RÉSUMÉ
Most drugs used to treat viral disease target a virus-coded product. They inhibit a single virus or virus family, and the pathogen can readily evolve resistance. Host-targeted antivirals can overcome these limitations. The broad-spectrum activity achieved by host targeting can be especially useful in combating emerging viruses and for treatment of diseases caused by multiple viral pathogens, such as opportunistic agents in immunosuppressed patients. We have developed a family of compounds that modulate sirtuin 2, an NAD+-dependent deacylase, and now report the properties of a member of that family, FLS-359. Biochemical and x-ray structural studies show that the drug binds to sirtuin 2 and allosterically inhibits its deacetylase activity. FLS-359 inhibits the growth of RNA and DNA viruses, including members of the coronavirus, orthomyxovirus, flavivirus, hepadnavirus, and herpesvirus families. FLS-359 acts at multiple levels to antagonize cytomegalovirus replication in fibroblasts, causing modest reductions in viral RNAs and DNA, together with a much greater reduction in infectious progeny, and it exhibits antiviral activity in humanized mouse models of infection. Our results highlight the potential of sirtuin 2 inhibitors as broad-spectrum antivirals and set the stage for further understanding of how host epigenetic mechanisms impact the growth and spread of viral pathogens.
Sujet(s)
Infections à coronavirus , Coronavirus , Animaux , Souris , Antiviraux/pharmacologie , Sirtuine-2/génétique , ARN viralRÉSUMÉ
New therapeutic agents for cryptosporidiosis are a critical medical need. The marine organic compound, tartrolon E (trtE), is highly effective against multiple apicomplexan parasites, including Cryptosporidium. Understanding the mechanism of action of trtE is required to advance in the drug development pipeline. Here, we validate using Nluc C. parvum parasites for the study of trtE and pinpoint the life stage targeted by trtE. Results show that trtE kills Nluc and wild type C. parvum with equal efficiency, confirming the use of the Nluc C. parvum to study this compound. Results revealed that trtE kills the parasite within an hour of treatment and while the compound has no effect on viability of sporozoites, trtE does inhibit establishment of infection. Targeting treatment at particular life cycle stages demonstrated that trtE is effective against asexual of the parasite but has reduced efficacy against mature sexual stages. Gene expression analysis shows that trtE inhibits the early sexual stage of the parasite. Results from these studies will aid the development of trtE as a therapeutic for cryptosporidiosis.
RÉSUMÉ
Listeria monocytogenes is a foodborne pathogen responsible for about 1600 illnesses each year in the United States (US) and about 2500 confirmed invasive human cases in European Union (EU) countries. Several technologies and antimicrobials are applied to control the presence of L. monocytogenes in food. Among these, the use of natural antimicrobials is preferred by consumers. This is due to their ability to inhibit the growth of foodborne pathogens but not prompt negative safety concerns. Among natural antimicrobials, plant extracts are used to inactivate L. monocytogenes. However, there is a large amount of these types of extracts, and their active compounds remain unexplored. The aim of this study was to evaluate the antibacterial activity against L. monocytogenes of about 800 plant extracts derived from plants native to different countries worldwide. The minimal inhibitory concentrations (MICs) were determined, and scanning electron microscopy (SEM) was used to verify how the plant extracts affected L. monocytogenes at the microscopic level. Results showed that 12 of the plant extracts had inhibitory activity against L. monocytogenes. Future applications of this study could include the use of these plant extracts as new preservatives to reduce the risk of growth of pathogens and contamination in the food industry from L. monocytogenes.
RÉSUMÉ
Human carbonic anhydrase (CA) I and II are cytosolic proteins, where their expression disorders can cause diseases such as glaucoma, edema, epilepsy or cancer. There are numerous inhibitors that target these isozymes, but it is difficult to design compounds that could bind to one of these proteins specifically. The binding of sulfonamide inhibitor to a CA is linked to several protonation reactions, namely, deprotonation of the sulfonamide group, protonation of the active site zinc hydroxide and the compensating protonation-deprotonation of buffer. By performing binding experiments at various pHs and buffers, all those contributions were dissected and the "intrinsic" binding parameters were calculated. Intrinsic thermodynamic binding parameters to CA I and II were determined for such widely studied drugs as acetazolamide, ethoxzolamide, methazolamide, trifluoromethanesulfonamide and dichlorophenamide. The assignment of all contributions should enhance our understanding of the underlying energetics and increase our capability to design more potent and specific CA inhibitors.
Sujet(s)
Carbonic anhydrase II/antagonistes et inhibiteurs , Carbonic anhydrase I/antagonistes et inhibiteurs , Inhibiteurs de l'anhydrase carbonique/synthèse chimique , Sulfonamides/synthèse chimique , Carbonic anhydrase I/isolement et purification , Carbonic anhydrase II/isolement et purification , Inhibiteurs de l'anhydrase carbonique/composition chimique , Inhibiteurs de l'anhydrase carbonique/pharmacologie , Érythrocytes/enzymologie , Humains , Structure moléculaire , Liaison aux protéines , Protons , Relation structure-activité , Sulfonamides/composition chimique , Sulfonamides/pharmacologie , ThermodynamiqueRÉSUMÉ
Diacylglycerol acyltransferase (DGAT) catalyzes the final step in triglyceride (TG) synthesis. There are two isoforms, DGAT1 and DGAT2, with distinct protein sequences and potentially different physiological functions. To date, the ability to determine clear functional differences between DGAT1 and DGAT2, especially with respect to hepatic TG synthesis, has been elusive. To dissect the roles of these two key enzymes, we pretreated HepG2 hepatoma cells with (13)C(3)-D(5)-glycerol or (13)C(18)-oleic acid, and profiled the major isotope-labeled TG species by liquid chromatography tandem mass spectrometry. Selective DGAT1 and DGAT2 inhibitors demonstrated that (13)C(3)-D(5)-glycerol-incorporated TG synthesis was mediated by DGAT2, not DGAT1. Conversely, (13)C(18)-oleoyl-incorporated TG synthesis was predominantly mediated by DGAT1. To trace hepatic TG synthesis and VLDL triglyceride (VLDL-TG) secretion in vivo, we administered D(5)-glycerol to mice and measured plasma levels of D(5)-glycerol-incorporated TG. Treatment with an antisense oligonucleotide (ASO) to DGAT2 led to a significant reduction in D(5)-glycerol incorporation into VLDL-TG. In contrast, the DGAT2 ASO had no effect on the incorporation of exogenously administered (13)C(18)-oleic acid into VLDL-TG. Thus, our results indicate that DGAT1 and DGAT2 mediate distinct hepatic functions: DGAT2 is primarily responsible for incorporating endogenously synthesized FAs into TG, whereas DGAT1 plays a greater role in esterifying exogenous FAs to glycerol.
Sujet(s)
Diacylglycerol O-acyltransferase/métabolisme , Dosages enzymatiques/méthodes , Glycérol/métabolisme , Foie/enzymologie , Acide oléique/métabolisme , Animaux , Diacylglycerol O-acyltransferase/antagonistes et inhibiteurs , Diacylglycerol O-acyltransferase/génétique , Antienzymes/pharmacologie , Estérification/effets des médicaments et des substances chimiques , Acides gras/biosynthèse , Acides gras/métabolisme , Cellules HepG2 , Humains , Marquage isotopique , Lipoprotéines VLDL/métabolisme , Mâle , Souris , Oligonucléotides antisens/génétique , Triglycéride/biosynthèseRÉSUMÉ
Endocannabinoids such as 2-arachidonylglycerol (2-AG) are ligands for cannabinoid receptors that contribute to the transmission and modulation of pain signals. The antinociceptive effect of exogenous 2-AG suggests that inhibition of monoglyceride lipase (MGLL), the enzyme responsible for degrading 2-AG and arresting signaling, may be a target for pain modulation. Here we describe the characterization of MGLL ligands following a high-throughput screening campaign. Ligands were discovered using ThermoFluor, a label-free affinity-based screening tool that measures ligand binding via modulation of protein thermal stability. A kinetic fluorescent assay using the substrate 4-methylcoumarin butyrate was used to counterscreen confirmed HTS positives. A comparison of results from binding and inhibition assays allowed elucidation of compound mechanism of action. We demonstrate the limit of each technology and the benefits of using orthogonal assay techniques in profiling compounds.
Sujet(s)
Domaine catalytique/effets des médicaments et des substances chimiques , Dosages enzymatiques/méthodes , Antienzymes/pharmacologie , Acylglycerol lipase/antagonistes et inhibiteurs , Acides arachidoniques/composition chimique , Endocannabinoïdes , Antienzymes/composition chimique , Glycérides/composition chimique , Tests de criblage à haut débit , Humains , Hydrolyse , Concentration inhibitrice 50 , Cinétique , Acylglycerol lipase/composition chimique , Acylglycerol lipase/métabolisme , Liaison aux protéines , Solubilité , Spécificité du substratRÉSUMÉ
Endothelial lipase (EL) is a phospholipase A1 (PLA1) enzyme that hydrolyzes phospholipids at the sn-1 position to produce lysophospholipids and free fatty acids. Measurement of the PLA1 activity of EL is usually accomplished by the use of substrates that are also hydrolyzed by lipases in other subfamilies such as PLA2 enzymes. In order to distinguish PLA1 activity of EL from PLA2 enzymatic activity in cell-based assays, cell supernatants, and other nonhomogeneous systems, a novel fluorogenic substrate with selectivity toward PLA1 hydrolysis was conceived and characterized. This substrate was preferred by PLA1 enzymes, such as EL and hepatic lipase, and was cleaved with much lower efficiency by lipases that exhibit primarily triglyceride lipase activity, such as LPL or a lipase with PLA2 activity. The phospholipase activity detected by the PLA1 substrate could be inhibited with the small molecule esterase inhibitor ebelactone B. Furthermore, the PLA1 substrate was able to detect EL activity in human umbilical vein endothelial cells in a cell-based assay. This substrate is a useful reagent for identifying modulators of PLA1 enzymes, such as EL, and aiding in characterizing their mechanisms of action.
Sujet(s)
Composés du bore/métabolisme , Endothélium/enzymologie , Lysophospholipides/métabolisme , Phospholipases A1/analyse , Animaux , Colorants fluorescents/métabolisme , Humains , Lactones/pharmacologie , Triacylglycerol lipase/antagonistes et inhibiteurs , Triacylglycerol lipase/métabolisme , Souris , Phospholipases A1/antagonistes et inhibiteursRÉSUMÉ
The marketed drug topiramate ( 1) is a moderate inhibitor of carbonic anhydrase-II (CA-II) ( K i or K d = 0.3-0.6 microM), whereas sulfamide cognate 2 is a comparatively weak inhibitor ( K i or K d = 25-650 microM). From an X-ray cocrystal structure of 2.CA-II, Winum et al. ( J. Med. Chem. 2006, 49, 7024) proposed that an adverse steric interaction between the C8 methyl group in 2 and Ala-65 of CA-II is responsible for the diminished CA-II inhibitory potency of 2. We performed a straightforward test of this Ala-65 effect by synthesizing and examining ligand 3, which lacks the offending (pro- S or C8) methyl substituent in 2. We also prepared and evaluated related sulfamides 5, 7, and 9. In a CA-II inhibition assay (4-nitrophenyl acetate), the K i for 3 was approximately 300 microM, indicating very weak inhibition, close to that for 2 (4NPA, K i = 340 microM). In a CA-II binding assay (ThermoFluor), the K d for 3 was >57 microM, indicating very weak binding, lower than the affinity of 2 ( K d = 25 microM). Our results draw into question the proposed steric interaction between the C8 methyl of 2 and Ala-65 of CA-II.
Sujet(s)
Carbonic anhydrase II/antagonistes et inhibiteurs , Inhibiteurs de l'anhydrase carbonique/pharmacologie , Fructose/analogues et dérivés , Inhibiteurs de l'anhydrase carbonique/composition chimique , Cristallographie aux rayons X , Fructose/composition chimique , Fructose/pharmacologie , Cinétique , Spectroscopie par résonance magnétique , TopiramateRÉSUMÉ
The no-wash calcium assay kits developed by Molecular Devices Corporation have greatly enhanced the throughput of cell-based calcium mobilization high-throughput screening (HTS) assays and enabled screening using nonadherent cells. The fluorescent imaging plate reader (FLIPR) Calcium 3 Assay Kit, optimal for targets that have proteins or peptides as agonists, has 2 potential drawbacks: 1) a significant downward spike in fluorescence signal upon liquid transfer that can be the same magnitude as the agonist response, making data analysis difficult; and 2) medium removal is required for some targets, which essentially reintroduces a wash step. Several no-wash products were introduced in 2005. The authors compare the Fluo-4 NW Calcium Assay Kit and the BD Calcium Assay Kit with the FLIPR Calcium 3 Assay Kit using human native rhabdomyosarcoma cells expressing the urotensin-II receptor (UT). The BDtrade mark Calcium Assay Kit gives the best performance in the true no-wash mode, in which both agonist and antagonist activity are easily quantified. Although these new products provide additional options for measuring calcium mobilization, the different results observed with each kit, using the UT receptor as an example, suggest that one should characterize all dyes against each target in a systematic way prior to choosing one for HTS.
Sujet(s)
Calcium/analyse , Trousses de réactifs pour diagnostic , Récepteurs couplés aux protéines G/agonistes , Récepteurs couplés aux protéines G/antagonistes et inhibiteurs , Dérivés de l'aniline , Calcium/métabolisme , Cellules cultivées , Clones cellulaires , Évaluation préclinique de médicament , Colorants fluorescents , Fluorimétrie/instrumentation , Fluorimétrie/méthodes , Humains , Concentration inhibitrice 50 , Récepteurs couplés aux protéines G/métabolisme , Rhabdomyosarcome/métabolisme , Rhabdomyosarcome/anatomopathologie , Urotensines/métabolisme , Urotensines/pharmacologie , XanthènesRÉSUMÉ
The reliable production of large amounts of stable, high-quality proteins is a major challenge facing pharmaceutical protein biochemists, necessary for fulfilling demands from structural biology, for high-throughput screening, and for assay purposes throughout early discovery. One strategy for bypassing purification challenges in problematic systems is to engineer multiple forms of a particular protein to optimize expression, purification, and stability, often resulting in a nonphysiological sub-domain. An alternative strategy is to alter process conditions to maximize wild-type construct stability, based on a specific protein stability profile (PSP). ThermoFluor, a miniaturized 384-well thermal stability assay, has been implemented as a means of monitoring solution-dependent changes in protein stability, complementing the protein engineering and purification processes. A systematic analysis of pH, buffer or salt identity and concentration, biological metals, surfactants, and common excipients in terms of an effect on protein stability rapidly identifies conditions that might be used (or avoided) during protein production. Two PSPs are presented for the kinase catalytic domains of Akt-3 and cFMS, in which information derived from a ThermoFluor PSP led to an altered purification strategy, improving the yield and quality of the protein using the primary sequences of the catalytic domains.
Sujet(s)
Protéines proto-oncogènes c-akt/biosynthèse , Protéines proto-oncogènes c-akt/composition chimique , Récepteur du facteur de stimulation des colonies de macrophages/biosynthèse , Récepteur du facteur de stimulation des colonies de macrophages/composition chimique , Protéines recombinantes/biosynthèse , Protéines recombinantes/composition chimique , Substances tampon , Fluorescence , Concentration en ions d'hydrogène , Métaux/pharmacologie , Concentration osmolaire , Structure quaternaire des protéines/effets des médicaments et des substances chimiques , Protéines proto-oncogènes c-akt/isolement et purification , Récepteur du facteur de stimulation des colonies de macrophages/isolement et purification , Protéines recombinantes/isolement et purification , Protéines recombinantes/normes , Sels/pharmacologie , Solutions/pharmacologie , ThermodynamiqueRÉSUMÉ
The thermal denaturation of Klenow DNA polymerase has been characterized over a wide variety of solution conditions to obtain a relative stability landscape for the protein. Measurements were conducted utilizing a miniaturized fluorescence assay that measures Tm based on the increase in the fluorescence of 1,8-anilinonaphthalene sulfonate (ANS) when the protein denatures. The melting temperature (Tm) for Klenow increases as the salt concentration is increased and as the pH is decreased. Klenow's Tm spans a range of over 20 degrees C, from 40 to 62 degrees C, depending upon the solution conditions. The landscape reconciles and extends previously measured Tm values for Klenow. Salt effects on the stability of Klenow show strong cation dependence overlaid onto a more typical Hofmeister anion type dependence. Cationic stabilization of proteins has been far less frequently documented than anionic stabilization. The monovalent cations tested stabilize Klenow with the following hierarchy: NH4+>Na+>Li+>K+. Of the divalent cations tested: Mg+2 and Mn+2 significantly stabilize the protein, while Ni+2 dramatically destabilizes the protein. Stability measurements performed in combined Mg+2 plus Na+ salts suggest that the stabilizing effects of these monovalent and divalent cations are synergistic. The cationic stabilization of Klenow can be well explained by a model postulating dampening of repulsion within surface anionic patches on the protein.
Sujet(s)
DNA polymerase I/composition chimique , Anilino-naphtalènesulfonates/composition chimique , Cations divalents/composition chimique , Cations monovalents/composition chimique , Stabilité enzymatique , Fluorescence , Concentration en ions d'hydrogène , Conformation des protéines , Sels/composition chimique , Spectrométrie de fluorescence , Température de transitionRÉSUMÉ
Heat shock protein 90 (Hsp90) is critical for the maturation of numerous client proteins, many of which are involved in cellular transformation and oncogenesis. The ansamycins, geldanamycin (GA) and its derivative, 17-allylaminogeldanamycin (17-AAG), inhibit Hsp90. As such, the prototypical Hsp90 inhibitor, 17-AAG, has advanced into clinical oncology trials. GA and 17-AAG potently inhibit tumor cell proliferation and survival but have been reported to bind weakly to Hsp90 in vitro. Recent studies have suggested that the in vitro potency of ansamycins against Hsp90 may be enhanced in the presence of cochaperones. Here, we present evidence of an alternative explanation. Ansamycins reduced to their dihydroquinones in the presence of common reducing agents in vitro have approximately 40-fold greater affinity than the corresponding oxidized quinones. The dihydroquinone of 17-AAG is not generated in an aqueous environment in the absence of reducing agents but is produced in both tumor and normal quiescent epithelial cells. The reduced form of 17-AAG is differentiated from its oxidized form not only by the higher affinity for Hsp90 but also by a protracted K(off) rate. Therefore, the in vivo accumulation of the high-affinity dihydroquinone ansamycins in tumor cells contributes to the antitumor activity of these compounds and alters our understanding of the active species driving the efficacy of this class of compounds.
Sujet(s)
Protéines du choc thermique HSP90/antagonistes et inhibiteurs , Quinones/métabolisme , Quinones/pharmacologie , Antinéoplasiques , Benzoquinones , Lignée cellulaire tumorale/effets des médicaments et des substances chimiques , Cellules cultivées , Stabilité de médicament , Humains , Lactames macrocycliques , Liaison aux protéines/effets des médicaments et des substances chimiques , Réducteurs/pharmacologie , Rifabutine/analogues et dérivés , Rifabutine/métabolisme , Rifabutine/pharmacologie , SolubilitéRÉSUMÉ
The N-type calcium channel is a member of the voltage-sensitive calcium channel family and plays a major role in the regulation of neurotransmitter release in the central and peripheral nervous systems. Inhibition of the N-type calcium channel by intrathecal administration of the channel-specific blocker omega-conotoxin MVIIA (ziconotide) is efficacious in the treatment of severe chronic pain. While no orally active small molecules that block the N-type calcium channel are currently available, the discovery of such potentially valuable therapeutics would benefit from a reliable, high throughput assay. However, the assay of N-type calcium channel activity by measuring calcium influx using nonadherent cells in a high throughput fashion has not been achieved before, likely owing to a number of technical hurdles. For example, the measurement of calcium levels in nonadherent cells using conventional calcium indicators, such as Fluo-3 or Fluo-4, requires dyeloading the cells in suspension and subsequent removal of extracellular dye. This limits plate throughput and requires constant handling of the cells. To assay the N-type calcium channel activity using a nonadherent cell line in a high throughput manner, we investigated the application of no-wash calcium assay kits from Molecular Devices Corp. (Sunnyvale, CA): FLIPR Calcium, FLIPR Calcium Plus, and FLIPR Calcium 3. We show here that the FLIPR Calcium 3 assay kit can be used with nonadherent IMR-32 cells to measure potassium-evoked, omega-conotoxin MVIIA-reversible calcium flux with high throughput (15,000 data points/day), high quality (Z approximately 0.6), and minimal handling of the cells. Thus, this assay can be used to reliably and efficiently screen large compound libraries in the search for small molecule N-type calcium channel blockers.
Sujet(s)
Inhibiteurs des canaux calciques/pharmacologie , Canaux calciques de type N/effets des médicaments et des substances chimiques , Calcium/métabolisme , Trousses de réactifs pour diagnostic , Lignée cellulaire tumorale , Agents colorants , Humains , Conotoxines-oméga/pharmacologieRÉSUMÉ
ThermoFluor (a miniaturized high-throughput protein stability assay) was used to analyze the linkage between protein thermal stability and ligand binding. Equilibrium binding ligands increase protein thermal stability by an amount proportional to the concentration and affinity of the ligand. Binding constants (K(b)) were measured by examining the systematic effect of ligand concentration on protein stability. The precise ligand effects depend on the thermodynamics of protein stability: in particular, the unfolding enthalpy. An extension of current theoretical treatments was developed for tight binding inhibitors, where ligand effect on T(m) can also reveal binding stoichiometry. A thermodynamic analysis of carbonic anhydrase by differential scanning calorimetry (DSC) enabled a dissection of the Gibbs free energy of stability into enthalpic and entropic components. Under certain conditions, thermal stability increased by over 30 degrees C; the heat capacity of protein unfolding was estimated from the dependence of calorimetric enthalpy on T(m). The binding affinity of six sulfonamide inhibitors to two isozymes (human type 1 and bovine type 2) was analyzed by both ThermoFluor and isothermal titration calorimetry (ITC), resulting in a good correlation in the rank ordering of ligand affinity. This combined investigation by ThermoFluor, ITC, and DSC provides a detailed picture of the linkage between ligand binding and protein stability. The systematic effect of ligands on stability is shown to be a general tool to measure affinity.
Sujet(s)
Carbonic anhydrases/composition chimique , Carbonic anhydrases/métabolisme , Animaux , Calorimétrie différentielle à balayage , Carbonic anhydrase I/composition chimique , Carbonic anhydrase I/métabolisme , Carbonic anhydrase II/composition chimique , Carbonic anhydrase II/métabolisme , Inhibiteurs de l'anhydrase carbonique/composition chimique , Bovins , Stabilité enzymatique , Humains , Techniques in vitro , Cinétique , Dénaturation des protéines , Spectrométrie de fluorescence , ThermodynamiqueRÉSUMÉ
The protein product of an essential gene of unknown function from Streptococcus pneumoniae was expressed and purified for screening in the ThermoFluor affinity screening assay. This assay can detect ligand binding to proteins of unknown function. The recombinant protein was found to be in a dimeric, native-like folded state and to unfold cooperatively. ThermoFluor was used to screen the protein against a library of 3000 compounds that were specifically selected to provide information about possible biological functions. The results of this screen identified pyridoxal phosphate and pyridoxamine phosphate as equilibrium binding ligands (K(d) approximately 50 pM, K(d) approximately 2.5 microM, respectively), consistent with an enzymatic cofactor function. Several nucleotides and nucleotide sugars were also identified as ligands of this protein. Sequence comparison with two enzymes of known structure but relatively low overall sequence homology established that several key residues directly involved in pyridoxal phosphate binding were strictly conserved. Screening a collection of generic drugs and natural products identified the antifungal compound canescin A as an irreversible covalent modifier of the enzyme. Our investigation of this protein indicates that its probable biological role is that of a nucleoside diphospho-keto-sugar aminotransferase, although the preferred keto-sugar substrate remains unknown. These experiments demonstrate the utility of a generic affinity-based ligand binding technology in decrypting possible biological functions of a protein, an approach that is both independent of and complementary to existing genomic and proteomic technologies.
Sujet(s)
Protéines bactériennes/physiologie , Gènes essentiels/physiologie , Nucléoside diphosphate ose/métabolisme , Streptococcus pneumoniae/génétique , Transaminases/physiologie , Séquence d'acides aminés , Benzopyranes/métabolisme , Dimérisation , Furanes/métabolisme , Ligands , Données de séquences moléculaires , Phosphate de pyridoxal/métabolisme , Pyridoxamine/métabolisme , Streptococcus pneumoniae/enzymologieRÉSUMÉ
The application of label-independent biophysical assays (affinity assays) to carefully selected libraries of biochemicals, natural products and known bioactive molecules generates a powerful tool to identify ligands indicative of protein function. Detection of ligand binding does not require the measurement of enzyme activity, thus every cofactor, ion, reactant or product might be identified separately. One such assay, ThermoFluor(®), monitors ligand perturbation of protein thermostability. In conjunction with a functional probe library (FPL), this assay has been used to decrypt the function of orphan proteins.:
RÉSUMÉ
Productive cis folding by the chaperonin GroEL is triggered by the binding of ATP but not ADP, along with cochaperonin GroES, to the same ring as non-native polypeptide, ejecting polypeptide into an encapsulated hydrophilic chamber. We examined the specific contribution of the gamma-phosphate of ATP to this activation process using complexes of ADP and aluminium or beryllium fluoride. These ATP analogues supported productive cis folding of the substrate protein, rhodanese, even when added to already-formed, folding-inactive cis ADP ternary complexes, essentially introducing the gamma-phosphate of ATP in an independent step. Aluminium fluoride was observed to stabilize the association of GroES with GroEL, with a substantial release of free energy (-46 kcal/mol). To understand the basis of such activation and stabilization, a crystal structure of GroEL-GroES-ADP.AlF3 was determined at 2.8 A. A trigonal AlF3 metal complex was observed in the gamma-phosphate position of the nucleotide pocket of the cis ring. Surprisingly, when this structure was compared with that of the previously determined GroEL-GroES-ADP complex, no other differences were observed. We discuss the likely basis of the ability of gamma-phosphate binding to convert preformed GroEL-GroES-ADP-polypeptide complexes into the folding-active state.