Imaging active site chemistry and protonation states: NMR crystallography of the tryptophan synthase α-aminoacrylate intermediate.

NMR-assisted crystallography-the integrated application of solid-state NMR, X-ray crystallography, and first-principles computational chemistry-holds significant promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates in enzyme active sites, including hydrogen atom locations and tautomeric equilibria, NMR crystallography offers insight into both structure and chemical dynamics. Here, this integrated approach is used to characterize the tryptophan synthase α-aminoacrylate intermediate, a defining species for pyridoxal-5'-phosphate-dependent enzymes that catalyze ß-elimination and replacement reactions. For this intermediate, NMR-assisted crystallography is able to identify the protonation states of the ionizable sites on the cofactor, substrate, and catalytic side chains as well as the location and orientation of crystallographic waters within the active site. Most notable is the water molecule immediately adjacent to the substrate ß-carbon, which serves as a hydrogen bond donor to the ε-amino group of the acid-base catalytic residue ßLys87. From this analysis, a detailed three-dimensional picture of structure and reactivity emerges, highlighting the fate of the L-serine hydroxyl leaving group and the reaction pathway back to the preceding transition state. Reaction of the α-aminoacrylate intermediate with benzimidazole, an isostere of the natural substrate indole, shows benzimidazole bound in the active site and poised for, but unable to initiate, the subsequent bond formation step. When modeled into the benzimidazole position, indole is positioned with C3 in contact with the α-aminoacrylate Cß and aligned for nucleophilic attack. Here, the chemically detailed, three-dimensional structure from NMR-assisted crystallography is key to understanding why benzimidazole does not react, while indole does.

Structural basis of the XPB helicase-Bax1 nuclease complex interacting with the repair bubble DNA.

Nucleotide excision repair (NER) removes various DNA lesions caused by UV light and chemical carcinogens. The DNA helicase XPB plays a key role in DNA opening and coordinating damage incision by nucleases during NER, but the underlying mechanisms remain unclear. Here, we report crystal structures of XPB from Sulfurisphaera tokodaii (St) bound to the nuclease Bax1 and their complex with a bubble DNA having one arm unwound in the crystal. StXPB and Bax1 together spirally encircle 10 base pairs of duplex DNA at the double-/single-stranded (ds-ss) junction. Furthermore, StXPB has its ThM motif intruding between the two DNA strands and gripping the 3'-overhang while Bax1 interacts with the 5'-overhang. This ternary complex likely reflects the state of repair bubble extension by the XPB and nuclease machine. ATP binding and hydrolysis by StXPB could lead to a spiral translocation along dsDNA and DNA strand separation by the ThM motif, revealing an unconventional DNA unwinding mechanism. Interestingly, the DNA is kept away from the nuclease domain of Bax1, potentially preventing DNA incision by Bax1 during repair bubble extension.

Structural basis of the XPB-Bax1 complex as a dynamic helicase-nuclease machinery for DNA repair.

Nucleotide excision repair (NER) is a major DNA repair pathway for a variety of DNA lesions. XPB plays a key role in DNA opening at damage sites and coordinating damage incision by nucleases. XPB is conserved from archaea to human. In archaea, XPB is associated with a nuclease Bax1. Here we report crystal structures of XPB in complex with Bax1 from Archaeoglobus fulgidus (Af) and Sulfolobus tokodaii (St). These structures reveal for the first time four domains in Bax1, which interacts with XPB mainly through its N-terminal domain. A Cas2-like domain likely helps to position Bax1 at the forked DNA allowing the nuclease domain to incise one arm of the fork. Bax1 exists in monomer or homodimer but forms a heterodimer exclusively with XPB. StBax1 keeps StXPB in a closed conformation and stimulates ATP hydrolysis by XPB while AfBax1 maintains AfXPB in the open conformation and reduces its ATPase activity. Bax1 contains two distinguished nuclease active sites to presumably incise DNA damage. Our results demonstrate that protein-protein interactions regulate the activities of XPB ATPase and Bax1 nuclease. These structures provide a platform to understand the XPB-nuclease interactions important for the coordination of DNA unwinding and damage incision in eukaryotic NER.

Mutation of ßGln114 to Ala Alters the Stabilities of Allosteric States in Tryptophan Synthase Catalysis.

The tryptophan synthase (TS) bienzyme complexes found in bacteria, yeasts, and molds are pyridoxal 5'-phosphate (PLP)-requiring enzymes that synthesize l-Trp. In the TS catalytic cycle, switching between the open and closed states of the α- and ß-subunits via allosteric interactions is key to the efficient conversion of 3-indole-d-glycerol-3'-phosphate and l-Ser to l-Trp. In this process, the roles played by ß-site residues proximal to the PLP cofactor have not yet been fully established. ßGln114 is one such residue. To explore the roles played by ßQ114, we conducted a detailed investigation of the ßQ114A mutation on the structure and function of tryptophan synthase. Initial steady-state kinetic and static ultraviolet-visible spectroscopic analyses showed the Q to A mutation impairs catalytic activity and alters the stabilities of intermediates in the ß-reaction. Therefore, we conducted X-ray structural and solid-state nuclear magnetic resonance spectroscopic studies to compare the wild-type and ßQ114A mutant enzymes. These comparisons establish that the protein structural changes are limited to the Gln to Ala replacement, the loss of hydrogen bonds among the side chains of ßGln114, ßAsn145, and ßArg148, and the inclusion of waters in the cavity created by substitution of the smaller Ala side chain. Because the conformations of the open and closed allosteric states are not changed by the mutation, we hypothesize that the altered properties arise from the lost hydrogen bonds that alter the relative stabilities of the open (ßT state) and closed (ßR state) conformations of the ß-subunit and consequently alter the distribution of intermediates along the ß-subunit catalytic path.

Toho-1 ß-lactamase: backbone chemical shift assignments and changes in dynamics upon binding with avibactam.

Backbone chemical shift assignments for the Toho-1 ß-lactamase (263 amino acids, 28.9 kDa) are reported based on triple resonance solution-state NMR experiments performed on a uniformly 2H,13C,15N-labeled sample. These assignments allow for subsequent site-specific characterization at the chemical, structural, and dynamical levels. At the chemical level, titration with the non-ß-lactam ß-lactamase inhibitor avibactam is found to give chemical shift perturbations indicative of tight covalent binding that allow for mapping of the inhibitor binding site. At the structural level, protein secondary structure is predicted based on the backbone chemical shifts and protein residue sequence using TALOS-N and found to agree well with structural characterization from X-ray crystallography. At the dynamical level, model-free analysis of 15N relaxation data at a single field of 16.4 T reveals well-ordered structures for the ligand-free and avibactam-bound enzymes with generalized order parameters of ~ 0.85. Complementary relaxation dispersion experiments indicate that there is an escalation in motions on the millisecond timescale in the vicinity of the active site upon substrate binding. The combination of high rigidity on short timescales and active site flexibility on longer timescales is consistent with hypotheses for achieving both high catalytic efficiency and broad substrate specificity: the induced active site dynamics allows variously sized substrates to be accommodated and increases the probability that the optimal conformation for catalysis will be sampled.

Backbone assignments and conformational dynamics in the S. typhimurium tryptophan synthase α-subunit from solution-state NMR.

Backbone assignments for the isolated α-subunit of Salmonella typhimurium tryptophan synthase (TS) are reported based on triple resonance solution-state NMR experiments on a uniformly 2H,13C,15N-labeled sample. From the backbone chemical shifts, secondary structure and random coil index order parameters (RCI-S2) are predicted. Titration with the 3-indole-D-glycerol 3'-phosphate analog, N-(4'-trifluoromethoxybenzenesulfonyl)-2-aminoethyl phosphate (F9), leads to chemical shift perturbations indicative of conformational changes from which an estimate of the dissociation constant is obtained. Comparisons of the backbone chemical-shifts, RCI-S2 values, and site-specific relaxation times with and without F9 reveal allosteric changes including modulation in secondary structures and loop rigidity induced upon ligand binding. A comparison is made to the X-ray crystal structure of the α-subunit in the full TS αßßα bi-enzyme complex and to two new X-ray crystal structures of the isolated TS α-subunit reported in this work.

The copper-inducible copAB operon in Xanthomonas citri subsp. citri is regulated at transcriptional and translational levels.

Upstream open reading frames (ORFs) are frequently found in the 5'-flanking regions of genes and may have a regulatory role in gene expression. A small ORF (named cohL here) was identified upstream from the copAB copper operon in Xanthomonascitri subsp. citri (Xac). We previously demonstrated that copAB expression was induced by copper and that gene inactivation produced a mutant strain that was unable to grow in the presence of copper. Here, we address the role of cohL in copAB expression control. We demonstrate that cohL expression is induced by copper in a copAB-independent manner. Although cohL is transcribed, the CohL protein is either not expressed in vivo or is synthesized at undetectable levels. Inactivation of cohL (X. citri cohL polar mutant strain) leads to an inability to synthesize cohL and copAB transcripts and consequently the inability to grow in the presence of copper. Bioinformatic tools predicted a stem-loop structure for the cohL-copAB intergenic region and revealed that this region may arrange itself in a secondary structure. Using in vitro gene expression, we found out that the structured 5'-UTR mRNA of copAB is responsible for sequestering the ribosome-binding site that drives the translation of copA. However, copper alone was not able to release the sequence. Based on the results, we speculate that cohL plays a role as a regulatory RNA rather than as a protein-coding gene.

Application of Electrochemical Devices to Characterize the Dynamic Actions of Helicases on DNA.

Much remains to be understood about the kinetics and thermodynamics of DNA helicase binding and activity. Here, we utilize probe-modified DNA monolayers on multiplexed gold electrodes as a sensitive recognition element and morphologically responsive transducer of helicase-DNA interactions. The electrochemical signals from these devices are highly sensitive to structural distortion of the DNA produced by the helicases. We used this DNA electrochemistry to distinguish the details of the DNA interactions of three distinct XPB helicases, which belong to the superfamily-2 of helicases. Clear changes in DNA melting temperature and duplex stability were observed upon helicase binding, shifts that could not be observed with conventional UV-visible absorption measurements. Binding dissociation constants were estimated in the range from 10 to 50 nM and correlated with observations of activity. ATP-stimulated DNA unwinding activity was also followed, revealing exponential time scales and distinct time constants associated with conventional and molecular wrench modes of operation further confirmed by crystal structures. These devices thus provide a sensitive measure of the structural thermodynamics and kinetics of helicase-DNA interactions.

Visualizing the tunnel in tryptophan synthase with crystallography: Insights into a selective filter for accommodating indole and rejecting water.

Four new X-ray structures of tryptophan synthase (TS) crystallized with varying numbers of the amphipathic N-(4'-trifluoromethoxybenzoyl)-2-aminoethyl phosphate (F6) molecule are presented. These structures show one of the F6 ligands threaded into the tunnel from the ß-site and reveal a distinct hydrophobic region. Over this expanse, the interactions between F6 and the tunnel are primarily nonpolar, while the F6 phosphoryl group fits into a polar pocket of the ß-subunit active site. Further examination of TS structures reveals that one portion of the tunnel (T1) binds clusters of water molecules, whereas waters are not observed in the nonpolar F6 binding region of the tunnel (T2). MD simulation of another TS structure with an unobstructed tunnel also indicates the T2 region of the tunnel excludes water, consistent with a dewetted state that presents a significant barrier to the transfer of water into the closed ß-site. We conclude that hydrophobic molecules can freely diffuse between the α- and ß-sites via the tunnel, while water does not. We propose that exclusion of water serves to inhibit reaction of water with the α-aminoacrylate intermediate to form ammonium ion and pyruvate, a deleterious side reaction in the αß-catalytic cycle. Finally, while most TS structures show ßPhe280 partially blocking the tunnel between the α- and ß-sites, new structures show an open tunnel, suggesting the flexibility of the ßPhe280 side chain. Flexible docking studies and MD simulations confirm that the dynamic behavior of ßPhe280 allows unhindered transfer of indole through the tunnel, therefore excluding a gating role for this residue.

Catalytic roles of ßLys87 in tryptophan synthase: (15)N solid state NMR studies.

The proposed mechanism for tryptophan synthase shows ßLys87 playing multiple catalytic roles: it bonds to the PLP cofactor, activates C4' for nucleophilic attack via a protonated Schiff base nitrogen, and abstracts and returns protons to PLP-bound substrates (i.e. acid-base catalysis). ε-¹5N-lysine TS was prepared to access the protonation state of ßLys87 using ¹5N solid-state nuclear magnetic resonance (SSNMR) spectroscopy for three quasi-stable intermediates along the reaction pathway. These experiments establish that the protonation state of the ε-amino group switches between protonated and neutral states as the ß-site undergoes conversion from one intermediate to the next during catalysis, corresponding to mechanistic steps where this lysine residue has been anticipated to play alternating acid and base catalytic roles that help steer reaction specificity in tryptophan synthase catalysis. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications. Guest Editors: Andrea Mozzarelli and Loredano Pollegioni.

NMR Crystallography of a Carbanionic Intermediate in Tryptophan Synthase: Chemical Structure, Tautomerization, and Reaction Specificity.

Carbanionic intermediates play a central role in the catalytic transformations of amino acids performed by pyridoxal-5'-phosphate (PLP)-dependent enzymes. Here, we make use of NMR crystallography-the synergistic combination of solid-state nuclear magnetic resonance, X-ray crystallography, and computational chemistry-to interrogate a carbanionic/quinonoid intermediate analogue in the ß-subunit active site of the PLP-requiring enzyme tryptophan synthase. The solid-state NMR chemical shifts of the PLP pyridine ring nitrogen and additional sites, coupled with first-principles computational models, allow a detailed model of protonation states for ionizable groups on the cofactor, substrates, and nearby catalytic residues to be established. Most significantly, we find that a deprotonated pyridine nitrogen on PLP precludes formation of a true quinonoid species and that there is an equilibrium between the phenolic and protonated Schiff base tautomeric forms of this intermediate. Natural bond orbital analysis indicates that the latter builds up negative charge at the substrate Cα and positive charge at C4' of the cofactor, consistent with its role as the catalytic tautomer. These findings support the hypothesis that the specificity for ß-elimination/replacement versus transamination is dictated in part by the protonation states of ionizable groups on PLP and the reacting substrates and underscore the essential role that NMR crystallography can play in characterizing both chemical structure and dynamics within functioning enzyme active sites.

Protonation states of the tryptophan synthase internal aldimine active site from solid-state NMR spectroscopy: direct observation of the protonated Schiff base linkage to pyridoxal-5'-phosphate.

The acid-base chemistry that drives catalysis in pyridoxal-5'-phosphate (PLP)-dependent enzymes has been the subject of intense interest and investigation since the initial identification of PLP's role as a coenzyme in this extensive class of enzymes. It was first proposed over 50 years ago that the initial step in the catalytic cycle is facilitated by a protonated Schiff base form of the holoenzyme in which the linking lysine ε-imine nitrogen, which covalently binds the coenzyme, is protonated. Here we provide the first (15)N NMR chemical shift measurements of such a Schiff base linkage in the resting holoenzyme form, the internal aldimine state of tryptophan synthase. Double-resonance experiments confirm the assignment of the Schiff base nitrogen, and additional (13)C, (15)N, and (31)P chemical shift measurements of sites on the PLP coenzyme allow a detailed model of coenzyme protonation states to be established.

Allostery and substrate channeling in the tryptophan synthase bienzyme complex: evidence for two subunit conformations and four quaternary states.

The allosteric regulation of substrate channeling in tryptophan synthase involves ligand-mediated allosteric signaling that switches the α- and ß-subunits between open (low activity) and closed (high activity) conformations. This switching prevents the escape of the common intermediate, indole, and synchronizes the α- and ß-catalytic cycles. (19)F NMR studies of bound α-site substrate analogues, N-(4'-trifluoromethoxybenzoyl)-2-aminoethyl phosphate (F6) and N-(4'-trifluoromethoxybenzenesulfonyl)-2-aminoethyl phosphate (F9), were found to be sensitive NMR probes of ß-subunit conformation. Both the internal and external aldimine F6 complexes gave a single bound peak at the same chemical shift, while α-aminoacrylate and quinonoid F6 complexes all gave a different bound peak shifted by +1.07 ppm. The F9 complexes exhibited similar behavior, but with a corresponding shift of -0.12 ppm. X-ray crystal structures show the F6 and F9 CF3 groups located at the α-ß subunit interface and report changes in both the ligand conformation and the surrounding protein microenvironment. Ab initio computational modeling suggests that the change in (19)F chemical shift results primarily from changes in the α-site ligand conformation. Structures of α-aminoacrylate F6 and F9 complexes and quinonoid F6 and F9 complexes show the α- and ß-subunits have closed conformations wherein access of ligands into the α- and ß-sites from solution is blocked. Internal and external aldimine structures show the α- and ß-subunits with closed and open global conformations, respectively. These results establish that ß-subunits exist in two global conformational states, designated open, where the ß-sites are freely accessible to substrates, and closed, where the ß-site portal into solution is blocked. Switching between these conformations is critically important for the αß-catalytic cycle.

Structure of the C-terminal half of human XPB helicase and the impact of the disease-causing mutation XP11BE.

XPB is a DNA-dependent helicase and a subunit of the TFIIH complex required for both transcription and DNA repair. XPB contains four domains: an N-terminal domain, two conserved helicase domains (HD1 and HD2) and a C-terminal extension. The C-terminal extension is important for DNA repair since the phosphorylation of Ser751 inhibits 5'-incision by ERCC1-XPF endonuclease. A disease-causing frameshift mutation (XP11BE) that changes the last 42 amino acids of XPB causes manifestations including impaired DNA repair and deficient transcription. Here, the crystal structure of the C-terminal half of XPB (residues 494-782) is reported at 1.8 Šresolution. The structure contained the conserved XPB HD2 and a C-terminal extension which shares structural similarity with RIG-I, leading to a structural model of the XPF-XPB-DNA complex for 5' incision during DNA repair. A mutation mimicking the XP11BE mutation produced the much less soluble mutant XPBm(494-781). Western blotting results confirmed that the intracellular levels of XPB and other TFIIH subunits in XP11BE patient cells were much lower than those from the healthy parents. Together, these results indicate that the XP11BE mutation not only divests the XPF-interaction motif, impairing DNA repair, but also reduces XPB solubility, leading to a lower intracellular level of TFIIH and deficient transcription.

The structure of a Xanthomonas general stress protein involved in citrus canker reveals its flavin-binding property.

Xanthomonas citri pv. citri (Xac) causes citrus canker and affects citrus agriculture worldwide. Functional genetic analysis has indicated that a putative general stress protein (XacGSP) encoded by the Xac2369 gene is involved in the bacterial infection. In this report, the crystal structure of XacGSP was determined to 2.5 Šresolution. There are four XacGSP molecules in the crystal asymmetric unit. Each XacGSP monomer folds into a six-stranded antiparallel ß-barrel flanked by five α-helices. A C-terminal extension protrudes from the sixth ß-strand of the ß-barrel and pairs with its counterpart from another monomer to form a bridge between the two subunits of an XacGSP dimer. Two XacGSP dimers cross over each other to form a tetramer; the ß-barrels from one dimer contact the ß-barrels of the other, while the two bridges are distant from each other and do not make contacts. The three-dimensional structure of the XacGSP monomer is very similar to those of pyridoxine 5-phosphate oxidases, a group of enzymes that use flavin mononucleotide (FMN) as a cofactor. Consistent with this, purified XacGSP protein binds to both FMN and flavin adenine dinucleotide (FAD), suggesting that XacGSP may help the bacteria to react against the oxidative stress induced by the defense mechanisms of the plant.

Allosteric regulation of substrate channeling: Salmonella typhimurium tryptophan synthase.

The regulation of the synthesis of L-tryptophan (L-Trp) in enteric bacteria begins at the level of gene expression where the cellular concentration of L-Trp tightly controls expression of the five enzymes of the Trp operon responsible for the synthesis of L-Trp. Two of these enzymes, trpA and trpB, form an αßßα bienzyme complex, designated as tryptophan synthase (TS). TS carries out the last two enzymatic processes comprising the synthesis of L-Trp. The TS α-subunits catalyze the cleavage of 3-indole D-glyceraldehyde 3'-phosphate to indole and D-glyceraldehyde 3-phosphate; the pyridoxal phosphate-requiring ß-subunits catalyze a nine-step reaction sequence to replace the L-Ser hydroxyl by indole giving L-Trp and a water molecule. Within αß dimeric units of the αßßα bienzyme complex, the common intermediate indole is channeled from the α site to the ß site via an interconnecting 25 Å-long tunnel. The TS system provides an unusual example of allosteric control wherein the structures of the nine different covalent intermediates along the ß-reaction catalytic path and substrate binding to the α-site provide the allosteric triggers for switching the αßßα system between the open (T) and closed (R) allosteric states. This triggering provides a linkage that couples the allosteric conformational coordinate to the covalent chemical reaction coordinates at the α- and ß-sites. This coupling drives the α- and ß-sites between T and R conformations to achieve regulation of substrate binding and/or product release, modulation of the α- and ß-site catalytic activities, prevention of indole escape from the confines of the active sites and the interconnecting tunnel, and synchronization of the α- and ß-site catalytic activities. Here we review recent advances in the understanding of the relationships between structure, function, and allosteric regulation of the complex found in Salmonella typhimurium.

Discovery of antimicrobial agent targeting tryptophan synthase.

Antibiotic resistance is a continually growing challenge in the treatment of various bacterial infections worldwide. New drugs and new drug targets are necessary to curb the threat of infectious diseases caused by multidrug-resistant pathogens. The tryptophan biosynthesis pathway is essential for bacterial growth but is absent in higher animals and humans. Drugs that can inhibit the bacterial biosynthesis of tryptophan offer a new class of antibiotics. In this work, we combined a structure-based strategy using in silico docking screening and molecular dynamics (MD) simulations to identify compounds targeting the α subunit of tryptophan synthase with experimental methods involving the whole-cell minimum inhibitory concentration (MIC) test, solution state NMR, and crystallography to confirm the inhibition of L-tryptophan biosynthesis. Screening 1,800 compounds from the National Cancer Institute Diversity Set I against α subunit revealed 28 compounds for experimental validation; four of the 28 hit compounds showed promising activity in MIC testing. We performed solution state NMR experiments to demonstrate that a one successful inhibitor, 3-amino-3-imino-2-phenyldiazenylpropanamide (Compound 1) binds to the α subunit. We also report a crystal structure of Salmonella enterica serotype Typhimurium tryptophan synthase in complex with Compound 1 which revealed a binding site at the αß interface of the dimeric enzyme. MD simulations were carried out to examine two binding sites for the compound. Our results show that this small molecule inhibitor could be a promising lead for future drug development.

Structural and biochemical characterization of a glutathione transferase from the citrus canker pathogen Xanthomonas.

The genus Xanthomonas comprises several cosmopolitan plant-pathogenic bacteria that affect more than 400 plant species, most of which are of economic interest. Citrus canker is a bacterial disease that affects citrus species, reducing fruit yield and quality, and is caused by the bacterium Xanthomonas citri subsp. citri (Xac). The Xac3819 gene, which has previously been reported to be important for citrus canker infection, encodes an uncharacterized glutathione S-transferase (GST) of 207 amino-acid residues in length (XacGST). Bacterial GSTs are implicated in a variety of metabolic processes such as protection against chemical and oxidative stresses. XacGST shares high sequence identity (45%) with the GstB dehalogenase from Escherichia coli O6:H1 strain CFT073 (EcGstB). Here, XacGST is reported to be able to conjugate glutathione (GSH) with bromoacetate with a Km of 6.67 ± 0.77 mM, a kcat of 42.69 ± 0.32 s-1 and a kcat/Km of 6.40 ± 0.72 mM-1 s-1 under a saturated GSH concentration (3.6 mM). These values are comparable to those previously reported for EcGstB. In addition, crystal structures of XacGST were determined in the apo form (PDB entry 6nxv) and in a GSH-bound complex (PDB entry 6nv6). XacGST has a canonical GST-like fold with a conserved serine residue (Ser12) at the GSH-binding site near the N-terminus, indicating XacGST to be a serine-type GST that probably belongs to the theta-class GSTs. GSH binding stabilizes a loop of about 20 residues containing a helix that is disordered in the apo XacGST structure.

PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase.

Structural studies with tryptophan synthase (TS) bienzyme complex (α2ß2 TS) from Salmonella typhimurium have been performed to better understand its catalytic mechanism, allosteric behavior, and details of the enzymatic transformation of substrate to product in PLP-dependent enzymes. In this work, a novel expression system to produce the isolated α- and isolated ß-subunit allowed the purification of high amounts of pure subunits and α2ß2 StTS complex from the isolated subunits within 2 days. Purification was carried out by affinity chromatography followed by cleavage of the affinity tag, ammonium sulfate precipitation, and size exclusion chromatography (SEC). To better understand the role of key residues at the enzyme ß-site, site-direct mutagenesis was performed in prior structural studies. Another protocol was created to purify the wild type and mutant α2ß2 StTS complexes. A simple, fast and efficient protocol using ammonium sulfate fractionation and SEC allowed purification of α2ß2 StTS complex in a single day. Both purification protocols described in this work have considerable advantages when compared with previous protocols to purify the same complex using PEG 8000 and spermine to crystalize the α2ß2 StTS complex along the purification protocol. Crystallization of wild type and some mutant forms occurs under slightly different conditions, impairing the purification of some mutants using PEG 8000 and spermine. To prepare crystals suitable for x-ray crystallographic studies several efforts were made to optimize crystallization, crystal quality and cryoprotection. The methods presented here should be generally applicable for purification of tryptophan synthase subunits and wild type and mutant α2ß2 StTS complexes.

Crystallization and preliminary X-ray diffraction analysis of XAC1151, a small heat-shock protein from Xanthomonas axonopodis pv. citri belonging to the alpha-crystallin family.

The hspA gene (XAC1151) from Xanthomonas axonopodis pv. citri encodes a protein of 158 amino acids that belongs to the small heat-shock protein (sHSP) family of proteins. These proteins function as molecular chaperones by preventing protein aggregation. The protein was crystallized using the sitting-drop vapour-diffusion method in the presence of ammonium phosphate. X-ray diffraction data were collected to 1.65 angstroms resolution using a synchrotron-radiation source. The crystal belongs to the rhombohedral space group R3, with unit-cell parameters a = b = 128.7, c = 55.3 angstroms. The crystal structure was solved by molecular-replacement methods. Structure refinement is in progress.