Changes In Intrinsic Tryptophan Fluorescence Research Articles

Investigation of structural factors controlling loop dynamics in acyl protein thioesterases

Acyl protein thioesterases catalyze the depalmitoylation of key signaling proteins attached to the plasma membrane. Recently, human APTs were shown to use a two‐step catalytic mechanism involving initial membrane binding of an exposed flexible loop followed by substrate binding and cleavage. This two‐step catalytic mechanism is likely conserved as a bacterial homologue of human APTs is also regulated by the large‐scale movement of an essential dynamic loop upon membrane binding. However, the causes for the movement of this dynamic loop in APTs are unknown. Herein, we investigated factors that trigger loop movement in human and bacterial APTs. In particular, APTs were assayed with inhibitors structurally similar to long chain lipids and to membrane‐mimicking surfactants to determine if these factors induce loop movement. Loop movement was detected by measuring the change in intrinsic tryptophan fluorescence of a central tryptophan on the dynamic loop before and after exposure to these conditions. Although inhibitors and substrate mimics induced minor changes in loop dynamics, larger shifts were measured with various membrane‐mimicking surfactants, suggesting that APT loop dynamics are mainly regulated by proximity to the plasma membrane. Ultimately, understanding the factors controlling APT function could provide novel mechanisms for controlling cell signaling.

Investigation of Factors that Trigger Essential Conformational Changes in FTT258

FTT258 is a close bacterial relative of human acyl protein thioesterase (hAPT), a protein linked to cancer. hAPT removes long chain lipids from key cancer signaling proteins. Removal of these lipids requires their insertion into a pocket within hAPT, which is only exposed after the movement of a flexible loop, a common feature between hAPT and FTT258. However, the causes for the movement of this loop are unknown. I investigated factors that may trigger loop movement in APTs like hAPT and FTT258. In particular, I exposed FTT258 to inhibitors structurally similar to long chain lipids and to membrane‐mimicking surfactants in order to determine if these factors induce loop movement. Loop movement was detected by measuring the change in intrinsic tryptophan fluorescence before and after exposure to these conditions. Ultimately, understanding the factors controlling FTT258 and hAPT function can provide novel mechanisms for inhibiting cancer progression.Support or Funding InformationNSF MCB‐1812971

Investigation of structural factors controlling loop dynamics in acyl protein thioesterases

FTT258 is a close bacterial relative of human acyl protein thioesterases (APTs). Human APTs catalyze the depalmitoylation of key signaling proteins attached to the plasma membrane. For FTT258, catalysis was hypothesized to be regulated by the large‐scale movement of an essential dynamic loop, which transitioned the protein between active and inactive states. However, the causes for the movement of this loop are unknown. Herein, we investigated factors that trigger loop movement in APTs like FTT258. In particular, FTT258 was exposed to inhibitors structurally similar to long chain lipids and to membrane‐mimicking surfactants to determine if these factors induce loop movement. Loop movement was detected by measuring the change in intrinsic tryptophan fluorescence of a central tryptophan on the dynamic loop before and after exposure to these conditions. Although inhibitors and substrate mimics induced minor changes in loop dynamics, larger shifts were measured with various membrane‐mimicking surfactants, suggesting that APT loop dynamics are mainly regulated by proximity to the plasma membrane. Ultimately, understanding the factors controlling APT function could provide novel mechanisms for controlling cell signaling.Support or Funding InformationFunded by NSF MCB‐1812971This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Monitoring GPCR Conformational Changes during Agonist Release in Real-Time: Evidence that Transmembrane Helix 6 (TM6) Movement in Rhodopsin Lags Behind Retinal Release

GPCR activation involves an outward movement of the cytoplasmic end of TM6, which exposes a binding site for G proteins, arrestins, and kinases. This movement, observed for several GPCRs (including rhodopsin and the β2 adrenergic receptor), appears critical for formation of the active receptor state. Previously, we showed real-time monitoring of TM6 movement using Tryptophan-Induced Quenching (TrIQ) of fluorescence of a bimane probe on TM6 by a tryptophan on TM3 (Schafer, Janz, and Farrens Biophysics Abstract 2014). The TrIQ data are consistent with an outward TM6 movement during activation; which then returns to its original position as the agonist, all-trans retinal (ATR), exits the receptor. We initially concluded that this TM6 “resetting” occurs simultaneously with agonist release, as the rates are similar to changes in intrinsic tryptophan fluorescence we measure during ATR dissociation. However, our more recent and precise measurements reveal a small, but reproducible time-lag between ATR release and the TM6 resetting. Interestingly, this lag slightly increases for a constitutively inactive mutant, as well as for another mutation inside the retinal binding pocket. Similarly, dramatically increasing the agonist dissociation rate by chemical attack (hydroxylamine) exacerbates the lag. Although the molecular mechanism underlying the delay between ATR release and TM6 movement is still unknown, the similar activation energies (∼21kcal/mol) they exhibit by Arrhenius analysis suggest a common mechanism for both. We propose these results suggest a heretofore unidentified empty opsin is present immediately after retinal release, one that differs from the “traditional” inactive opsin conformation, in which TM6 adopts an inward position. Our further investigations into this phenomenon and the implications they have on the process of retinal binding and release will be discussed along with potential caveats.

Constitutive Nuclear Localization of an Alternatively Spliced Sirtuin-2 Isoform

Sirtuin-2 (SIRT2), the cytoplasmic member of the sirtuin family, has been implicated in the deacetylation of nuclear proteins. Although the enzyme has been reported to be located to the nucleus during G2/M phase, its spectrum of targets suggests functions in the nucleus throughout the cell cycle. While a nucleocytoplasmic shuttling mechanism has been proposed for SIRT2, recent studies have indicated the presence of a constitutively nuclear isoform. Here we report the identification of a novel splice variant (isoform 5) of SIRT2 that lacks a nuclear export signal and encodes a predominantly nuclear isoform. This novel isoform 5 fails to show deacetylase activity using several assays, both in vitro and in vivo, and we are led to conclude that this isoform is catalytically inactive. Nevertheless, it retains the ability to interact with p300, a known interaction partner. Moreover, changes in intrinsic tryptophan fluorescence upon denaturation indicate that the protein is properly folded. These data, together with computational analyses, confirm the structural integrity of the catalytic domain. Our results suggest an activity-independent nuclear function of the novel isoform.

Substrate Activation and Conformational Dynamics of Guanosine 5′-Monophosphate Synthetase

Glutamine amidotransferases catalyze the amination of a wide range of molecules using the amide nitrogen of glutamine. The family provides numerous examples for study of multi-active-site regulation and interdomain communication in proteins. Guanosine 5'-monophosphate synthetase (GMPS) is one of three glutamine amidotransferases in de novo purine biosynthesis and is responsible for the last step in the guanosine branch of the pathway, the amination of xanthosine 5'-monophosphate (XMP). In several amidotransferases, the intramolecular path of ammonia from glutamine to substrate is understood; however, the crystal structure of GMPS only hinted at the details of such transfer. Rapid kinetics studies provide insight into the mechanism of the substrate-induced changes in this complex enzyme. Rapid mixing of GMPS with substrates also manifests absorbance changes that report on the kinetics of formation of a reactive intermediate as well as steps in the process of rapid transfer of ammonia to this intermediate. Isolation and use of the adenylylated nucleotide intermediate allowed the study of the amido transfer reaction distinct from the ATP-dependent reaction. Changes in intrinsic tryptophan fluorescence upon mixing of enzyme with XMP suggest a conformational change upon substrate binding, likely the ordering of a highly conserved loop in addition to global domain motions. In the GMPS reaction, all forward rates before product release appear to be faster than steady-state turnover, implying that release is likely rate-limiting. These studies establish the functional role of a substrate-induced conformational change in the GMPS catalytic cycle and provide a kinetic context for the formation of an ammonia channel linking the distinct active sites.

Enhancement of NEIL1 Protein-initiated Oxidized DNA Base Excision Repair by Heterogeneous Nuclear Ribonucleoprotein U (hnRNP-U) via Direct Interaction

Repair of oxidized base lesions in the human genome, initiated by DNA glycosylases, occurs via the base excision repair pathway using conserved repair and some non-repair proteins. However, the functions of the latter noncanonical proteins in base excision repair are unclear. Here we elucidated the role of heterogeneous nuclear ribonucleoprotein-U (hnRNP-U), identified in the immunoprecipitate of human NEIL1, a major DNA glycosylase responsible for oxidized base repair. hnRNP-U directly interacts with NEIL1 in vitro via the NEIL1 common interacting C-terminal domain, which is dispensable for its enzymatic activity. Their in-cell association increases after oxidative stress. hnRNP-U stimulates the NEIL1 in vitro base excision activity for 5-hydroxyuracil in duplex, bubble, forked, or single-stranded DNA substrate, primarily by enhancing product release. Using eluates from FLAG-NEIL1 immunoprecipitates from human cells, we observed 3-fold enhancement in complete repair activity after oxidant treatment. The lack of such enhancement in hnRNP-U-depleted cells suggests its involvement in repairing enhanced base damage after oxidative stress. The NEIL1 disordered C-terminal region binds to hnRNP-U at equimolar ratio with high affinity (K(d) = ∼54 nm). The interacting regions in hnRNP-U, mapped to both termini, suggest their proximity in the native protein; these are also disordered, based on PONDR (Predictor of Naturally Disordered Regions) prediction and circular dichroism spectra. Finally, depletion of hnRNP-U and NEIL1 epistatically sensitized human cells at low oxidative genome damage, suggesting that the hnRNP-U protection of cells after oxidative stress is largely due to enhancement of NEIL1-mediated repair.

The V30M Amyloidogenic Mutation Decreases the Rate of Refolding Kinetics of the Tetrameric Protein Transthyretin

Transthyretin (TTR) is a homotetrameric protein implicated in several amyloid diseases. The mechanism by which TTR is converted into elongated fibrillar assemblies has been extensively investigated, and numerous studies showed that dissociation of the native tetrameric structure into partially unfolded monomeric species precedes amyloid formation. The small differences observed in the crystal structures of different TTR variants, as well as the thermodynamics and kinetics of tetramer dissociation, do not seem to completely justify the amyloidogenic potential of different TTR variants. With this in mind, we have studied the refolding kinetics of WT-TTR and its most common amyloidogenic variant V30M-TTR, monitoring changes in intrinsic tryptophan fluorescence at different urea and protein concentrations. Our results demonstrate that thein vitrorefolding mechanisms of WT- and V30M-TTR are similar, involving a dimeric intermediate. However, there are large differences in the refolding rate constants for the two variants, specially close to physiological conditions. Interestingly, tetramer formation occurs at a much slower rate in the amyloidogenic variant V30M-TTR than in WT-TTR, which in thein vivosetting may promote the accumulation of monomeric species in the extracellular environment, resulting in higher susceptibility for aggregation and amyloid formation instead of spontaneous refolding.

Ligand-Binding Domain of Type 1 Metabotropic Glutamate Receptor is Fully Functional in iTs Monomeric Form

The type 1 metabotropic glutamate receptor (mGluR1) is an archetypal class C G-protein coupled receptor (GPCR) that modulates neurotransmission and synaptic plasticity in the brain. It has been demonstrated that mGluR1 carries out its biological function as a covalently linked dimer. All class C GPCRs investigated to date undergo dimerization, yet the precise biological significance of this phenomenon remains unclear. We sought to test the effect of dimerization on the activation properties of the ligand-binding domain (LBD) of mGluR1, expressed in HEK293 cells as a truncated soluble domain. Two variants of the protein were created: a wild-type construct and the C140S mutant, which lacks covalent linkage between subunits. The extent of dimerization in solution under non-denaturing conditions was examined by using static light scattering (SLS) to measure weight-average molecular weights for both constructs. These measurements support full dimerization in the wild type and permit quantification of the monomer-dimer equilibrium in the C140S mutant. Structural rearrangements of the purified LBDs were probed in vitro by monitoring changes in intrinsic tryptophan fluorescence; fluorescence was titrated with the native ligand L-glutamate to quantify biological activity. These titrations were carried out at low protein concentration at which preliminary SLS results show that the C140S mutant exists in its monomeric state, in contrast to the covalently linked wild-type dimer. We found that the monomeric mGluR1 LBD has equal or greater activity compared to the wild type. Our results indicate that dimerization is not a prerequisite of function, but likely plays a modulatory role in the ligand-binding domain of this receptor. This approach will allow future investigations to reveal emergent properties arising from dimerization in mGluR1 and similar GPCRs, with implications for our understanding of chemical signaling in the nervous system.

Identification of farnesoid X receptor modulators by a fluorescence polarization-based interaction assay

Farnesoid X receptor (FXR) serves as a receptor for chenodeoxycholic acid (CDCA) and other bile acids, and it coordinates cholesterol and lipid metabolism. Because targeting the FXR–CDCA interaction might provide a way to regulate lipid homeostasis, we developed an FXR binding assay based on fluorescence polarization. Employing a fluorescently labeled CDCA (CDCA-F), we showed that CDCA-F selectively bound to the ligand binding domain of FXR (FXR–LBD) among nuclear receptors. The assay was then used for screening inhibitors against the FXR–CDCA interaction, thereby discovering four relatively potent inhibitors. The selected inhibitors were further studied for changes in intrinsic tryptophan fluorescence of FXR–LBD to gain structural insights into the interaction. Furthermore, transactivation effects of the inhibitors on the human bile salt excretory pump (BSEP) promoter were examined to reveal their cellular activities in the FXR-mediated pathway. Therefore, we demonstrated that the developed assay would offer an efficient primary screening tool for identifying FXR modulators.

Inhibition of Heterotrimeric G Protein Signaling by a Small Molecule Acting on Gα Subunit

The simultaneous activation of many distinct G protein-coupled receptors (GPCRs) and heterotrimeric G proteins play a major role in various pathological conditions. Pan-inhibition of GPCR signaling by small molecules thus represents a novel strategy to treat various diseases. To better understand such therapeutic approach, we have characterized the biomolecular target of BIM-46187, a small molecule pan-inhibitor of GPCR signaling. Combining bioluminescence and fluorescence resonance energy transfer techniques in living cells as well as in reconstituted receptor-G protein complexes, we observed that, by direct binding to the Galpha subunit, BIM-46187 prevents the conformational changes of the receptor-G protein complex associated with GPCR activation. Such a binding prevents the proper interaction of receptors with the G protein heterotrimer and inhibits the agonist-promoted GDP/GTP exchange. These observations bring further evidence that inhibiting G protein activation through direct binding to the Galpha subunit is feasible and should constitute a new strategy for therapeutic intervention.

Conformational Change in the Chromatin Remodelling Protein MENT

Chromatin condensation to heterochromatin is a mechanism essential for widespread suppression of gene transcription, and the means by which a chromatin-associated protein, MENT, induces a terminally differentiated state in cells. MENT, a protease inhibitor of the serpin superfamily, is able to undergo conformational change in order to effect enzyme inhibition. Here, we sought to investigate whether conformational change in MENT is ‘fine-tuned’ in the presence of a bound ligand in an analogous manner to other serpins, such as antithrombin where such movements are reflected by a change in intrinsic tryptophan fluorescence. Using this technique, MENT was found to undergo structural shifts in the presence of DNA packaged into nucleosomes, but not naked DNA. The contribution of the four Trp residues of MENT to the fluorescence change was mapped using deconvolution analysis of variants containing single Trp to Phe mutations. The analysis indicated that the overall emission spectra is dominated by a helix-H tryptophan, but this residue did not dominate the conformational change in the presence of chromatin, suggesting that other Trp residues contained in the A-sheet and RCL regions contribute to the conformational change. Mutagenesis revealed that the conformational change requires the presence of the DNA-binding ‘M-loop’ and D-helix of MENT, but is independent of the protease specificity determining ‘reactive centre loop’. The D-helix mutant of MENT, which is unable to condense chromatin, does not undergo a conformational change, despite being able to bind chromatin, indicating that the conformational change may contribute to chromatin condensation by the serpin.

Urea Unfolding of Opsin in Phospholipid Bicelles†

Opsin is the unstable apo-protein of the light-activated G protein-coupled receptor rhodopsin. We investigated the stability of bovine opsin, solubilized in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/detergent bicelles, against urea-induced unfolding. A single irreversible protein unfolding transition was observed from changes in intrinsic tryptophan fluorescence and far-UV circular dichroism. This unfolding transition correlated with loss of protein activity. Changes in tertiary structure, as indicated by fluorescence measurements, were concomitant with an approximate 50% reduction in alpha-helical content of opsin, indicating that global unfolding had been induced by urea. The urea concentration at the midpoint of unfolding was dependent on the lipid/detergent environment, occurring at approximately 1.2 m urea in DMPC/1,2-dihexanoyl-sn-glycero-3-phosphocholine bicelles, while being significantly stabilized to approximately 3.5 m urea in DMPC/3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonate bicelles. These findings demonstrate that interactions with the surrounding lipids and detergent are highly influential in the unfolding of membrane protein structure. The urea/bicelle system offers the possibility for a more detailed understanding of the structural changes that take place upon irreversible unfolding of opsin.

Structural and Biochemical Characterization of the Oxidoreductase NmDsbA3 from Neisseria meningitidis

DsbA is an enzyme found in the periplasm of Gram-negative bacteria that catalyzes the formation of disulfide bonds in a diverse array of protein substrates, many of which are involved in bacterial pathogenesis. Although most bacteria possess only a single essential DsbA, Neisseria meningitidis is unusual in that it possesses three DsbAs, although the reason for this additional redundancy is unclear. Two of these N. meningitidis enzymes (NmDsbA1 and NmDsbA2) play an important role in meningococcal attachment to human epithelial cells, whereas NmDsbA3 is considered to have a narrow substrate repertoire. To begin to address the role of DsbAs in the pathogenesis of N. meningitidis, we have determined the structure of NmDsbA3 to 2.3-A resolution. Although the sequence identity between NmDsbA3 and other DsbAs is low, the NmDsbA3 structure adopted a DsbA-like fold. Consistent with this finding, we demonstrated that NmDsbA3 acts as a thiol-disulfide oxidoreductase in vitro and is reoxidized by Escherichia coli DsbB (EcDsbB). However, pronounced differences in the structures between DsbA3 and EcDsbA, which are clustered around the active site of the enzyme, suggested a structural basis for the unusual substrate specificity that is observed for NmDsbA3.

Characterization of Molecular Interactions between Eosinophil Cationic Protein and Heparin

Eosinophil cationic protein (ECP) is currently used as a biomarker for airway inflammation. It is a heparin-binding ribonuclease released by activated eosinophils. Its cytotoxicity toward cancer cell lines is blocked by heparin. The objective of this study was to locate the heparin binding site of ECP by site-directed mutagenesis and construction of a synthetic peptide derived from this region. Synthetic heparin with > or =5 monosaccharide units showed strong inhibition of ECP binding to the cell surface. Analysis of ECP mt1 (R34A/W35A/R36A/K38A) showed that these charged and aromatic residues were involved in ECP binding to heparin and the cell surface. A potential binding motif is located in the loop L3 region between helix alpha2 and strand beta1, outside the RNA binding domain. The synthetic peptide derived from the loop L3 region displayed strong pentasaccharide binding affinity and blocked ECP binding to cells. In addition, ECP mt1 showed reduced cytotoxicity. Thus, the tight interaction between ECP and heparin acts as the primary step for ECP endocytosis. These results provide new insights into the structure and function of ECP for anti-asthma therapy.

Molecular Features Governing the Stability and Specificity of Functional Complex Formation by Mycobacterium tuberculosis CFP-10/ESAT-6 Family Proteins

The Mycobacterium tuberculosis complex CFP-10/ESAT-6 family proteins play essential but poorly defined roles in tuberculosis pathogenesis. In this article we report the results of detailed spectroscopic studies of several members of the CFP-10/ESAT-6 family. This work shows that the CFP-10/ESAT-6 related proteins, Rv0287 and Rv0288, form a tight 1:1 complex, which is predominantly helical in structure and is predicted to closely resemble the complex formed by CFP-10 and ESAT-6. In addition, the Rv0287.Rv0288 complex was found to be significantly more stable to both chemical and temperature induced denaturation than CFP-10.ESAT-6. This approach demonstrated that neither Rv0287.Rv0288 nor the CFP-10.ESAT-6 complexes are destabilized at low pH (4.5), indicating that even in low pH environments, such as the mature phagosome, both Rv0287.Rv0288 and CFP-10.ESAT-6 undoubtedly function as complexes rather than individual proteins. Analysis of the structure of the CFP-10.ESAT-6 complex and optimized amino acid sequence alignments of M. tuberculosis CFP-10/ESAT-6 family proteins revealed that residues involved in the intramolecular contacts between helices are conserved across the CFP-10/ESAT-6 family, but not those involved in primarily intermolecular contacts. This analysis identified the molecular basis for the specificity and stability of complex formation between CFP-10/ESAT-6 family proteins, and indicates that the formation of functional complexes with key roles in pathogenesis will be limited to genome partners, or very closely related family members, such as Rv0287/Rv0288 and Rv3019c/Rv3020c.

Characterization of the Folding and Unfolding Reactions of Single-Chain Monellin: Evidence for Multiple Intermediates and Competing Pathways

The mechanisms of folding and unfolding of the small plant protein monellin have been delineated in detail. For this study, a single-chain variant of the natively two-chain monellin, MNEI, was used, in which the C terminus of chain B was connected to the N terminus of chain A by a Gly-Phe linker. Equilibrium guanidine hydrochloride (GdnHCl)-induced unfolding experiments failed to detect any partially folded intermediate that is stable enough to be populated at equilibrium to a significant extent. Kinetic experiments in which the refolding of GdnHCl-unfolded protein was monitored by measurement of the change in the intrinsic tryptophan fluorescence of the protein indicated the accumulation of three transient partially structured folding intermediates. The fluorescence change occurred in three kinetic phases: very fast, fast, and slow. It appears that the fast and slow changes in fluorescence occur on competing folding pathways originating from one unfolded form and that the very fast change in fluorescence occurs on a third parallel pathway originating from a second unfolded form of the protein. Kinetic experiments in which the refolding of alkali-unfolded protein was monitored by the change in the fluorescence of the hydrophobic dye 8-anilino-1-naphthalenesulfonic acid (ANS), consequent to the dye binding to the refolding protein, as well as by the change in intrinsic tryptophan fluorescence, not only confirmed the presence of the three kinetic intermediates but also indicated the accumulation of one or more early intermediates at a few milliseconds of refolding. These experiments also exposed a very slow kinetic phase of refolding, which was silent to any change in the intrinsic tryptophan fluorescence of the protein. Hence, the spectroscopic studies indicated that refolding of single-chain monellin occurs in five distinct kinetic phases. Double-jump, interrupted-folding experiments, in which the accumulation of folding intermediates and native protein during the folding process could be determined quantitatively by an unfolding assay, indicated that the fast phase of fluorescence change corresponds to the accumulation of two intermediates of differing stabilities on competing folding pathways. They also indicated that the very slow kinetic phase of refolding, identified by ANS binding, corresponds to the formation of native protein. Kinetic experiments in which the unfolding of native protein in GdnHCl was monitored by the change in intrinsic tryptophan fluorescence indicated that this change occurs in two kinetic phases. Double-jump, interrupted-unfolding experiments, in which the accumulation of unfolding intermediates and native protein during the unfolding process could be determined quantitatively by a refolding assay, indicated that the fast unfolding phase corresponds to the formation of fully unfolded protein via one unfolding pathway and that the slow unfolding phase corresponds to a separate unfolding pathway populated by partially unfolded intermediates. It is shown that the unfolded form produced by the fast unfolding pathway is the one which gives rise to the very fast folding pathway and that the unfolded form produced by the slower unfolding pathway is the one which gives rise to the slow and fast folding pathways.

Urea-induced Inactivation and Unfolding of Recombinant Phospholipid Hydroperoxide Glutathione Peroxidase from Oryza sativa

Abstract Phospholipid hydroperoxide glutathione peroxidase is an antioxidant enzyme that has the highest capability of reducing membrane-bound hydroperoxy lipids as compared to free organic and inorganic hydroperoxides amongst the glutathione peroxidases. In this study, urea-induced effects on the inactivation and unfolding of a recombinant phospholipid hydroperoxide glutathione peroxidase (PHGPx) from Oryza sativa were investigated by means of circular dichroism and fluorescence spectroscopy. With the increase of urea concentration, the residual activity of OsPHGPx decreases correspondingly. When the urea concentration is above 5.0 mol/L, there was no residual activity. In addition, the observed changes in intrinsic tryptophan fluorescence, the binding of the hydrophobic fluorescence probe ANS, and the far UV CD describe a common dependence on the concentration of urea suggesting that the conformational features of the native OsPHGPx are lost in a highly cooperative single transition. The unfolding process comprises of three zones: the native base-line zone between 0 and 2.5 mol/L urea, the transition zone between 2.5 and 5.5 mol/L urea, and the denatured base-line zone above 5.5 mol/L urea. The transition zone has a midpoint at about 4.0 mol/L urea.

Src Tail Phosphorylation Is Limited by Structural Changes in the Regulatory Tyrosine Kinase Csk

Src family tyrosine kinases are down-regulated through phosphorylation of a single C-terminal tyrosine by the nonreceptor tyrosine kinase Csk. Despite the fundamental role of Csk in controlling cell growth and differentiation, it is unclear what limits this key signaling reaction and controls the production of catalytically repressed Src. To investigate this issue, stopped-flow fluorescence experiments were performed to determine which steps modulate catalysis. Both Src binding and phosphorylation can be monitored by changes in intrinsic tryptophan fluorescence. Association kinetics are biphasic with the initial phase corresponding to the bimolecular interaction of both proteins and the second phase representing a slow conformational change that coincides with the rate of maximum turnover. The kinetic transients for the phosphorylation reaction are also biphasic with the initial phase corresponding to the rapid phosphorylation and the release of phospho-Src. These data, along with equilibrium sedimentation and product inhibition experiments, suggest that steps involving Src association, phosphorylation, and product release are fast and that a structural change in Csk participates in limiting the catalytic cycle.

Heterotrimeric G-protein α-Subunit Adopts a “Preactivated” Conformation When Associated with βγ-Subunits

Heterotrimeric G-protein α-Subunit Adopts a “Preactivated” Conformation When Associated with βγ-Subunits