Changes In Intrinsic Fluorescence Research Articles

Site directed mutagenesis at position 193 of human trypsin 4 alters the rate of conformational change during activation: Role of local internal viscosity in protein dynamics

Upon activation of trypsinogen four peptide segments flanked by hinge glycine residues undergo conformational changes. To test whether the degree of conformational freedom of hinge regions affects the rate of activation, we introduced amino acid side chains of different characters at one of the hinges (position 193) and studied their effects on the rate constant of the conformational change. This structural rearrangement leading to activation was triggered by a pH-jump and monitored by intrinsic fluorescence change in the stopped-flow apparatus. We found that an increase in the size of the side chain at position 193 is associated with the decrease of the reaction rate constant. To analyze the thermodynamics of the reaction, temperature dependence of the reaction rate constants was examined in a wide temperature range (5-60 degrees C) using a novel temperature-jump/stopped-flow apparatus developed in our laboratory. Our data show that the mutations do not affect the activation energy (the exponential term) of the reaction, but they significantly alter the preexponential term of the Arrhenius equation. The effect of solvent viscosity on the rate constants of the conformational change during activation of the wild type enzyme and its R193G and R193A mutants was determined and evaluated on the basis of Kramers' theory. Based on this we propose that the reaction rate of this conformational transition is regulated by the internal molecular friction, which can be specifically modulated by mutagenesis in the hinge region.

Monomeric Creatine Kinase Aggregation and Sodium Dodecyl Sulfate-cyclodextrin Assisted Refolding

The monomelic state of creatine kinase (CK) was stably captured at the equilibrium state by employing cysteine residue modifications in the presence of a dénaturant, and at a partially folded state. The partially folded monomeric CK (PF-CK) was aggregated with kinetic order, which was mainly caused by the hydrophobic surface interactions between the CK subunits. The artificial chaperone, described as a SDS-cyclodextrin, was applied to prevent aggregation as well as to refold the PF-CK: SDS treatment onto the monomeric CK can significantly block aggregation and can be successfully refolded in the solutions containing cyclodextrins and DTT. Three types of cyclodextrins such as α-, β-, and γ-cyclodextrins were applied to strip SDS from the enzyme molecule, and each kinetic course was measured. The intrinsic fluorescence changes showed that reactivation occurred and this accompanied the conformational changes. The size exclusion chromatography detected the variously trapped monomeric CKs such as the thiol residue modified PF-CK, the SDS- binding PF-CK, the cyclodextrin treated PF-CK, and the DTT treated SDS-binding PF-CK. Our study demonstrated monomer CK aggregation for the first time; we also demonstrated the complex reassociation of CK during refolding with the aid of the SDS-cyclodextrin, and these pathways followed first-order kinetics.

A Pre-Steady State Analysis of Ligand Binding to Human Glucokinase: Evidence for a Preexisting Equilibrium

Cooperativity with glucose is a key feature of human glucokinase (GK), allowing its crucial role as a glucose sensor in hepatic and pancreatic cells. We studied the changes in enzyme intrinsic tryptophan fluorescence induced by binding of different ligands to this monomeric enzyme using stopped-flow and equilibrium binding methods. Glucose binding data under pre-steady state conditions suggest that the free enzyme in solution is in a preexisting equilibrium between at least two conformers (super-open and open) which differ in their affinity for glucose (Kd* = 0.17 +/- 0.02 mM and Kd = 73 +/- 18 mM). Increasing the glucose concentration changes the ratio of the two conformers, thus yielding an apparent Kd of 3 mM (different from a Km of 7-10 mM). The rates of conformational transitions of free and GK complexed with sugar are slow and during catalysis are most likely affected by ATP binding, phosphate transfer, and product release steps to allow the kcat to be 60 s-1. The ATP analogue PNP-AMP binds to free GK (super-open) and GK-glucose (open) complexes with comparable affinities (Kd = 0.23 +/- 0.02 and 0.19 +/- 0.08 mM, respectively). However, cooperativity with PNP-AMP observed under equilibrium binding conditions in the presence of glucose (Hill slope of 1.6) is indicative of further complex tightening to the closed conformation. Another physiological modulator (inhibitor), palmitoyl-CoA, binds to GK with similar characteristics, suggesting that conformational changes induced upon ligand binding are not restricted by an active site ligand. In conclusion, our data support control of GK activity and Km through the ratio of distinct conformers (super-open, open, and closed) through either substrate or other ligand binding and/or dissociation.

Intrinsic fluorescence changes associated with the conformational state of silk fibroin in biomaterial matrices

Silk fibroin is emerging as an important biomaterial for tissue engineering applications. The ability to monitor non-invasively the structural conformation of silk matrices prior to and following cell seeding could provide important insights with regards to matrix remodeling and cell-matrix interactions that are critical for the functional development of silk-based engineered tissues. Thus, we examined the potential of intrinsic fluorescence as a tool for assessing the structural conformation of silk proteins. Specifically, we characterized the intrinsic fluorescence spectra of silk in solution, gel and scaffold configurations for excitation in the 250 to 335 nm range and emission from 265 to 600 nm. We have identified spectral components that are attributed to tyrosine, tryptophan and crosslinks based on their excitation-emission profiles. We have discovered significant spectral shifts in the emission profiles and relative contributions of these components among the silk solution, gel and scaffold samples that represent enhancements in the levels of crosslinking, hydrophobic and intermolecular interactions that are consistent with an increase in the levels of ss-sheet formation and stacking. This information can be easily utilized for the development of simple, non-invasive, ratiometric methods to assess and monitor the structural conformation of silk in engineered tissues.

Quenching-enhanced fluorescence titration protocol for accurate determination of free energy of membrane binding

Quenching-enhanced fluorescence titration protocol for accurate determination of free energy of membrane binding

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.

Stored Ca2+ Depletion-induced Oligomerization of Stromal Interaction Molecule 1 (STIM1) via the EF-SAM Region

Stromal interaction molecule 1 (STIM1) has recently been identified as a key player in store-operated Ca2+ entry. Endoplasmic reticulum (ER) luminal Ca2+ depletion results in STIM1 redistribution from ER membrane homogeneity to distinctly localized aggregates near the plasma membrane; these changes precede and are linked to cytoplasmic Ca2+ influx via Ca2+ release-activated channels (CRACs). The molecular mechanisms initiating ER STIM1 redistribution and plasma membrane CRAC activity are not well understood. We recombinantly expressed the Ca2+-sensing region of STIM1 consisting of the EF-hand together with the sterile alpha-motif (SAM) domain (EF-SAM) to investigate its Ca2+-related conformational and biochemical features. We demonstrate that Ca2+-loaded EF-SAM (holo) contains high alpha-helicity, whereas EF-SAM in the absence of Ca2+ (apo) is much less compact. Accordingly, the melting temperature (Tm) of the holoform is approximately 25 degrees C higher than apoform; heat and urea-derived thermodynamic parameters indicate a Ca2+-induced stabilization of 3.2 kcal mol(-1). We show that holoEF-SAM exists as a monomer, whereas apoEF-SAM readily forms a dimer and/or oligomer, and that oligomer to monomer transitions and vice versa are at least in part mediated by changes in surface hydrophobicity. Additionally, we find that the Ca2+ binding affinity of EF-SAM is relatively low with an apparent dissociation constant (Kd) of approximately 0.2-0.6 mM and a binding stoichiometry of 1. Our results suggest that EF-SAM actively participates in and is the likely the molecular trigger initiating STIM1 punctae formation via large conformational changes. The low Ca2+ affinity of EF-SAM is reconciled with the confirmed role of STIM1 as an ER Ca2+ sensor.

Protective and Immunochemical Activities of Monoclonal Antibodies Reactive with theBacillus anthracisPolypeptide Capsule

Bacillus anthracis is surrounded by a polypeptide capsule composed of poly-gamma-d-glutamic acid (gammaDPGA). In a previous study, we reported that a monoclonal antibody (MAb F26G3) reactive with the capsular polypeptide is protective in a murine model of pulmonary anthrax. The present study examined a library of six MAbs generated from mice immunized with gammaDPGA. Evaluation of MAb binding to the capsule by a capsular "quellung" type reaction showed a striking diversity in capsular effects. Most MAbs produced a rim type reaction that was characterized by a sharp increase followed directly by a decrease in refractive index at the capsular edge. Some MAbs produced a second capsular reaction well beneath the capsular edge, suggesting complexity in capsular architecture. Binding of MAbs to soluble gammaDPGA was assessed by a fluorescence perturbation assay in which a change in the MAb intrinsic fluorescence produced by ligand binding was used as a reporter for antigen-antibody interaction. The MAbs differed considerably in the complexity of the binding curves. MAbs producing rim type capsule reactions typically produced the more complex binding isotherms. Finally, the protective activity of the MAbs was compared in a murine model of pulmonary anthrax. One MAb was markedly less protective than the remaining five MAbs. Characteristics of the more protective MAbs included a relatively high affinity, an immunoglobulin G3 isotype, and a complex binding isotherm in the fluorescence perturbation assay. Given the relatively monotonous structure of gammaDPGA, the results demonstrate a striking diversity in the antigen binding behavior of gammaDPGA antibodies.

Two conserved tryptophan residues are responsible for intrinsic fluorescence enhancement in Rubisco activase upon ATP binding

Two species-invariant tryptophan residues at positions 109 and 250 of tobacco Rubisco activase were identified by site-directed mutagenesis as being responsible for the increase in intrinsic fluorescence upon addition of ATP, which has been previously attributed to increased self-association. Substitution of W109, which is immediately prior to a 'P-loop' sequence in the ATP catalytic motif, with aromatic residues (Tyr or Phe), Cys or Lys eliminated both ATP hydrolysis and the intrinsic fluorescence enhancement. Although the W109 mutants bound ATP, ATP did not provide a partial protection against proteolysis by trypsin that was observed with the recombinant wild-type enzyme. In contrast, substitution of W250 with Tyr or Phe abolished about half (44%) of the increase in intrinsic fluorescence with ATP, but had little effect on ATP hydrolysis, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activation or proteolytic protection with ATP. The substitution of the other tryptophan residues, W16 and W305, with phenylalanine did not significantly alter the change in intrinsic fluorescence upon addition of ATP. Therefore, W109 and W250 are the residues reporting the conformational change that increases the intrinsic fluorescence.

Discovery of Different Types of Inhibition between the Human and Thermotoga maritima α-Fucosidases by Fuconojirimycin-Based Derivatives

An efficient method for examining the selectivity of inhibitors on two alpha-fucosidases, one from Thermotoga maritima and the other from human, was established. The X-ray crystal structure of the former enzyme makes possible the homology modeling of the human alpha-fucosidase, indicating the major difference between both enzymes in the periphery of the catalytic site. To investigate the difference at the molecular level, a variety of fuconojirimycin (FNJ) derivatives with substitution at C1, C2, C6, or N were rapidly prepared in microplates and screened without purification for the inhibition activities of the two alpha-fucosidases. Among the molecules that were tested, only the substitution at C1 can significantly enhance the inhibitory potency, in contrast to the control (no substitution) and compounds with substitution at other positions. The majority of C1-substituted FNJs were found to be slow tight-binding inhibitors of the Thermotoga enzyme, while acting as the reversible inhibitors of the human fucosidase. The best inhibitor exhibited 13,700-fold difference in affinity between the two enzymes, which was attributed to the dissimilar aglycon binding site. Further investigations were carried out, including site-directed mutagenesis, the comparison of K(i) values among the wild type and mutants, and the intrinsic fluorescence change upon inhibitor titration, all supporting the idea that Tyr64 and Tyr267 of the Thermotoga alpha-fucosidase are critically involved in closely interacting with the aglycon of inhibitors. The increased level of contact thus induced conformational change, leading to the observed slow tight-binding inhibition.

Direct binding of recombinant plasminogen kringle 1–3 to angiogenin inhibits angiogenin-induced angiogenesis in the chick embryo CAM

Angiogenin is one of the most potent angiogenesis-inducing proteins. Angiostatin is one of the most potent angiogenesis inhibitors, and it contains the first four kringle domains of plasminogen (K1–4). Recombinant human plasminogen kringle 1–3 (rK1–3) was expressed in Escherichia coli and purified to homogeneity. The binding of t-4-aminomethylcyclohexanecarboxylic acid with the purified kringle 1–3 was determined by changes in intrinsic fluorescence. rK1–3 exhibits comparable ligand-binding properties as native human plasminogen kringle 1–3. The purified rK1–3 inhibits neovascularization in the chick embryo chorioallantoic membrane (CAM) assay. Interaction of angiogenin with rK1–3 was examined by immunological binding assay and surface plasmon resonance kinetic analysis, and the equilibrium dissociation constants for the complex, K d, are 0.89 and 0.18 μM, respectively. rK1–3 inhibits angiogenin-induced angiogenesis in the chick embryo CAM in a concentration-dependent manner. These results indicate that rK1–3 directly binds to angiogenin and thus rK1–3 inhibits the angiogenic activity of angiogenin.

Several Archaeal Homologs of Putative Oligopeptide-Binding Proteins Encoded byThermotoga maritimaBind Sugars

The hyperthermophilic bacterium Thermotoga maritima has shared many genes with archaea through horizontal gene transfer. Several of these encode putative oligopeptide ATP binding cassette (ABC) transporters. We sought to test the hypothesis that these transporters actually transport sugars by measuring the substrate affinities of their encoded substrate-binding proteins (SBPs). This information will increase our understanding of the selective pressures that allowed this organism to retain these archaeal homologs. By measuring changes in intrinsic fluorescence of these SBPs in response to exposure to various sugars, we found that five of the eight proteins examined bind to sugars. We could not identify the ligands of the SBPs TM0460, TM1150, and TM1199. The ligands for the archaeal SBPs are TM0031 (BglE), the beta-glucosides cellobiose and laminaribiose; TM0071 (XloE), xylobiose and xylotriose; TM0300 (GloE), large glucose oligosaccharides represented by xyloglucans; TM1223 (ManE), beta-1,4-mannobiose; and TM1226 (ManD), beta-1,4-mannobiose, beta-1,4-mannotriose, beta-1,4-mannotetraose, beta-1,4-galactosyl mannobiose, and cellobiose. For comparison, seven bacterial putative sugar-binding proteins were examined and ligands for three (TM0595, TM0810, and TM1855) were not identified. The ligands for these bacterial SBPs are TM0114 (XylE), xylose; TM0418 (InoE), myo-inositol; TM0432 (AguE), alpha-1,4-digalactouronic acid; and TM0958 (RbsB), ribose. We found that T. maritima does not grow on several complex polypeptide mixtures as sole sources of carbon and nitrogen, so it is unlikely that these archaeal ABC transporters are used primarily for oligopeptide transport. Since these SBPs bind oligosaccharides with micromolar to nanomolar affinities, we propose that they are used primarily for oligosaccharide transport.

Fast kinetics of calcium dissociation from calsequestrin

We measured the kinetics of calcium dissociation from calsequestrin in solution or forming part of isolated junctional sarcoplasmic reticulum membranes by mixing calsequestrin equilibrated with calcium with calcium-free solutions in a stopped-flow system. In parallel, we measured the kinetics of the intrinsic fluorescence changes that take place following calcium dissociation from calsequestrin. We found that at 25 degrees C calcium dissociation was 10-fold faster for calsequestrin attached to junctional membranes (k = 109 s(-1)) than in solution. These results imply that calcium dissociation from calsequestrin in vivo is not rate limiting during excitation-contraction coupling. In addition, we found that the intrinsic fluorescence decrease for calsequestrin in solution or forming part of junctional membranes was significantly slower than the rates of calcium dissociation. The kinetics of intrinsic fluorescence changes had two components for calsequestrin associated to junctional membranes and only one for calsequestrin in solution; the faster component was 8-fold faster (k = 54.1 s(-1)) than the slower component (k = 6.9 s(-1)), which had the same k value as for calsequestrin in solution. These combined results suggest that the presence of calsequestrin at high concentrations in a restricted space, such as when bound to the junctional membrane, accelerates calcium dissociation and the resulting structural changes, presumably as a result of cooperative molecular interactions.

Intrinsic fluorescence and redox changes associated with apoptosis of primary human epithelial cells

Apoptosis plays a key role in the development and maintenance of human tissues. This process has been studied traditionally in cells that are stained with exogenous fluorophores. These approaches affect cell viability, and thus are ill-suited for in vivo applications. We present an imaging approach that can identify apoptotic cells in living cell populations based on detection and quantification of distinct changes in the intensity and localization of cellular autofluorescence. Specifically, we acquire NAD(P)H, FAD, and redox ratio autofluorescence images of primary keratinocytes following 1, 9, 14, and 18 h of treatment with cisplatin, a known apoptosis-inducing chemotherapy agent. We find that intense autofluorescence combined with a low redox fluorescence ratio is progressively confined to a gradually smaller perinuclear cytoplasmic region with cisplatin treatment. Studies with exogenous nuclear fluorophores demonstrate that these autofluorescence changes occur at early stages of apoptosis. Additional costaining experiments suggest that this strongly autofluorescent, highly metabolically active perinuclear ring represents a subpopulation of mitochondria that are mobilized in response to the apoptotic stimulus and may provide the energy required to execute the final apoptotic steps. Thus, autofluorescence localization changes could serve as a sensitive, noninvasive indicator of early apoptosis in vivo.

Changes in FAD and NADH Fluorescence in Neurosecretory Terminals Are Triggered by Calcium Entry and by ADP Production

We measured changes in the intrinsic fluorescence (IF) of the neurosecretory terminals of the mouse neurohypophysis during brief (1-2 s) trains of stimuli. With fluorescence excitation at either 350 +/- 20 or 450 +/- 50 nm, and with emission measured, respectively, at 450 +/- 50 or > or = 520 nm, DeltaF/F(o) was approximately 5-8 % for a 2 s train of 30 action potentials. The IF changes lagged the onset of stimulation by approximately 100 ms and were eliminated by 1 microM tetrodotoxin (TTX). The signals were partially inhibited by 500 microM Cd(2+), by substitution of Mg(2+) for Ca(2+), by Ca(2+)-free Ringer's with 0.5 mM EGTA, and by 50 microM ouabain. The IF signals were also sensitive to the mitochondrial metabolic inhibitors CCCP (0.3 microM), FCCP (0.3 microM), and NaN(3) (0.3 mM), and their amplitude reflected the partial pressure of oxygen (pO(2)) in the bath. Resting fluorescence at both 350 nm and 450 nm exhibited significant bleaching. Flavin adenine dinucleotide (FAD) is fluorescent, while its reduced form FADH(2) is relatively non-fluorescent; conversely, NADH is fluorescent, while its oxidized form NAD is non-fluorescent. Thus, our experiments suggest that the stimulus-coupled rise in [Ca(2+)](i) triggers an increase in FAD and NAD as FADH(2) and NADH are oxidized, but that elevation of [Ca(2+)](i), alone cannot account for the totality of changes in intrinsic fluorescence.

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

Vertebrate Myosin VIIb Is a High Duty Ratio Motor Adapted for Generating and Maintaining Tension

Kinetic adaptation of muscle and non-muscle myosins plays a central role in defining the unique cellular functions of these molecular motor enzymes. The unconventional vertebrate class VII myosin, myosin VIIb, is highly expressed in polarized cells and localizes to highly ordered actin filament bundles such as those found in the microvilli of the intestinal brush border and kidney. We have cloned mouse myosin VIIb from a cDNA library, expressed and purified the catalytic motor domain, and characterized its actin-activated ATPase cycle using quantitative equilibrium and kinetic methods. The myosin VIIb steady-state ATPase activity is slow (approximately 1 s(-1)), activated by very low actin filament concentrations (K(ATPase) approximately 0.7 microm), and limited by ADP release from actomyosin. The slow ADP dissociation rate constant generates a long lifetime of the strong binding actomyosin.ADP states. ADP and actin binding is uncoupled, which enables myosin VIIb to remain strongly bound to actin and ADP at very low actin concentrations. In the presence of 2 mm ATP and 2 microm actin, the duty ratio of myosin VIIb is approximately 0.8. The enzymatic properties of actomyosin VIIb are suited for generating and maintaining tension and favor a role for myosin VIIb in anchoring membrane surface receptors to the actin cytoskeleton. Given the high conservation of vertebrate class VII myosins, deafness phenotypes arising from disruption of normal myosin VIIa function are likely to reflect a loss of tension in the stereocilia of inner ear hair cells.

A new type of uncompetitive inhibition of tyrosinase induced by Cl – binding

A new type of Cl – induced inhibition of mushroom derived tyrosinase has been detected in this study, and it is defined as the reversible partial hyperbolic uncompetitive inhibition. The Cl – binding site was only induced at the state of the enzyme–substrate complex, and this was confirmed with the intrinsic fluorescence changes. As the oxygen bridge is broken by L-DOPA binding, Cl – simultaneously binds to the ES state to form the ESI complex. It is worth noticing that tyrosinase reacts sensitively to Cl – in the manner of a complex interaction, and this indicates that Cl – might be physiologically involved in the regulation of tyrosinase activity.

Dimethyl sulfoxide targets phage RNA polymerases to promote transcription

Dimethyl sulfoxide (DMSO) is a “universal” solvent in pharmaceutical sciences and cell biology. DMSO was previously reported to facilitate in vitro transcription of chromatin and supercoiled plasmid, with the underlying mechanism being attributed to the alteration of the template structure. Here, we demonstrated that low concentrations of DMSO significantly increase the phage polymerase-catalyzed RNA synthesis when the naked and short PCR products were used as templates, suggesting that DMSO promotes transcription through additional mechanism(s). Interestingly, SP6 RNA polymerase was more sensitive to the DMSO stimulation than T7 RNA polymerase, suggesting that the polymerase is an important target for DMSO stimulation of RNA synthesis. Consistent with this finding, we also showed that DMSO dramatically elevated the RNA polymerase activity. This elevated activity is explained by altered polymerase structure as reflected by a change in intrinsic fluorescence. Furthermore, DMSO was shown to simultaneously accumulate both the abortive and full-length transcripts, leading us to conclude that the DMSO-altered polymerase structure primarily encodes an enhanced activity at the stage of transcription initiation. DMSO-induced alteration of the polymerase structure and function highlights a potentially generalized mechanism in interpreting the molecular action of this popular solvent.

Probing the Mechanism of the Archaeoglobus fulgidus Inositol-1-phosphate Synthase

myo-Inositol-1-phosphate synthase (mIPS) catalyzes the conversion of glucose-6-phosphate (G-6-P) to inositol-1-phosphate. In the sulfate-reducing archaeon Archaeoglobus fulgidus it is a metal-dependent thermozyme that catalyzes the first step in the biosynthetic pathway of the unusual osmolyte di-myo-inositol-1,1'-phosphate. Several site-specific mutants of the archaeal mIPS were prepared and characterized to probe the details of the catalytic mechanism that was suggested by the recently solved crystal structure and by the comparison to the yeast mIPS. Six charged residues in the active site (Asp225, Lys274, Lys278, Lys306, Asp332, and Lys367) and two noncharged residues (Asn255 and Leu257) have been changed to alanine. The charged residues are located at the active site and were proposed to play binding and/or direct catalytic roles, whereas noncharged residues are likely to be involved in proper binding of the substrate. Kinetic studies showed that only N255A retains any measurable activity, whereas two other mutants, K306A and D332A, can carry out the initial oxidation of G-6-P and reduction of NAD+ to NADH. The rest of the mutant enzymes show major changes in binding of G-6-P (monitored by the 31P line width of inorganic phosphate when G-6-P is added in the presence of EDTA) or NAD+ (detected via changes in the protein intrinsic fluorescence). Characterization of these mutants provides new twists on the catalytic mechanism previously proposed for this enzyme.