkpA To identify the Cma prolyl bonds targeted by FkpA we replaced the proline residues individually with alanine Seven mutant proteins were fully active Cma P A Cma P A and Cma P A displayed and Cma P A displayed of the wild-type activity Cma P A Cma P A and Cma P A but not Cma P A killed cells after entering the periplasm via osmotic shock indicating that the former mutants were ... More
Colicin M (Cma) is specifically imported into the periplasm of Escherichia coli and kills the cells. Killing depends on the periplasmic peptidyl prolyl cis-trans isomerase/chaperone FkpA. To identify the Cma prolyl bonds targeted by FkpA, we replaced the 15 proline residues individually with alanine. Seven mutant proteins were fully active; Cma(P129A), Cma(P176A), and Cma(P260A) displayed 1%, and Cma(P107A) displayed 10% of the wild-type activity. Cma(P107A), Cma(P129A), and Cma(P260A), but not Cma(P176A), killed cells after entering the periplasm via osmotic shock, indicating that the former mutants were translocation-deficient; Cma(P129A) did not bind to the FhuA outer membrane receptor. The crystal structures of Cma and Cma(P176A) were identical, excluding inactivation of the activity domain located far from Pro-176. In a new peptidyl prolyl cis-trans isomerase assay, FkpA isomerized the Cma prolyl bond in peptide Phe-Pro-176 at a high rate, but Lys-Pro-107 and Leu-Pro-260 isomerized at only <10% of that rate. The four mutant proteins secreted into the periplasm via a fused signal sequence were toxic but much less than wild-type Cma. Wild-type and mutant Cma proteins secreted or translocated across the outer membrane by energy-coupled import or unspecific osmotic shock were only active in the presence of FkpA. We propose that Cma unfolds during transfer across the outer or cytoplasmic membrane and refolds to the active form in the periplasm assisted by FkpA. Weak refolding of Cma(P176A) would explain its low activity in all assays. Of the four proline residues identified as being important for Cma activity, Phe-Pro-176 is most likely targeted by FkpA. Less
In Escherichia coli the -barrel assembly machinery or BAM complex mediates the recognition insertion and assembly of outer membrane proteins The complex consists of the integral membrane protein BamA an Omp -family member and the lipoproteins BamB BamC BamD and BamE The purification and crystallization of BamC BamD and BamE each lacking the N- terminal membrane anchor is described While the smallest protein BamE yielded crystals under conventional conditions BamD only crystallized after stabilization with urea Full-length BamC did not crystallize but was cleaved by subtilisin into two domains which were subsequently crystallized independently High-resolution data were acquired from all ... More
In Escherichia coli, the �-barrel assembly machinery (or BAM complex) mediates the recognition, insertion and assembly of outer membrane proteins. The complex consists of the integral membrane protein BamA (an Omp85-family member) and the lipoproteins BamB, BamC, BamD and BamE. The purification and crystallization of BamC, BamD and BamE, each lacking the N-�terminal membrane anchor, is described. While the smallest protein BamE yielded crystals under conventional conditions, BamD only crystallized after stabilization with urea. Full-length BamC did not crystallize, but was cleaved by subtilisin into two domains which were subsequently crystallized independently. High-resolution data were acquired from all proteins. Less
P II proteins control key processes of nitrogen metabolism in bacteria archaea and plants in response to the central metabolites ATP ADP and -oxoglutarate -OG signaling cellular energy and carbon and nitrogen abundance This metabolic information is integrated by P II and transmitted to regulatory targets key enzymes transporters and transcription factors modulating their activity In oxygenic phototrophs the controlling enzyme of arginine synthesis N-acetyl-glutamate kinase NAGK is a major P II target whose activity responds to -OG via P II Here we show structures of the Synechococcus elongatus P II protein in complex with ATP Mg and -OG which ... More
P II proteins control key processes of nitrogen metabolism in bacteria, archaea, and plants in response to the central metabolites ATP, ADP, and 2-oxoglutarate (2-OG), signaling cellular energy and carbon and nitrogen abundance. This metabolic information is integrated by P II and transmitted to regulatory targets (key enzymes, transporters, and transcription factors), modulating their activity. In oxygenic phototrophs, the controlling enzyme of arginine synthesis, N-acetyl-glutamate kinase (NAGK), is a major P II target, whose activity responds to 2-OG via P II . Here we show structures of the Synechococcus elongatus P II protein in complex with ATP, Mg??, and 2-OG, which clarify how 2-OG affects P II -NAGK interaction. P II trimers with all three sites fully occupied were obtained as well as structures with one or two 2-OG molecules per P II trimer. These structures identify the site of 2-OG located in the vicinity between the subunit clefts and the base of the T loop. The 2-OG is bound to a Mg?? ion, which is coordinated by three phosphates of ATP, and by ionic interactions with the highly conserved residues K58 and Q39 together with B- and T-loop backbone interactions. These interactions impose a unique T-loop conformation that affects the interactions with the P II target. Structures of P II trimers with one or two bound 2-OG molecules reveal the basis for anticooperative 2-OG binding and shed light on the intersubunit signaling mechanism by which P II senses effectors in a wide range of concentrations. Less
Fungal human pathogens such as Cryptococcus neoformans are becoming an increasingly prevalent cause of human morbidity and mortality owing to the increasing numbers of susceptible individuals The few antimycotics available to combat these pathogens usually target fungal-specific cell-wall or membrane-related components however the number of these targets is limited In the search for new targets and lead compounds C neoformans has been found to be susceptible to mycophenolic acid through its target inosine monophosphate dehydrogenase IMPDH in contrast a rare subtype of the related C gattii is naturally resistant Here the expression purification crystallization and preliminary crystallographic analysis of IMPDH ... More
Fungal human pathogens such as Cryptococcus neoformans are becoming an increasingly prevalent cause of human morbidity and mortality owing to the increasing numbers of susceptible individuals. The few antimycotics available to combat these pathogens usually target fungal-specific cell-wall or membrane-related components; however, the number of these targets is limited. In the search for new targets and lead compounds, C. neoformans has been found to be susceptible to mycophenolic acid through its target inosine monophosphate dehydrogenase (IMPDH); in contrast, a rare subtype of the related C. gattii is naturally resistant. Here, the expression, purification, crystallization and preliminary crystallographic analysis of IMPDH complexed with IMP and NAD+ is reported for both of these Cryptococcus species. The crystals of IMPDH from both sources had the symmetry of the tetragonal space group I422 and diffracted to a resolution of 2.5 Å for C. neoformans and 2.6 Å for C. gattii. Less
PII signal transduction proteins are highly conserved in bacteria archaea and plants and have key functions in coordination of central metabolism by integrating signals from the carbon nitrogen and energy status of the cell In the cyanobacterium Synechococcus elongatus PCC PII binds ATP and -oxoglutarate -OG in a synergistic manner with the ATP binding sites also accepting ADP Depending on its effector molecule binding status PII from this cyanobacterium and other oxygenic phototrophs complexes and regulates the arginine-controlled enzyme of the cyclic ornithine pathway N-acetyl-l-glutamate kinase NAGK to control arginine biosynthesis To gain deeper insights into the process of PII ... More
PII signal transduction proteins are highly conserved in bacteria, archaea and plants and have key functions in coordination of central metabolism by integrating signals from the carbon, nitrogen and energy status of the cell. In the cyanobacterium Synechococcus elongatus PCC 7942, PII binds ATP and 2-oxoglutarate (2-OG) in a synergistic manner, with the ATP binding sites also accepting ADP. Depending on its effector molecule binding status, PII (from this cyanobacterium and other oxygenic phototrophs) complexes and regulates the arginine-controlled enzyme of the cyclic ornithine pathway, N-acetyl-l-glutamate kinase (NAGK), to control arginine biosynthesis. To gain deeper insights into the process of PII binding to NAGK, we searched for PII variants with altered binding characteristics and found PII variants I86N and I86T to be able to bind to an NAGK variant (R233A) that was previously shown to be unable to bind wild-type PII protein. Analysis of interactions between these PII variants and wild-type NAGK as well as with the NAGK R233A variant suggested that the PII I86N variant was a superactive NAGK binder. To reveal the structural basis of this property, we solved the crystal structure of the PII I86N variant at atomic resolution. The large T-loop, which prevails in most receptor interactions of PII proteins, is present in a tightly bended conformation that mimics the T-loop of S. elongatus PII after having latched onto NAGK. Moreover, both PII I86 variants display a specific defect in 2-OG binding, implying a role of residue I86 in 2-OG binding. We propose a two-step model for the mechanism of PII–NAGK complex formation: in an initiating step, a contact between R233 of NAGK and E85 of PII initiates the bending of the extended T-loop of PII, followed by a second step, where a bended T-loop deeply inserts into the NAGK clefts to form the tight complex. Less
Pathogens require protein-folding enzymes to produce functional virulence determinants These foldases include the Dsb family of proteins which catalyze oxidative folding in bacteria Bacterial disulfide catalytic processes have been well characterized in Escherichia coli K- and these mechanisms have been extrapolated to other organisms However recent research indicates that the K- complement of Dsb proteins is not common to all bacteria Importantly many pathogenic bacteria have an extended arsenal of Dsb catalysts that is linked to their virulence To help to elucidate the process of oxidative folding in pathogens containing a wide repertoire of Dsb proteins Salmonella enterica serovar Typhimurium ... More
Pathogens require protein-folding enzymes to produce functional virulence determinants. These foldases include the Dsb family of proteins, which catalyze oxidative folding in bacteria. Bacterial disulfide catalytic processes have been well characterized in Escherichia coli K-12 and these mechanisms have been extrapolated to other organisms. However, recent research indicates that the K-�12 complement of Dsb proteins is not common to all bacteria. Importantly, many pathogenic bacteria have an extended arsenal of Dsb catalysts that is linked to their virulence. To help to elucidate the process of oxidative folding in pathogens containing a wide repertoire of Dsb proteins, Salmonella enterica serovar Typhimurium has been focused on. This Gram-negative bacterium contains three DsbA proteins: SeDsbA, SeDsbL and SeSrgA. Here, the expression, purification, crystallization and preliminary diffraction analysis of these three proteins are reported. SeDsbA, SeDsbL and SeSrgA crystals diffracted to resolution limits of 1.55, 1.57 and 2.6 � and belonged to space groups P21, P21212 and C2, respectively. Less
The environment of individual tryptophans in known protein structures and the effectiveness of four commercial robotic UV microscopes to illuminate tryptophan-containing protein crystals by either tryptophan fluorescence epi-illumination or absorbance transmission are evaluated In agreement with other studies tryptophan residues are found on average to be largely buried in protein structures with of their surface area buried and to be surrounded by partially polar microenvironments with of their surface area covered by polar residues which suggests an inherent degree of fluorescence signal quenching In bacterial genomes up to one-third on average of open reading frames are deficient in tryptophan In ... More
The environment of individual tryptophans in known protein structures and the effectiveness of four commercial robotic UV microscopes to illuminate tryptophan-containing protein crystals by either tryptophan fluorescence (epi-illumination) or absorbance (transmission) are evaluated. In agreement with other studies, tryptophan residues are found on average to be largely buried in protein structures (with ~84% of their surface area buried) and to be surrounded by partially polar microenvironments (with ~43% of their surface area covered by polar residues), which suggests an inherent degree of fluorescence signal quenching. In bacterial genomes, up to one-third (~18.5% on average) of open reading frames are deficient in tryptophan. In the laboratory, because of the attenuation of UV light by the media commonly used in sitting-drop and hanging-drop crystallization trials, it was often necessary to simplify the light path by manually removing or inverting the supporting media. Prolonged exposure (minutes) to UV light precipitates some protein samples. The absorbance spectra of many commercially available media in crystallization trials are presented. The advantages of using tryptophan absorbance over fluorescence for characterizing crystals are discussed. Less
Second order nonlinear optical imaging of chiral crystals SONICC is explored for selective detection of integral membrane protein crystals grown in opaque and turbid environments High turbidity is a hallmark of membrane protein crystallization due to the extensive use of detergent and or lipids that often form various mesophases Detection of crystals in such media by conventional optical methods e g intrinsic UV fluorescence birefringence bright-field image analysis etc is often complicated by optical scattering and by the small sizes of the crystals that routinely form SONICC is shown to be well-suited for this application by nature of its compatibility ... More
Second order nonlinear optical imaging of chiral crystals (SONICC) is explored for selective detection of integral membrane protein crystals grown in opaque and turbid environments. High turbidity is a hallmark of membrane protein crystallization due to the extensive use of detergent and/or lipids that often form various mesophases. Detection of crystals in such media by conventional optical methods (e.g., intrinsic UV fluorescence, birefringence, bright-field image analysis, etc.) is often complicated by optical scattering and by the small sizes of the crystals that routinely form. SONICC is shown to be well-suited for this application, by nature of its compatibility with imaging in scattering media and its high selectivity for protein crystals. Bright second harmonic generation (SHG) (up to 18 million counts/s) was observed from even relatively small crystals (5 micron) with a minimal background due to the surrounding lipid mesophase (~1 thousand counts/s). The low background nature of the resulting protein crystal images permitted the use of a relatively simple, particle counting analysis for preliminary scoring. Comparisons between a particle counting analysis of SONICC images and protocols based on the human expert analysis of conventional bright-field and birefringence images were performed. Less
The use of design of experiments DOE in assay development AD has the potential to speed up assay optimisation ie reduce assay development bottlenecks and to facilitate a more thorough evaluation of assay variables Only one liquid handling vendor currently offers application specific software and support for investigating DOE in biological assays Although standalone DOE software packages are available these were not written specifically for biological applications and they vary in their suitability for AD DOE needs to be simpler to implement to make a major impact on AD A market opportunity exists for a turnkey solution that directly links ... More
The use of design of experiments (DOE) in assay development (AD) has the potential to speed up assay optimisation (ie reduce assay development bottlenecks) and to facilitate a more thorough evaluation of assay variables. Only one liquid handling vendor currently offers application specific software and support for investigating DOE in biological assays. Although standalone DOE software packages are available, these were not written specifically for biological applications and they vary in their suitability for AD. DOE needs to be simpler to implement to make a major impact on AD. A market opportunity exists for a turnkey solution that directly links statistical design with automated liquid handler programming and also feeds the assay readout directly into the statistical analysis, to suggest and facilitate further iterative retesting. Until new tools or more encompassing solutions emerge, the full impact of DOE on AD is unlikely to be realised. Less
Background Protein crystallization screening involves the parallel testing of large numbers of candidate conditions with the aim of identifying conditions suitable as a starting point for the production of diffraction quality crystals Generally condition screening is performed in -well plates While previous studies have examined the effects of protein construct protein purity or crystallisation condition ingredients on protein crystallisation few have examined the effect of the crystallisation plate Methodology Principal Findings We performed a statistically rigorous examination of protein crystallisation and evaluated interactions between crystallisation success and plate row column different plates of same make different plate makes and different ... More
Background Protein crystallization screening involves the parallel testing of large numbers of candidate conditions with the aim of identifying conditions suitable as a starting point for the production of diffraction quality crystals. Generally, condition screening is performed in 96-well plates. While previous studies have examined the effects of protein construct, protein purity, or crystallisation condition ingredients on protein crystallisation, few have examined the effect of the crystallisation plate. Methodology/Principal Findings We performed a statistically rigorous examination of protein crystallisation, and evaluated interactions between crystallisation success and plate row/column, different plates of same make, different plate makes and different proteins. From our analysis of protein crystallisation, we found a significant interaction between plate make and the specific protein being crystallised. Conclusions/Significance Protein crystal structure determination is the principal method for determining protein structure but is limited by the need to produce crystals of the protein under study. Many important proteins are difficult to crystallise, so that identification of factors that assist crystallisation could open up the structure determination of these more challenging targets. Our findings suggest that protein crystallisation success may be improved by matching a protein with its optimal plate make. Less
T-cell receptors TCRs are membrane proteins which recognize antigens with high specificity forming the basis of the cellular immune response The study of these receptors has been limited by the challenges in expressing sufficient quantities of stable soluble protein Here we report our systematic approach for generating soluble -TCRs for X-ray crystallographic studies By using small-scale expression screens novel standardized quality control mechanisms and crystallization and imaging robots we were able to add significantly to the current TCR structural database Our success in crystallizing both isolated TCRs and Major histocompatibility complex MHC TCR complexes has provided us with sufficient data ... More
T-cell receptors (TCRs) are membrane proteins which recognize antigens with high specificity forming the basis of the cellular immune response. The study of these receptors has been limited by the challenges in expressing sufficient quantities of stable soluble protein. Here we report our systematic approach for generating soluble, αβ-TCRs, for X-ray crystallographic studies. By using small-scale expression screens, novel standardized quality control mechanisms and crystallization and imaging robots we were able to add significantly to the current TCR structural database. Our success in crystallizing both isolated TCRs and Major histocompatibility complex (MHC):TCR complexes has provided us with sufficient data to develop focused crystallization screens, which have proved generically useful for the crystallization of this family of proteins and complexes. Less
RNA silencing is a conserved regulatory mechanism in fungi plants and animals that regulates gene expression and defence against viruses and transgenes Small silencing RNAs of nucleotides and their associated effector proteins the Argonaute family proteins are the central components in RNA silencing A subset of small RNAs such as microRNAs and small interfering RNAs siRNAs in plants Piwi-interacting RNAs in animals and siRNAs in Drosophila requires an additional crucial step for their maturation that is '-O-methylation on the ' terminal nucleotide A conserved S-adenosyl-l-methionine-dependent RNA methyltransferase HUA ENHANCER HEN and its homologues are responsible for this specific modification Here ... More
RNA silencing is a conserved regulatory mechanism in fungi, plants and animals that regulates gene expression and defence against viruses and transgenes1. Small silencing RNAs of ~20�30 nucleotides and their associated effector proteins, the Argonaute family proteins, are the central components in RNA silencing2. A subset of small RNAs, such as microRNAs and small interfering RNAs (siRNAs) in plants, Piwi-interacting RNAs in animals and siRNAs in Drosophila, requires an additional crucial step for their maturation; that is, 2'-O-methylation on the 3' terminal nucleotide3�6. A conserved S-adenosyl-l-methionine-dependent RNA methyltransferase, HUA ENHANCER 1 (HEN1), and its homologues are responsible for this specific modification3�5,7,8. Here we report the 3.1 � crystal structure of full-length HEN1 from Arabidopsis in complex with a 22-nucleotide small RNA duplex and cofactor product S-adenosyl-l-homocysteine. Highly cooperative recognition of the small RNA substrate by multiple RNA binding domains and the methyltransferase domain in HEN1 measures the length of the RNA duplex and determines the substrate specificity. Metal ion coordination by both 2' and 3' hydroxyls on the 3'-terminal nucleotide and four invariant residues in the active site of the methyltransferase domain suggests a novel Mg2+-dependent 2'-O-methylation mechanism. Less
Crystallization of human membrane proteins in lipidic cubic phase often results in very small but highly ordered crystals Advent of the sub- m minibeam at the APS GM CA CAT has enabled the collection of high quality diffraction data from such microcrystals Herein we describe the challenges and solutions related to growing manipulating and collecting data from optically invisible microcrystals embedded in an opaque frozen in meso material Of critical importance is the use of the intense and small synchrotron beam to raster through and locate the crystal sample in an efficient and reliable manner The resulting diffraction patterns have ... More
Crystallization of human membrane proteins in lipidic cubic phase often results in very small but highly ordered crystals. Advent of the sub-10 �m minibeam at the APS GM/CA CAT has enabled the collection of high quality diffraction data from such microcrystals. Herein we describe the challenges and solutions related to growing, manipulating and collecting data from optically invisible microcrystals embedded in an opaque frozen in meso material. Of critical importance is the use of the intense and small synchrotron beam to raster through and locate the crystal sample in an efficient and reliable manner. The resulting diffraction patterns have a significant reduction in background, with strong intensity and improvement in diffraction resolution compared with larger beam sizes. Three high-resolution structures of human G protein-coupled receptors serve as evidence of the utility of these techniques that will likely be useful for future structural determination efforts. We anticipate that further innovations of the technologies applied to microcrystallography will enable the solving of structures of ever more challenging targets. Less
A detailed protocol for crystallizing membrane proteins that makes use of lipidic mesophases is described This has variously been referred to as the lipid cubic phase or in meso method The method has been shown to be quite general in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins proteins that are monomeric homo- and hetero-multimeric chromophore-containing and chromophore-free and a-helical and -barrel proteins Its most recent successes are the human engineered -adrenergic and adenosine A A G protein-coupled receptors Protocols are provided for preparing and characterizing the lipidic mesophase for reconstituting the protein ... More
A detailed protocol for crystallizing membrane proteins that makes use of lipidic mesophases is described. This has variously been referred to as the lipid cubic phase or in meso method. The method has been shown to be quite general in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and a-helical and �-barrel proteins. Its most recent successes are the human engineered �2-adrenergic and adenosine A2A G protein-coupled receptors. Protocols are provided for preparing and characterizing the lipidic mesophase, for reconstituting the protein into the monoolein-based mesophase, for functional assay of the protein in the mesophase, and for setting up crystallizations in manual mode. Methods for harvesting micro-crystals are also described. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour. Less
VERNALIZATION VRN is required in the model plant Arabidopsis thaliana for the epigenetic suppression of the floral repressor FLC by prolonged cold treatment Stable suppression of FLC accelerates flowering a physiological process known as vernalization VRN is a -residue DNA-binding protein that contains two plant-specific B domains B a and B b a putative nuclear localization sequence NLS and two putative PEST domains VRN includes the second B domain and a region upstream that is highly conserved in the VRN orthologues of other dicotyledonous plants VRN was crystallized by the hanging-drop method in M sodium acetate pH containing M NaCl ... More
VERNALIZATION1 (VRN1) is required in the model plant Arabidopsis thaliana for the epigenetic suppression of the floral repressor FLC by prolonged cold treatment. Stable suppression of FLC accelerates flowering, a physiological process known as vernalization. VRN1 is a 341-residue DNA-binding protein that contains two plant-specific B3 domains (B3a and B3b), a putative nuclear localization sequence (NLS) and two putative PEST domains. VRN1208�341 includes the second B3 domain and a region upstream that is highly conserved in the VRN1 orthologues of other dicotyledonous plants. VRN1208�341 was crystallized by the hanging-drop method in 0.05 M sodium acetate pH 6.0 containing 1.0 M NaCl and 18%(w/v) PEG 3350. Preliminary X-ray diffraction data analysis revealed that the VRN1208�341 crystal diffracted to 2.1 � and belonged to space group C2, with unit-cell parameters a = 105.2, b = 47.9, c = 61.2 �, a = 90.0, � = 115.4, ? = 90.0�. Assuming that two molecules occupy the asymmetric unit, a Matthews coefficient of 2.05 �3 Da-1 and a solvent content of 40.1% were calculated. Less
The stimulatory RNA of the Visna-Maedi virus VMV - ribosomal frameshifting signal has not previously been characterized but can be modeled either as a two-stem helix reminiscent of the HIV- frameshift-stimulatory RNA or as an RNA pseudoknot The pseudoknot is unusual in that it would include a nucleotide loop termed here an interstem element ISE between the two stems In almost all frameshift-promoting pseudoknots ISEs are absent or comprise a single adenosine residue Using a combination of RNA structure probing site directed mutagenesis NMR and phylogenetic sequence comparisons we show here that the VMV stimulatory RNA is indeed a pseudoknot ... More
The stimulatory RNA of the Visna-Maedi virus (VMV) -1 ribosomal frameshifting signal has not previously been characterized but can be modeled either as a two-stem helix, reminiscent of the HIV-1 frameshift-stimulatory RNA, or as an RNA pseudoknot. The pseudoknot is unusual in that it would include a 7 nucleotide loop (termed here an interstem element [ISE]) between the two stems. In almost all frameshift-promoting pseudoknots, ISEs are absent or comprise a single adenosine residue. Using a combination of RNA structure probing, site directed mutagenesis, NMR, and phylogenetic sequence comparisons, we show here that the VMV stimulatory RNA is indeed a pseudoknot, conforming closely to the modeled structure, and that the ISE is essential for frameshifting. Pseudoknot function was predictably sensitive to changes in the length of the ISE, yet altering its sequence to alternate pyrimidine/purine bases was also detrimental to frameshifting, perhaps through modulation of local tertiary interactions. How the ISE is placed in the context of an appropriate helical junction conformation is not known, but its presence impacts on other elements of the pseudoknot, for example, the necessity for a longer than expected loop 1. This may be required to accommodate an increased flexibility of the pseudoknot brought about by the ISE. In support of this, 1H NMR analysis at increasing temperatures revealed that stem 2 of the VMV pseudoknot is more labile than stem 1, perhaps as a consequence of its connection to stem 1 solely via flexible single-stranded loops. Less
In this article we describe for the first time the high-resolution crystal structure of a phenylalanine tRNA synthetase from the pathogenic bacterium Staphylococcus haemolyticus We demonstrate the subtle yet important structural differences between this enzyme and the previously described Thermus thermophilus ortholog We also explain the structure-activity relationship of several recently reported inhibitors The native enzyme crystals were of poor quality they only diffracted X-rays to resolution Therefore we have executed a rational surface mutagenesis strategy that has yielded crystals of this -amino acid multidomain protein diffracting to or better This methodology is discussed and contrasted with the more traditional ... More
In this article, we describe for the first time the high-resolution crystal structure of a phenylalanine tRNA synthetase from the pathogenic bacterium Staphylococcus haemolyticus. We demonstrate the subtle yet important structural differences between this enzyme and the previously described Thermus thermophilus ortholog. We also explain the structure-activity relationship of several recently reported inhibitors. The native enzyme crystals were of poor quality—they only diffracted X-rays to 3–5 Å resolution. Therefore, we have executed a rational surface mutagenesis strategy that has yielded crystals of this 2300-amino acid multidomain protein, diffracting to 2 Å or better. This methodology is discussed and contrasted with the more traditional domain truncation approach. Less
Plastic microchannel crystallization template designs made from inexpensive cyclic olefin copolymers have been shown to be low-birefringent X-ray transmissive and compatible with microfluidic fabrication in restricted geometry The model proteins thaumatin lysozyme and bacteriorhodopsin demonstrated the feasibility of conducting counter-diffusion equilibration within the new plastic configuration Crystals of each of these proteins were directly evaluated in situ using synchrotron radiation and their diffraction quality was evaluated without invasive manipulation or cryofreezing Protein crystals able to produce complete X-ray data sets were used to calculate electron-density maps for structure determination Fluidic crystallization in the plastic platform was also coupled with a ... More
Plastic microchannel crystallization template designs made from inexpensive cyclic olefin copolymers have been shown to be low-birefringent, X-ray transmissive and compatible with microfluidic fabrication in restricted geometry. The model proteins thaumatin, lysozyme and bacteriorhodopsin demonstrated the feasibility of conducting counter-diffusion equilibration within the new plastic configuration. Crystals of each of these proteins were directly evaluated in situ using synchrotron radiation and their diffraction quality was evaluated without invasive manipulation or cryofreezing. Protein crystals able to produce complete X-ray data sets were used to calculate electron-density maps for structure determination. Fluidic crystallization in the plastic platform was also coupled with a commercialized automated imager and an in situ X-ray scanner that allowed optical and X-ray inspection of crystallization hits. The results demonstrate the feasibility of rapid nanovolume counter-diffusion crystallization experiments without the need for additional instrumentation. Less
Protein disorder can plague protein crystallography where the first important goal is to identify conditions that grow stable ordered and well diffracting crystals Initial crystal trials for protease X produced numerous crystals in a myriad of conditions many of which did not produce well diffracting crystals Optimizing all of the most obvious parameters pH temperature salt and precipitant concentration continued to produce poor crystals The fast growth rate and consistently poor diffraction gave an indication that the protein may have some stability issues that could stem from the choice of storage buffer Recent studies have shown that incorporating thermal melting ... More
"Protein disorder can plague protein crystallography where the first important goal is to identify conditions that grow stable, ordered, and well diffracting crystals. Initial crystal trials for protease X produced numerous crystals in a myriad of conditions; many of which did not produce well diffracting crystals. Optimizing all of the most obvious parameters: pH, temperature, salt and precipitant concentration continued to produce poor crystals. The fast growth rate and consistently poor diffraction gave an indication that the protein may have some stability issues that could stem from the choice of storage buffer. Recent studies have shown that incorporating thermal melting (Tm) assays into the crystallization screening experiments completely opens up a greater opportunity to explore the protein?s stability prior to crystal trials. This poster will look at the use of thermal shift and the use of automation to screen, characterize and optimize poorly diffracting crystals to well ordered crystals diffracting to beyond 2 angstrom resolution" Less
Proteins of the cradle-loop barrel metafold are formed by duplication of a conserved -element suggesting a common evolutionary origin from an ancestral group of nucleic acid-binding proteins The basal fold within this metafold the RIFT barrel is also found in a wide range of enzymes whose homologous relationship with the nucleic acid-binding group is unclear We have characterized a protein family that is intermediate in sequence and structure between the basal group of cradle-loop barrels and one family of RIFT-barrel enzymes the riboflavin kinases We report the structure substrate-binding mode and catalytic activity for one of these proteins Methanocaldococcus jannaschii ... More
Proteins of the cradle-loop barrel metafold are formed by duplication of a conserved βαβ-element, suggesting a common evolutionary origin from an ancestral group of nucleic acid-binding proteins. The basal fold within this metafold, the RIFT barrel, is also found in a wide range of enzymes, whose homologous relationship with the nucleic acid-binding group is unclear. We have characterized a protein family that is intermediate in sequence and structure between the basal group of cradle-loop barrels and one family of RIFT-barrel enzymes, the riboflavin kinases. We report the structure, substrate-binding mode, and catalytic activity for one of these proteins, Methanocaldococcus jannaschii Mj0056, which is an archaeal riboflavin kinase. Mj0056 is unusual in utilizing CTP rather than ATP as the donor nucleotide, and sequence conservation in the relevant residues suggests that this is a general feature of archaeal riboflavin kinases. Less
Bacterial over-expression of proteins is a powerful tool to obtain soluble protein amenable to biochemical biophysical and or structural characterization However it is well established that many recombinant proteins cannot be produced in a soluble form Several theoretical and empirical methods to improve soluble production have been suggested although there is to date no universally accepted protocol This report describes and quantitatively analyses a systematic multi-construct approach to obtain soluble protein Although commonly used in several laboratories quantitative analyses of the merits of the strategy applied to a larger number of target proteins are missing from the literature In this ... More
Bacterial over-expression of proteins is a powerful tool to obtain soluble protein amenable to biochemical, biophysical and/or structural characterization. However, it is well established that many recombinant proteins cannot be produced in a soluble form. Several theoretical and empirical methods to improve soluble production have been suggested, although there is to date no universally accepted protocol. This report describes, and quantitatively analyses, a systematic multi-construct approach to obtain soluble protein. Although commonly used in several laboratories, quantitative analyses of the merits of the strategy applied to a larger number of target proteins are missing from the literature. In this study, typically 10 different protein constructs were tested for each targeted domain of nearly 400 human proteins. Overall, soluble expression was obtained for nearly 50% of the human target proteins upon over-expression in Escherichia coli. The chance of obtaining soluble expression was almost doubled using the multi-construct method as compared to more traditional approaches. Soluble protein constructs were subsequently subjected to crystallization trials and the multi-construct approach yielded a more than fourfold increase, from 15 proteins to 65, for the likelihood of obtaining well-diffracting crystals. The results also demonstrate the value of testing multiple constructs in crystallization trials. Finally, a retrospective analysis of gel filtration profiles indicates that these could be used with caution to prioritize protein targets for crystallization trials. Less
A microfluidic device denoted the Phase Chip has been designed to measure and manipulate the phase diagram of multi-component fluid mixtures The Phase Chip exploits the permeation of water through poly dimethylsiloxane PDMS in order to controllably vary the concentration of solutes in aqueous nanoliter volume microdrops stored in wells The permeation of water in the Phase Chip is modeled using the diffusion equation and good agreement between experiment and theory is obtained The Phase Chip operates by first creating drops of the water solute mixture whose composition varies sequentially Next drops are transported down channels and guided into storage ... More
A microfluidic device denoted the Phase Chip has been designed to measure and manipulate the phase diagram of multi-component fluid mixtures. The Phase Chip exploits the permeation of water through poly(dimethylsiloxane) (PDMS) in order to controllably vary the concentration of solutes in aqueous nanoliter volume microdrops stored in wells. The permeation of water in the Phase Chip is modeled using the diffusion equation and good agreement between experiment and theory is obtained. The Phase Chip operates by first creating drops of the water/solute mixture whose composition varies sequentially. Next, drops are transported down channels and guided into storage wells using surface tension forces. Finally, the solute concentration of each stored drop is simultaneously varied and measured. Two applications of the Phase Chip are presented. First, the phase diagram of a polymer/salt mixture is measured on-chip and validated off-chip and second, protein crystallization rates are enhanced through the manipulation of the kinetics of nucleation and growth. Less
The structure of human inosine triphosphate pyrophosphohydrolase ITPA has been determined using diffraction data to resolution ITPA contributes to the accurate replication of DNA by cleansing cellular dNTP pools of mutagenic nucleotide purine analogs such as dITP or dXTP A similar high-resolution unpublished structure has been deposited in the Protein Data Bank from a monoclinic and pseudo-merohedrally twinned crystal Here cocrystallization of ITPA with a molar ratio of XTP appears to have improved the crystals by eliminating twinning and resulted in an orthorhombic space group However there was no evidence for bound XTP in the structure Comparison with substrate-bound NTPase ... More
The structure of human inosine triphosphate pyrophosphohydrolase (ITPA) has been determined using diffraction data to 1.6 � resolution. ITPA contributes to the accurate replication of DNA by cleansing cellular dNTP pools of mutagenic nucleotide purine analogs such as dITP or dXTP. A similar high-resolution unpublished structure has been deposited in the Protein Data Bank from a monoclinic and pseudo-merohedrally twinned crystal. Here, cocrystallization of ITPA with a molar ratio of XTP appears to have improved the crystals by eliminating twinning and resulted in an orthorhombic space group. However, there was no evidence for bound XTP in the structure. Comparison with substrate-bound NTPase from a thermophilic organism predicts the movement of residues within helix a1, the loop before a6 and helix a7 to cap off the active site when substrate is bound. Less
Crystals of the apo-form of the vitamin B and colicin transporter BtuB that diffract to have been grown by the membrane-based in meso technique The structure of the protein differs in several details from that of its counterpart grown by the more traditional detergent-based in surfo method Some of these differences include i the five N-terminal residues are resolved in meso ii residues in the hatch domain and residues in loop are disordered in meso and are ordered in surfo iii residues in loop are resolved in meso iv residues in loop in loop in loop and in loop have ... More
Crystals of the apo-form of the vitamin B12 and colicin transporter, BtuB, that diffract to 1.95 Å have been grown by the membrane-based in meso technique. The structure of the protein differs in several details from that of its counterpart grown by the more traditional, detergent-based (in surfo) method. Some of these differences include i) the five N-terminal residues are resolved in meso, ii) residues 57–62 in the hatch domain and residues 574–581 in loop 21–22 are disordered in meso and are ordered in surfo, iii) residues 278–287 in loop 7–8 are resolved in meso, iv) residues 324–331 in loop 9–10, 396–411 in loop 13–14, 442–458 in loop 15–16 and 526–541 in loop 19–20 have large differences in position between the two crystal forms, as have residues 86–96 in the hatch domain, and v) the conformation of residues 6 and 7 in the Ton box (considered critical to signal transduction and substrate transport) are entirely different in the two structures. Importantly, the in meso orientation of residues 6 and 7 is similar to that of the vitamin B12-charged state. These data suggest that the 'substrate-induced' 180-degree rotation of residues 6 and 7 reported in the literature may not be a unique signaling event. The extent to which these findings agree with structural, dynamic and functional insights gleaned from site-directed spin labeling and electron paramagnetic resonance measurements is evaluated. Packing in in meso-grown crystals is dense and layered, consistent with the current model for crystallogenesis of membrane proteins in lipidic mesophases. Layered packing has been used to locate the transmembrane hydrophobic surface of the protein. Generally, this is consistent with tryptophan, tyrosine, lipid and Cα-B-factor distributions in the protein, and with predictions based on transfer free energy calculations. Less
The cubic phase or in meso crystallization method is responsible for almost solved integral membrane protein structures Most of these are small and compact proteins A model for how crystals form by the in meso method has been proposed that invokes a transition between mesophases In light of this model we speculated that a more hydrated and open mesophase of reduced interfacial curvature would support facile crystallization of bigger and bulkier proteins The proposal was explored here by performing crystallization in the presence of additives that swell the cubic phase The additive concentration inducing swelling as quantified by small-angle X-ray ... More
The cubic phase or in meso crystallization method is responsible for almost 40 solved integral membrane protein structures. Most of these are small and compact proteins. A model for how crystals form by the in meso method has been proposed that invokes a transition between mesophases. In light of this model, we speculated that a more hydrated and open mesophase, of reduced interfacial curvature, would support facile crystallization of bigger and bulkier proteins. The proposal was explored here by performing crystallization in the presence of additives that swell the cubic phase. The additive concentration inducing swelling, as quantified by small-angle X-ray diffraction, coincided with a "crystallization window" in which two, very different transmembranal proteins produced crystals. That the swollen mesophase can grow structure-grade crystals was proven with one of these, the light-harvesting II complex. In most regards, the structural details of the corresponding complex resembled those of crystals grown by the conventional vapour diffusion method, with some important differences. In particular, packing density in the in meso-grown crystals was dramatically higher, more akin to that seen with water-soluble proteins, which accounts for their enhanced diffracting power. The layered and close in-plane packing observed has been rationalized in a model for nucleation and crystal growth by the in meso method that involves swollen mesophases. These results present a rational case for including mesophase-swelling additives in screens for in meso crystallogenesis. Their use will contribute to broadening the range of membrane proteins that yield to structure determination. Less
Protein crystallization is a difficult and time-consuming task because to obtain a crystal optimization steps are required almost systematically A tool that simplifies the optimization of crystallization conditions and that can be used by any crystallographer to design a crystallization plate and to visualize its content has become a paramount necessity A free and open-source application has been developed to automate this task It is based on a graphical user interface GUI that allows a personalized crystallization plate to be designed All data used and generated are saved in XML documents which allow reuse of the information The steps involved ... More
Protein crystallization is a difficult and time-consuming task, because to obtain a
crystal, optimization steps are required almost systematically. A tool that
simplifies the optimization of crystallization conditions, and that can be used by
any crystallographer to design a crystallization plate and to visualize its content,
has become a paramount necessity. A free and open-source application has been
developed to automate this task. It is based on a graphical user interface (GUI)
that allows a personalized crystallization plate to be designed. All data used and
generated are saved in XML documents, which allow reuse of the information.
The steps involved in preparing a crystallization plate and the functions of the
GUI designed to perform these steps are described. Plans for future
development are presented. The program was written in Java. The application
and its documentation are available under CeCILL license, which is a Free
Software license agreement. Less
crystal, optimization steps are required almost systematically. A tool that
simplifies the optimization of crystallization conditions, and that can be used by
any crystallographer to design a crystallization plate and to visualize its content,
has become a paramount necessity. A free and open-source application has been
developed to automate this task. It is based on a graphical user interface (GUI)
that allows a personalized crystallization plate to be designed. All data used and
generated are saved in XML documents, which allow reuse of the information.
The steps involved in preparing a crystallization plate and the functions of the
GUI designed to perform these steps are described. Plans for future
development are presented. The program was written in Java. The application
and its documentation are available under CeCILL license, which is a Free
Software license agreement. Less