Intrinsic Fluorescence Measurements Research Articles

Skin Intrinsic Fluorescence Is Associated With Hemoglobin A1c and Hemoglobin Glycation Index but Not Mean Blood Glucose in Children With Type 1 Diabetes

OBJECTIVETo evaluate the relationship between skin advanced glycation end products (sAGEs) with mean blood glucose (MBG), hemoglobin A1c (HbA1c), and MBG-independent, between-patient differences in HbA1c among children with type 1 diabetes.RESEARCH DESIGN AND METHODSChildren aged 5 to 20 years with type 1 diabetes of at least 1 year duration participated. At a clinic visit, sAGE was estimated noninvasively by measurement of skin intrinsic fluorescence (SIF). SIF data were adjusted to correct for variation in skin pigmentation. MBG-independent, between-patient differences in HbA1c were examined by statistically controlling HbA1c for MBG or alternatively by use of a hemoglobin glycation index (HGI). Results were similar whether HbA1c, MBG, and HGI were analyzed as single values from the time of the SIF examination visit or as the mean values from all available visits of the patient.RESULTSHbA1c was correlated with MBG (r = 0.5; P < 0.001; n = 110). HbA1c and HGI, but not MBG, were statistically associated with SIF after adjustment for age, duration of diabetes, race, sex, and BMI z-score. SIF increased with age and duration of diabetes and was higher in girls than boys.CONCLUSIONSsAGE levels estimated by SIF increase with age, duration of diabetes, and female sex. sAGE is correlated with MBG-independent biological variation in HbA1c, but not with MBG itself. These results suggest that factors besides MBG that influence HbA1c levels also contribute to accumulation of sAGE.

Stability of haloalkaliphilic Geomicrobium sp. protease modulated by salt

A novel protease from the halophilic bacterium Geomicrobium sp. EMB2 (MTCC 10310) is described. The activity of the protease was modulated by salt, and it exhibited remarkable stability in organic solvents, at alkaline pH, and in other denaturing conditions. The structural changes under various denaturing conditions were analyzed by measurements of intrinsic fluorescence and circular dichroism spectroscopy. Circular dichroism showed that the secondary structure of the protease was predominantly α-helical but unfolded in salt-free medium. The structure is regained by inclusion of NaCl in the range of 2-5%. The presence of NaCl exerted a protective effect against thermal, organic solvent, and guanidine hydrochloride denaturation by preventing unfolding.

Glucuronoxylomannan from Cryptococcus neoformans Down-regulates the Enzyme 6-Phosphofructo-1-kinase of Macrophages

The encapsulated yeast Cryptococcus neoformans is the causative agent of cryptococosis, an opportunistic life-threatening infection. C. neoformans is coated by a polysaccharide capsule mainly composed of glucuronoxylomannan (GXM). GXM is considered a key virulence factor of this pathogen. The present work aimed at evaluating the effects of GXM on the key glycolytic enzyme, 6-phosphofructo-1-kinase (PFK). GXM inhibited PFK activity in cultured murine macrophages in both dose- and time-dependent manners, which occurred in parallel to cell viability decrease. The polysaccharide also inhibited purified PFK, promoting a decrease on the enzyme affinity for its substrates. In macrophages GXM and PFK partially co-localized, suggesting that internalized polysaccharide directly may interact with this enzyme. The mechanism of PFK inhibition involved dissociation of tetramers into weakly active dimers, as revealed by fluorescence spectroscopy. Allosteric modulators of the enzyme able to stabilize its tetrameric conformation attenuated the inhibition promoted by GXM. Altogether, our results suggest that the mechanism of GXM-induced cell death involves the inhibition of the glycolytic flux.

Tyrosinase inhibition by isophthalic acid: Kinetics and computational simulation

Using inhibition kinetics and computational simulation, we studied the reversible inhibition of tyrosinase by isophthalic acid (IPA). IPA inhibited tyrosinase in a complex manner with Ki=17.8±1.8mM. Measurements of intrinsic and ANS-binding fluorescence showed that IPA induced no changes in tertiary protein structure. For further insight, we predicted the 3D structure of tyrosinase and used a docking algorithm to simulate binding between tyrosinase and IPA. Simulation was successful (binding energies for Dock6.3: −25.19kcal/mol and for AutoDock4.2: −4.28kcal/mol), suggesting that IPA interacts with PRO175 or VAL190. This strategy of predicting tyrosinase inhibition based on hydroxyl group number and orientation may prove useful for the screening of potential tyrosinase inhibitors.

Parallel detection of intrinsic fluorescence from peptides and proteins for quantification during mass spectrometric analysis.

Direct mass spectrometric quantification of peptides and proteins is compromised by the wide variabilities in ionization efficiency which are hallmarks of both the MALDI and ESI ionization techniques. We describe here the implementation of a fluorescence detection system for measurement of the UV-excited intrinsic fluorescence (UV-IF) from peptides and proteins just prior to their exit and electrospray ionization from an ESI capillary. The fluorescence signal provides a quantifiable measure of the amount of protein or peptide present, while direct or tandem mass spectrometric analysis (MS/MS) on the ESI-generated ions provides information on identity. We fabricated an inexpensive, modular fluorescence excitation and detection device utilizing an ultraviolet light-emitting diode for excitation in a ∼300 nL fluorescence detection cell integrated into the fused-silica separation column. The fluorescence signal is linear over 3 orders of magnitude with on-column limits of detection in the low femtomole range. Chromatographically separated intact proteins analyzed using UV-IF prior to top-down mass spectrometry demonstrated sensitive detection of proteins as large as 77 kDa.

Role of N-Myristoylation of Camp-Dependent Protein Kinase a in Recognition and Phosphorylation of Membrane-Bound Substrates

The catalytic subunit of cAMP-dependent protein kinase A (PKA-C), controls the activation and deactivation of over 100 unique cytosolic and membrane bound substrates. Nearly all the PKA-C in mammals is subjected to myristoylation and myristoylation can be dependent on cell/tissue type. Although the function of myristoylation is not known, it has been hypothesized to mediate PKA-C/membrane and PKA-C/membrane protein interactions. Here, we developed an in vitro myristoylation method to obtain homogeneously myristoylated PKA-C (myrPKA-C) in quantities suitable for NMR studies. Using NMR spectroscopy, the interactions of membrane mimicking isotropic bicelles (DMPC/DHPC) with 15N-labeled myrPKA-C or 15N-labeled non-myristoylated PKA-C was monitored by [1H-15N]-TROSY-HSQC spectroscopy. Significant chemical shift perturbations were detected for the N-terminus and the myristate pocket of myrPKA-C, while both perturbations and line broadening was detected in remote regions far away from the myristoyl binding pocket. Such changes were not observed for the non-myristoylated form, suggesting that myristoylation drives the enzyme towards the membrane and may act allosterically on the enzyme. Membrane localization was further supported by intrinsic tryptophan fluorescence measurements which showed drastically different patterns between myrPKA-C and PKA-C. Finally, the insertion of the myristate group into lipid membranes was confirmed using 2H solid-state NMR spectroscopy on myrPKA-C with a deuterated myristoyl group. We propose that the myristoyl group of myrPKA-C steers the enzyme towards the membrane, even in the absence of its regulatory subunit or A-kinase anchoring proteins (AKAPs). These results provide an additional mechanism for PKA localization and recognition of membrane bound substrates. Preliminary data on PKA recognition of the integral membrane protein, phospholamban, are also presented.

Thermodynamic studies on the interaction of folic acid with bovine serum albumin

Binding of the vitamin folic acid with bovine serum albumin (BSA) has been studied using isothermal titration calorimetry (ITC) in combination with fluorescence and circular dichroism spectroscopies. The thermodynamic parameters of binding have been evaluated as a function of temperature, ionic strength, in the presence of nonionic surfactants triton X-100, tetrabutylammonium bromide, and sucrose. The values of the van’t Hoff enthalpy calculated from the temperature dependence of the binding constant agree with the calorimetric enthalpies indicating that the binding of folic acid to the BSA is a two state process without involving intermediates. These observations are supported by the intrinsic fluorescence and circular dichroism spectroscopic measurements. With increase in the ionic strength, reduction in the binding affinity of folic acid to BSA is observed suggesting predominance of electrostatic interactions in the binding. The contribution of hydrophobic interactions in the binding is also demonstrated by decrease in the binding affinity in the presence of tetrabutylammonium bromide (TBAB). The value of binding affinity in the presence of sucrose indicates that hydrogen bonding also plays a significant contribution in the complexation process. The calorimetric and spectroscopic results provide quantitative information on the binding of folic acid to BSA and suggest that the binding is dominated by electrostatic interactions with contribution from hydrogen bonding.

FYVE-Dependent Endosomal Targeting of an Arrestin-Related Protein in Amoeba

BackgroundVisual and β-arrestins are scaffolding proteins involved in the regulation of receptor-dependent intracellular signaling and their trafficking. The arrestin superfamilly includes several arrestin domain-containing proteins and the structurally related protein Vps26. In Dictyostelium discoideum, the arrestin-domain containing proteins form a family of six members, namely AdcA to -F. In contrast to canonical arrestins, Dictyostelium Adc proteins show a more complex architecture, as they possess, in addition to the arrestin core, other domains, such as C2, FYVE, LIM, MIT and SAM, which potentially mediate selective interactions with either lipids or proteins.Methodology and Principal FindingsA detailed analysis of AdcA has been performed. AdcA extends on both sides of the arrestin core, in particular by a FYVE domain which mediates selective interactions with PI(3)P, as disclosed by intrinsic fluorescence measurements and lipid overlay assays. Localization studies showed an enrichment of tagged- and endogenous AdcA on the rim of early macropinosomes and phagosomes. This vesicular distribution relies on a functional FYVE domain. Our data also show that the arrestin core binds the ADP-ribosylation factor ArfA, the unique amoebal Arf member, in its GDP-bound conformation.SignificanceThis work describes one of the 6 arrestin domain-containing proteins of Dictyostelium, a novel and atypical member of the arrestin clan. It provides the basis for a better understanding of arrestin-related protein involvement in trafficking processes and for further studies on the expanding roles of arrestins in eukaryotes.

Structural autonomy of a β-hairpin peptide derived from the pneumococcal choline-binding protein LytA

The cell wall of Streptococcus pneumoniae and several other micro-organisms is decorated with a number of the so-called choline-binding proteins (CBPs) that recognise the choline residues in the bacterial surface by means of highly conserved, concatenated 20-aa sequences termed choline-binding repeats (CBRs), that are composed of a loop and a β-hairpin structure. In this work, we have investigated the ability to fold in aqueous solution of a 14-aa peptide (LytA₁₉₇₋₂₁₀[wt]) and a single derivative of it, LytA₁₉₇₋₂₁₀[ND], corresponding to one of the six β-hairpins of the LytA pneumococcal amidase. Intrinsic fluorescence and circular dichroism spectroscopical measurements showed that both peptides spontaneously acquire a non-random conformation which is also able to bind the natural ligand choline. Furthermore, nuclear magnetic resonance techniques allowed the calculation of the structure of the LytA₁₉₇₋₂₁₀[ND] peptide, which displayed a β-hairpin conformation highly similar to that found within the full-length C-LytA module. These results provide a structural basis for the modular organisation of CBPs and suggest the use of CBRs as new templates for the design of stable β-hairpins.

Integrated biophysical studies implicate partial unfolding of NBD1 of CFTR in the molecular pathogenesis of F508del cystic fibrosis

The lethal genetic disease cystic fibrosis is caused predominantly by in-frame deletion of phenylalanine 508 in the cystic fibrosis transmembrane conductance regulator (CFTR). F508 is located in the first nucleotide-binding domain (NBD1) of CFTR, which functions as an ATP-gated chloride channel on the cell surface. The F508del mutation blocks CFTR export to the surface due to aberrant retention in the endoplasmic reticulum. While it was assumed that F508del interferes with NBD1 folding, biophysical studies of purified NBD1 have given conflicting results concerning the mutation's influence on domain folding and stability. We have conducted isothermal (this paper) and thermal (accompanying paper) denaturation studies of human NBD1 using a variety of biophysical techniques, including simultaneous circular dichroism, intrinsic fluorescence, and static light-scattering measurements. These studies show that, in the absence of ATP, NBD1 unfolds via two sequential conformational transitions. The first, which is strongly influenced by F508del, involves partial unfolding and leads to aggregation accompanied by an increase in tryptophan fluorescence. The second, which is not significantly influenced by F508del, involves full unfolding of NBD1. Mg-ATP binding delays the first transition, thereby offsetting the effect of F508del on domain stability. Evidence suggests that the initial partial unfolding transition is partially responsible for the poor in vitro solubility of human NBD1. Second-site mutations that increase the solubility of isolated F508del-NBD1 in vitro and suppress the trafficking defect of intact F508del-CFTR in vivo also stabilize the protein against this transition, supporting the hypothesize that it is responsible for the pathological trafficking of F508del-CFTR.

Spectroscopic studies on the pH-dependent structural dynamics of γ-conglutin, the blood glucose-lowering protein of lupin seeds

γ-Conglutin is a blood glucose-lowering protein purified from lupin ( Lupinus albus, L.) seed. Despite various features of this protein have already been studied, no function in the seed nor any mechanism of action as a hypoglycemic nutraceutical compound have been identified so far. The lupin protein was shown to exist both in monomeric and multimeric forms as a function of pH. However, a detailed description of the pH-dependent structural dynamics of this protein, as the basis to investigate the reason/s of its functional behaviour, is not available yet. In this study, multiple and independent spectroscopic approaches, including light scattering associated to size exclusion chromatography of both untreated and covalently cross-linked protein, near and far UV circular dichroism, intrinsic and extrinsic fluorescence measurements, have been used to monitor oligomeric and conformational modifications caused by pH changes. Altogether, the results revealed a tetramer–dimer–monomer transition between neutral to slightly acidic pH and a dramatic and abrupt conformational change below pH 3.5. According to these findings, a model depicting γ-conglutin structural dynamics was drawn. This model highlights the primary role of amino acid side group electrostatic interactions in the oligomer association/dissociation equilibria and in the pH-driven collapse of the native conformation.

Protein denaturation and protein:drugs interactions from intrinsic protein fluorescence measurements at the nanolitre scale

Protein stability and ligand-binding affinity measurements are widely required for the formulation of biopharmaceutical proteins, protein engineering and drug screening within life science research. Current techniques either consume too much of often precious biological or compound materials, in large sample volumes, or alternatively require chemical labeling with fluorescent tags to achieve measurements at submicrolitre volumes with less sample. Here we present a quantitative and accurate method for the determination of protein stability and the affinity for small molecules, at only 1.5-20 nL optical sample volumes without the need for fluorescent labeling, and that takes advantage of the intrinsic tryptophan fluorescence of most proteins. Coupled to appropriate microfluidic sample preparation methods, the sample requirements could thus be reduced 85,000-fold to just 10(8) molecules. The stability of wild-type FKBP-12 and a destabilizing binding-pocket mutant are studied in the presence and absence of rapamycin, to demonstrate the potential of the technique to both drug screening and protein engineering. The results show that 75% of the interaction energy between FKBP-12 and rapamycin originates from residue Phe99 in the binding site.

Zinc Modulates the Interaction of Protein C and Activated Protein C with Endothelial Cell Protein C Receptor

Zinc is an essential trace element for human nutrition and is critical to the structure, stability, and function of many proteins. Zinc ions were shown to enhance activation of the intrinsic pathway of coagulation but down-regulate the extrinsic pathway of coagulation. The protein C pathway plays a key role in blood coagulation and inflammation. At present there is no information on whether zinc modulates the protein C pathway. In the present study we found that Zn(2+) enhanced the binding of protein C/activated protein C (APC) to endothelial cell protein C receptor (EPCR) on endothelial cells. Binding kinetics revealed that Zn(2+) increased the binding affinities of protein C/APC to EPCR. Equilibrium dialysis with (65)Zn(2+) revealed that Zn(2+) bound to the Gla domain as well as sites outside of the Gla domain of protein C/APC. Intrinsic fluorescence measurements suggested that Zn(2+) binding induces conformational changes in protein C/APC. Zn(2+) binding to APC inhibited the amidolytic activity of APC, but the inhibition was reversed by Ca(2+). Zn(2+) increased the rate of APC generation on endothelial cells in the presence of physiological concentrations of Ca(2+) but did not further enhance increased APC generation obtained in the presence of physiological concentrations of Mg(2+) with Ca(2+). Zn(2+) had no effect on the anticoagulant activity of APC. Zn(2+) enhanced APC-mediated activation of protease activated receptor 1 and p44/42 MAPK. Overall, our data show that Zn(2+) binds to protein C/APC, which results in conformational changes in protein C/APC that favor their binding to EPCR.

Mutational analysis of the proposed calcium-binding aspartates of a truncated α-amylase from Bacillus sp. strain TS-23

The role of the aspartate residues Asp193, Asp217, Asp228, Asp234, Asp236, Asp238, Asp438, and Asp461 of a truncated Bacillus sp. strain TS-23 α-amylase (BACΔNC) was investigated by site-directed mutagenesis. These residues were replaced with Asn to generate D193N, D217N, D228N, D234N, D236N, D238N, D438N, and D461N, respectively. Parental and mutant amylases were purified to homogeneity by nickel-chelated chromatography, and the purified enzymes had a molecular mass of approximately 54 kDa. The amylolytic activity of purified BACΔNC was abolished completely upon the addition of 15 mM EDTA. D438N and D461N were very similar to BACΔNC in terms of specific activity, temperature–activity profiles, and kinetic parameters, whereas the remaining mutant enzymes showed a dramatic reduction in both catalytic efficiency (k cat/K M) and thermostability. Compared with BACΔNC, measurement of intrinsic tryptophan fluorescence revealed the minor alterations of the microenvironment of aromatic amino acid residues in all of mutant enzymes. Far-UV circular dichroism spectra were nearly identical for the parental and mutant enzymes, but D193N, D217N, D228N, D234N, D236N, and D238N exhibited a different sensitivity towards temperature-induced denaturation. This implicates that the rigidity of the enzyme has been changed as the consequence of Asp193, Asp217, Asp228, Asp234, Asp236, and Asp238 mutations.

Equilibrium unfolding of kinetically stable serine protease milin: the presence of various active and inactive dimeric intermediates

Kinetically stable homodimeric serine protease milin reveals high conformational stability against temperature, pH and chaotrope [urea, guanidine hydrochloride (GuHCl) and guanidine isothiocynate (GuSCN)] denaturation as probed by circular dichroism, fluorescence, differential scanning calorimetry and activity measurements. GuSCN induces complete unfolding in milin, whereas temperature, urea and GuHCl induce only partial unfolding even at low pH, through several intermediates with distinct characteristics. Some of these intermediates are partially active (viz. in urea and 2 M GuHCl at pH 7.0), and some exhibited strong ANS binding as well. All three tryptophans in the protein seem to be buried in a rigid, compact core as evident from intrinsic fluorescence measurements coupled to equilibrium unfolding experiments. The protein unfolds as a dimer, where the unfolding event precedes dimer dissociation as confirmed by hydrodynamic studies. The solution studies performed here along with previous biochemical characterization indicate that the protein has alpha-helix and beta-sheet rich regions or structural domains that unfold independently, and the monomer association is isologous. The complex unfolding pathway of milin and the intermediates has been characterized. The physical, physiological and probable therapeutic importance of the results has been discussed.

Energy transfer ligands of the GluR2 ligand binding core.

Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate excitatory signaling in the central nervous system. When a ligand binds to the extracellular domain of iGluRs, local conformational changes ensue and this motion is translated to the transmembrane domain, inducing channel opening. We have used an isolated ligand binding domain, GluR2-S1S2J (GluR2), as a model system to study the protein-ligand complex by steady-state and time-resolved intrinsic tryptophan fluorescence measurements. Specifically, we determined that the widely used and structurally characterized antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX), acts as an efficient fluorescence energy transfer (FET) acceptor for Trp. Consistent with crystallographic data, our results indicate that the four native tryptophans are within Forster's radius (R(o) approximately 33 A) of the bound ligand. Additionally, we demonstrate the broader value of this technique by identifying an original FET ligand, 3-nitrotyrosine (3NY), for GluR2 (R(o) approximately 24 A; apparent dissociation constant K(d) approximately 170 microM). Estimated average donor-acceptor (Trp-ligand) distances extracted from tryptophan excited-state decays are similar for both ligands (approximately 24 A), suggesting that 3NY binds in the structurally characterized ligand binding cleft. Moreover, an alternative competition assay utilizing Trp --> DNQX quenching for detection of ligand binding in GluR2 is described.

Recognition of B2GPI by the Lipoprotein Receptors.

Abstract 851Lipoprotein receptors of the LDLR family serve as clearance receptors for beta2-glycoprotein-I (B2GPI) and as signaling receptors for the B2GPI/antibody complexes in patients with antiphospholipid syndrome (APS). B2GPI binds lipoprotein receptors through its domain V (B2GPI-DV) and the lipoprotein receptors use the LA modules to interact with B2GPI. The LA modules, which are about 40 residues each, are arranged in sequence to form the ligand-binding domains of the receptors. The LA modules have little sequence homology both within a single receptor and between different receptors, but adopt a very similar three dimensional structure. We have investigated the structural features of the contact interface in the B2GPI-DV/LA complexes and the characteristic features of the LA modules required for efficient binding to B2GPI. Using solution NMR spectroscopy, we have demonstrated that three different LA modules bind to the same site on B2GPI-DV and that the binding site for B2GPI-DV on these LA modules is centered on the residues involved in coordination of the calcium ion. The dissociation constants measured for different LA complexes are in the range from 1 mM to approximately 100 mM. The difference in binding affinity is attributed to the auxiliary contacts that augment the interactions of the residues at the core of the binding interface. We have solved the structure of the B2GPI-DV/LA4 complex by molecular docking guided by NMR-derived restraints and have validated the structure by four independent experimental observations based on mutagenesis, solution NMR spectroscopy, fluorescence polarization and intrinsic tryptophan fluorescence measurements. The structure identified three lysine residues of B2GPI-DV, Lys308, Lys317 and Lys282, as critical for binding the LA modules. Our data shows that not all the LA modules can bind B2GPI-DV and that those LA modules capable of binding B2GPI-DV will interact with it in a similar fashion as observed in the B2GPI-DV/LA4 complex. Whether an LA module is capable of binding B2GPI could be distinguished based on the primary sequence in the vicinity of the calcium-coordinating residues. According to the current hypothetical model, the binding of B2GPI to anionic phospholipids in the presence of APS-related antibodies is the first event leading to the pathology of APS. However, we have shown that the LA modules interfere with binding of B2GPI-DV to cardiolipin indicating that B2GPI-DV cannot simultaneously bind to anionic phospholipids and lipoprotein receptors. Disclosures:No relevant conflicts of interest to declare.

Residues Leu52 and Leu134 are important for the structural integrity of a nucleotide exchange factor GrpE from Bacillus licheniformis

A DNA fragment encoding Bacillus licheniformis GrpE ( BlGrpE) with double mutations at codons 52 and 134 was obtained during PCR cloning. Leu52 and Leu134 in BlGrpE were individually replaced with Pro and His to generate BlGrpE-L52P and BlGrpE-L134H. BlGrpE and BlGrpE-L52P synergistically stimulated the ATPase activity of B. licheniformis DnaK ( BlDnaK); however, BlGrpE-L134H and the double-mutated protein ( BlGrpE-L52P/L134H) had no co-chaperone function. BlGrpE, BlGrpE-L52P, and BlGrpE-L134H mainly interacted with the monomer of BlDnaK but non-specific interaction was observed for BlGrpE-L52P/L134H. Measurement of intrinsic fluorescence revealed a significant alteration of the microenvironment of aromatic acid residues in the mutant proteins. As compared with BlGrpE, quenching of 208-nm and 222-nm signals were observed in the mutant BlGrpEs and the single-mutated proteins were more sensitive to thermal denaturation.

Deletion analysis of the C-terminal region of a molecular chaperone DnaK from Bacillus licheniformis

Bacillus licheniformis DnaK (BlDnaK) is predicted to consist of a 45-kDa N-terminal ATPase domain and a 25-kDa C-terminal substrate-binding domain. In this study, the full-length BlDnaK and its T86W and three C-terminally truncated mutants were constructed to evaluate the role of up to C-terminal 255 amino acids of the protein. The steady-state ATPase activity for BlDnaK, T86W, T86W/DeltaC120, T86W/DeltaC249, and T86W/DeltaC255 was 65.68, 53.21, 116.04, 321.38, and 90.59 nmol Pi/min per mg, respectively. In vivo, BldnaK, T86W and T86W/DeltaC120 genes allowed an E. coli dnaK756-ts mutant to grow at 44 degrees C. Except for T86W/DeltaC255, simultaneous addition of B. licheniformis DnaJ and GrpE, and NR-peptide synergistically stimulated the ATPase activity of BlDnaK, T86W, T86W/DeltaC120, and T86W/DeltaC249 by 16.9-, 13.9-, 33.9-, 9.9-fold, respectively. Measurement of intrinsic tryptophan fluorescence revealed significant alterations of microenvironment of aromatic amino acids in the C-terminally truncated mutants. The temperature-dependent signal in the far-UV region for T86W was consistent with that of BlDnaK, but the C-terminally truncated mutant proteins showed a higher sensitivity toward temperature-induced denaturation. These results suggest that C-terminal truncations alter the ATPase activity and thermal stability of BlDnaK and induce the conformation change of the ATPase domain.

Role of the Conserved Thr399 and Thr417 Residues of Bacillus licheniformis γ-Glutamyltranspeptidase as Evaluated by Mutational Analysis

Role of the conserved Thr399 and Thr417 residues of Bacillus licheniformis gamma-glutamyltranspeptidase (BlGGT) was investigated by site-directed mutagenesis. Substitutions of Thr399 and Thr417 of BlGGT with Ser resulted in a dramatic reduction in enzymatic activity. A complete loss of the GGT activity was observed in T399A, T399C, T417A, and T417K mutant enzymes. Furthermore, mutations on these two residues impaired the capability of autocatalytic processing of the enzyme. In vitro maturation experiments showed that BlGGT mutant precursors, pro-T399S, pro-T417S, and pro-T417A, could precede a time-dependent autocatalytic process to generate the 44.9- and 21.7-kDa subunits; however, the processed T417A had no enzymatic activity. Measurement of intrinsic tryptophan fluorescence revealed alteration of the microenvironment of aromatic amino acid residues, while Far-UV circular dichroism spectra were nearly identical for wild-type and mutant enzymes. These results suggest that residues Thr399 and Thr417 are important for BlGGT in the enzymatic maturation and reaction.