Tryptophan Fluorescence Emission Research Articles

Dipicolinic Acid Derivatives as Inhibitors of New Delhi Metallo-β-lactamase-1.

The efficacy of β-lactam antibiotics is threatened by the emergence and global spread of metallo-β-lactamase (MBL) mediated resistance, specifically New Delhi metallo-β-lactamase-1 (NDM-1). By utilization of fragment-based drug discovery (FBDD), a new class of inhibitors for NDM-1 and two related β-lactamases, IMP-1 and VIM-2, was identified. On the basis of 2,6-dipicolinic acid (DPA), several libraries were synthesized for structure-activity relationship (SAR) analysis. Inhibitor 36 (IC50 = 80 nM) was identified to be highly selective for MBLs when compared to other Zn(II) metalloenzymes. While DPA displayed a propensity to chelate metal ions from NDM-1, 36 formed a stable NDM-1:Zn(II):inhibitor ternary complex, as demonstrated by 1H NMR, electron paramagnetic resonance (EPR) spectroscopy, equilibrium dialysis, intrinsic tryptophan fluorescence emission, and UV-vis spectroscopy. When coadministered with 36 (at concentrations nontoxic to mammalian cells), the minimum inhibitory concentrations (MICs) of imipenem against clinical isolates of Eschericia coli and Klebsiella pneumoniae harboring NDM-1 were reduced to susceptible levels.

Synthesis, characterization and separation of chiral and achiral diastereomers of Schiff base Pd(II) complex: A comparative study of their DNA- and HSA-binding

A racemic mixture of a new chiral Schiff base ligand (HL: R/S-(1-phenylethylimino)methylnaphtalen-2-ol) has been utilized to prepare Pd(II) complex. Crystallization technique has been employed to separate diastereomeric pairs of Pd(II) complex: (mesoPdL2) and (racPdL2) that in this paper are known as PdL21 and PdL22, respectively. The synthesized complexes have been characterized by means of elemental analysis (CHN), FT-IR, 1H and 13C NMR spectroscopies. Moreover, PdL21 has been structurally characterized by single-crystal X-ray diffraction. The geometry around the metal center is square-planar. The interaction of two diastereomers of Pd(II) complex with FS-DNA has been explored, using UV–vis spectroscopy, fluorescence quenching, chemometrics and viscosity measurement methods. The PdL21 exhibited higher binding constant, about 10-fold, (1.0×106M−1) as compared to PdL22 (1.51.5×105M−1). Moreover, the human serum albumin (HSA) binding ability has been monitored by absorption, quenching of tryptophan fluorescence emission and circular dichroism (CD) studies. The slight difference is observed between HSA binding affinity with the complexes: PdL21 (6.2×104M−1) and PdL22 (3.3×104M−1). Also, the thermodynamic parameters were determined at three different temperatures (298, 308 and 318K).In this study, molecular docking was also carried out to confirm and illustrate the specific DNA- and HSA-binding of the Pd(II) complexes. In the PdL21-HSA system a T-shaped π-π interaction with PHE206 was observed. While in the PdL22-HSA system there are a hydrogen bond, a π-cation and two T-shaped π-π interactions with ASB324, LYS212 and PHE228, respectively. The groove binding mode of DNA interaction has been proposed for both diastereomers.

Beyond Dinitrophenol Interaction with Tryptophan-Based Compounds

Abstract The effects of 2,4-dinitrophenol (2,4-DNP) on the spectroscopic parameters (UV-Vis or FT-IR absorbance) or fluorescence emission of tryptophan and glycyl-tryptophan were studied. A quenching phenomenon of fluorescence was observed, attributed to interactions between the indole ring of the fluorophore and the aromatic ring of the quencher. The analysis of fluorescence spectra confirms that the quenching is dictated by 2,4-DNP concentration and pH. A combined mechanism of static and dynamic quenching was detected. The quenching phenomenon observed in this work could be employed to explain the mechanism of action of such compounds on large fluorescent peptides or proteins.

Application of Tryptophan Fluorescence Bandwidth-Maximum Plot in Analysis of Monoclonal Antibody Structure.

Monoclonal antibodies have become the fastest growing protein therapeutics in recent years. The stability and heterogeneity pertaining to its physical and chemical structures remain a big challenge. Tryptophan fluorescence has been proven to be a versatile tool to monitor protein tertiary structure. By modeling the tryptophan fluorescence emission envelope with log-normal distribution curves, the quantitative measure can be exercised for the routine characterization of monoclonal antibody overall tertiary structure. Furthermore, the log-normal deconvolution results can be presented as a two-dimensional plot with tryptophan emission bandwidth vs. emission maximum to enhance the resolution when comparing samples or as a function of applied perturbations. We demonstrate this by studying four different monoclonal antibodies, which show the distinction on emission bandwidth-maximum plot despite their similarity in overall amino acid sequences and tertiary structures. This strategy is also used to demonstrate the tertiary structure comparability between different lots manufactured for one of the monoclonal antibodies (mAb2). In addition, in the unfolding transition studies of mAb2 as a function of guanidine hydrochloride concentration, the evolution of the tertiary structure can be clearly traced in the emission bandwidth-maximum plot.

Elucidating the role of surfactant dispersed CNTs towards HSA fibrillation in vitro — A multiple spectroscopic approach

Nanoparticles could act as the efficient catalyst or the inhibitor towards the fibrillation pathway of biomolecules. In the present work, spectroscopic approaches including Thioflavin T fluorescence, Congo red dye absorbance, tryptophan fluorescence emission, three-dimensional fluorescence and circular dichroism spectra has been used to detect and evaluate the fibrillation induced conformational changes in HSA in the existence of CNTs. CR dye absorbance showed the red shift from 495 to 500nm upon binding with fibrils. Similarly, ThT fluorescence emission upon the excitation at 450nm showed the gradual increase in the intensity that conveyed the enhanced formation of fibrils in the presence of CNTs. Trp emission maxima obtained at 344nm gradually decreased with no prominent shift in the fibrillar samples. Changes in the 3D spectral peaks 1 and 2 of native and fibrillar solutions suggested the alteration in the aromatic amino acid residues micro-environment. CD results showed increased percentage contents of beta-sheet structures in the fibrillar solutions interacted with CNTs. Hence, the research outcome of the study conveys a promoting role of surfactant detached SW and MWCNTs against HSA fibrillation in vitro.

Arginine kinase shows nucleoside diphosphate kinase-like activity toward deoxythymidine diphosphate.

Arginine kinase (AK) (ATP: L-arginine phosphotransferase, E.C. 2.7.3.3) catalyzes the reversible transfer of ATP γ-phosphate group to L-arginine to synthetize phospho-arginine as a high-energy storage. Previous studies suggest additional roles for AK in cellular processes. Since AK is found only in invertebrates and it is homologous to creatine kinase from vertebrates, the objective of this work was to demonstrate nucleoside diphosphate kinase-like activity for shrimp AK. For this, AK from marine shrimp Litopenaeus vannamei (LvAK) was purified and its activity was assayed for phosphorylation of TDP using ATP as phosphate donor. Moreover, by using high-pressure liquid chromatography (HPLC) the phosphate transfer reaction was followed. Also, LvAK tryptophan fluorescence emission changes were detected by dTDP titration, suggesting that the hydrophobic environment of Trp 221, which is located in the top of the active site, is perturbed upon dTDP binding. The kinetic constants for both substrates Arg and dTDP were calculated by isothermal titration calorimetry (ITC). Besides, docking calculations suggested that dTDP could bind LvAK in the same cavity where ATP bind, and LvAK basic residues (Arg124, 126 and 309) stabilize the dTDP phosphate groups and the pyrimidine base interact with His284 and Ser122. These results suggest that LvAK bind and phosphorylate dTDP being ATP the phosphate donor, thus describing a novel alternate nucleoside diphosphate kinase-like activity for this enzyme.

High-level production of aquaporin Z in Escherichia coli using maltose-binding protein/polyhistidine dual-affinity tag fusion system

Aquaporin Z (AqpZ) is a water channel protein belonging to the major intrinsic protein (MIP) family and is found in the membrane of Escherichia coli. Due to its high water permeability and low activation energy, AqpZ is a potential candidate for developing biomimetic water filtration technologies. In the present study, membrane-associated AqpZ was expressed in E. coli using the maltose-binding protein (MBP)/polyhistidine (HIS) dual-affinity tag fusion system. The effects of different host strains and the expression conditions on the production of the fusion protein MBP–AqpZ–HIS were systematically studied. Furthermore, an efficient protocol was developed to purify AqpZ–HIS via affinity chromatography, and approximately 15–20mg of functional AqpZ–HIS was obtained from 1l of culture with a specific water transport activity of around 2×10−14cm3s−1 monomer−1. The related sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) and tryptophan fluorescence emission analyses indicated that purified AqpZ–HIS assembled as a homotetramer with high thermostability, in turn indicating the correct folding of AqpZ. Our results showed that the MBP/HIS dual-affinity tag system was highly efficient for the overexpression, membrane targeting, and purification of MIP channel proteins in E. coli.

Lactoferricin Peptides: The Importance of Methyl-Tryptophan and Glutamine for Structure and Activity

The rise in antibiotic-resistant strains of bacteria has led to an active search for more potent antimicrobial drugs. The hexapeptide (LfB6: RRWQWR-NH2) from the N-terminal of the protein lactoferricin was shown to be the antimicrobial ‘active core’ by Tomita (Acta Paediatr Jpn, 1994, 36:585). A heptapeptide synthesized in our lab, which contains 4 positively charged arginines and 2 methylated tryptophans (LfB7 MeTrp3,5: RRMeWQMeWRR-NH2), exhibits enhanced activity when compared to LfB6 against gram positive and gram negative bacteria. To determine if Trp-methylation is required for the increased efficacy, a control peptide without methylated Trp (LfB7: RRWQWRR-NH2) has been synthesized and characterized. In addition, glutamine was replaced with alanine in both peptides to examine the importance of H-bonding at the central position (LfB7 MeTrp3,5 Ala4: RRMeWAMeWRR-NH2and LfB7 Ala4: RRWAWRR-NH2). Replacement of the central Gln with Ala reduced activity 5-fold against E. coli. LfB7 MeTrp3,5 exhibits the highest activity, with a 4- and 6-fold increase compared to LfB7, against E. coli and S. aureus, respectively. The Ala, Trp and MeTrp residues were labeled with deuterium, and the peptide was studied by solid-state deuterium NMR in bacterial- and mammalian-like membranes. The 2H-quadrupolar splittings, whether from labeled Ala, MeTrp or Trp, are larger when MeTrp is present in the sequence, indicating a change in peptide conformation and/or dynamics upon Trp-methylation. Tryptophan emission fluorescence spectra reveal that the Trp and MeTrp residues are more deeply buried in anionic compared to neutral lipids, and circular dichroism spectra suggest that anionic lipids promote a conformational change that results in Trp-Trp (and MeTrp-MeTrp) interactions. These results emphasize the importance of Gln at the central position and of Trp-methylation for structure and activity of lactoferricin.

Comparisons of biophysical properties and bioactivities of mono-PEGylated endostatin and an endostatin analog

BackgroundEndostatin (ES) is a well-established potent endogenous antiangiogenic factor. An ES variant, called zinc-binding protein-ES (ZBP-ES), is clinically available; however, its use is limited by rapid renal clearance and short residence time. PEGylation has been exploited to overcome these shortcomings, and mono-PEGylated ES (called M2ES) as well as mono-PEGylated ZBP-ES (MZBP-ES) are developed in our study. This study aimed to compare the biophysical properties and biological effects of M2ES and MZBP-ES to evaluate their druggability.MethodsCircular dichroism and tryptophan emission fluorescence were used to monitor the conformational changes of M2ES and MZBP-ES. Their resistance to trypsin digestion and guanidinium chloride (GdmCl)-induced unfolding was examined by Coomassie staining and tryptophan emission fluorescence, respectively. The biological effects of M2ES and MZBP-ES on endothelial cell migration were evaluated using Transwell migration and wound healing assays, and the uptake of M2ES and MZBP-ES in endothelial cells was also compared by Western blotting and immunofluorescence.ResultsStructural analyses revealed that M2ES has a more compact tertiary structure than MZBP-ES. Moreover, M2ES was more resistant to trypsin digestion and GdmCl-induced unfolding compared with MZBP-ES. In addition, although M2ES and MZBP-ES showed comparable levels of inhibiting transwell migration and wound healing of endothelial cells, M2ES displayed an increased ability to enter cells compared with MZBP-ES, possibly caused by the enhanced interaction with nucleolin.ConclusionsM2ES has a more compact tertiary structure, is more stable for trypsin digestion and GdmCl-induced unfolding, exhibits increased cellular uptake and shows equivalent inhibitory effects on cell migration relative to MZBP-ES, indicating that M2ES is a more promising candidate for anticancer drug development compared with MZBP-ES.

Potential Antileukemia Effect and Structural Analyses of SRPK Inhibition by N-(2-(Piperidin-1-yl)-5-(Trifluoromethyl)Phenyl)Isonicotinamide (SRPIN340).

Dysregulation of pre-mRNA splicing machinery activity has been related to the biogenesis of several diseases. The serine/arginine-rich protein kinase family (SRPKs) plays a critical role in regulating pre-mRNA splicing events through the extensive phosphorylation of splicing factors from the family of serine/arginine-rich proteins (SR proteins). Previous investigations have described the overexpression of SRPK1 and SRPK2 in leukemia and other cancer types, suggesting that they would be useful targets for developing novel antitumor strategies. Herein, we evaluated the effect of selective pharmacological SRPK inhibition by N-(2-(piperidin-1-yl)-5-(trifluoromethyl)phenyl)isonicotinamide (SRPIN340) on the viability of lymphoid and myeloid leukemia cell lines. Along with significant cytotoxic activity, the effect of treatments in regulating the phosphorylation of the SR protein family and in altering the expression of MAP2K1, MAP2K2, VEGF and FAS genes were also assessed. Furthermore, we found that pharmacological inhibition of SRPKs can trigger early and late events of apoptosis. Finally, intrinsic tryptophan fluorescence emission, molecular docking and molecular dynamics were analyzed to gain structural information on the SRPK/SRPIN340 complex. These data suggest that SRPK pharmacological inhibition should be considered as an alternative therapeutic strategy for fighting leukemias. Moreover, the obtained SRPK-ligand interaction data provide useful structural information to guide further medicinal chemistry efforts towards the development of novel drug candidates.

A look into amyloid formation by transthyretin: aggregation pathway and a novel kinetic model.

The aggregation of proteins into insoluble amyloid fibrils is the hallmark of many, highly debilitating, human pathologies such as Alzheimer's or Parkinson's disease. Transthyretin (TTR) is a homotetrameric protein implicated in several amyloidoses like Senile Systemic Amyloidosis (SSA), Familial Amyloid Polyneuropathy (FAP), Familial Amyloid Cardiomyopathy (FAC), and the rare Central Nervous System selective Amyloidosis (CNSA). In this work, we have investigated the kinetics of TTR aggregation into amyloid fibrils produced by the addition of NaCl to acid-unfolded TTR monomers and we propose a mathematically simple kinetic mechanism to analyse the aggregation kinetics of TTR. We have conducted circular dichroism, intrinsic tryptophan fluorescence and thioflavin-T emission experiments to follow the conformational changes accompanying amyloid formation at different TTR concentrations. Kinetic traces were adjusted to a two-step model with the first step being second-order and the second being unimolecular. The molecular species present in the pathway of TTR oligomerization were characterized by size exclusion chromatography coupled to multi-angle light scattering and by transmission electron microscopy. The results show the transient accumulation of oligomers composed of 6 to 10 monomers in agreement with reports suggesting that these oligomers may be the causative agent of cell toxicity. The results obtained may prove to be useful in understanding the mode of action of different compounds in preventing fibril formation and, therefore, in designing new drugs against TTR amyloidosis.

Characterization of Membrane Interactions of Antimicrobial Lactoferricin Peptides with Central Residue Substitutions

The rise in antibiotic-resistant bacteria has led to an active search for new and more effective antimicrobial drugs. A hexapeptide (LfB6: RRWQWR-NH2) derived from the iron-binding protein lactoferrin exhibits antimicrobial activity (Tomita, Acta Paediatr Jpn, 1994, 36:585). A related heptapeptide produced in our lab, with 4 positively charged arginines and 2 methylated tryptophans (RRMeWQMeWRR-NH2; MeTrp-LfB7), exhibits enhanced activity against gram negative and gram positive bacteria. Substitutions of the central glutamine (Gln4;Q) residue that may alter peptide conformational flexibility are now being investigated. When Gln4 was changed to glycine (Gly;G) or proline (Pro;P), significant changes in peptide-membrane interactions were observed, although the antimicrobial activity was not increased. We now examine the effects of replacing Gln4 with gamma amino butyric acid (GABA), to introduce more flexibility; or D-Pro-Gly, to constrain the backbone into a β-hairpin turn (Stanger and Gellman, J. Am. Chem. Soc. 1998, 120:4236). The increased positive ellipticity at ∼230 nm observed in the CD spectrum of the D-Pro-Gly peptide in anionic membranes suggests significant Trp-Trp interactions. Tryptophan fluorescence emission spectra indicate that peptides with either GABA or D-Pro-Gly substitutions are more deeply buried in anionic membranes. Although the GABA and D-Pro-Gly peptides were both more active against gram negative (E. coli), compared to gram positive (S. aureus) bacteria, they were not as active as the Gln4 peptide. Investigations of the peptide-lipid interactions are being continued by means of solid-state 2H and 31P NMR spectra.

Correction to The Specialized Hsp70 (HscA) Interdomain Linker Binds to Its Nucleotide-Binding Domain and Stimulates ATP Hydrolysis in Both cis and trans Configurations

Proteins from the isc operon of Escherichia coli constitute the machinery used to synthesize iron-sulfur (Fe-S) clusters for delivery to recipient apoproteins. Efficient and rapid [2Fe-2S] cluster transfer from the holo-scaffold protein IscU depends on ATP hydrolysis in the nucleotide-binding domain (NBD) of HscA, a specialized Hsp70-type molecular chaperone with low intrinsic ATPase activity (0.02 min(-1) at 25 °C, henceforth reported in units of min(-1)). HscB, an Hsp40-type cochaperone, binds to HscA and stimulates ATP hydrolysis to promote cluster transfer, yet while the interactions between HscA and HscB have been investigated, the role of HscA's interdomain linker in modulating ATPase activity has not been explored. To address this issue, we created three variants of the 40 kDa NBD of HscA: NBD alone (HscA386), NBD with a partial linker (HscA389), and NBD with the full linker (HscA395). We found that the rate of ATP hydrolysis of HscA395 (0.45 min(-1)) is nearly 15-fold higher than that of HscA386 (0.035 min(-1)), although their apparent affinities for ATP are equivalent. HscA395, which contains the full covalently linked linker peptide, exhibited intrinsic tryptophan fluorescence emission and basal thermostability that were higher than those of HscA386. Furthermore, HscA395 displayed narrower (1)H(N) line widths in its two-dimensional (1)H-(15)N TROSY-HSQC spectrum in comparison to HscA386, indicating that the peptide in the cis configuration binds to and stabilizes the structure of the NBD. The addition to HscA386 of a synthetic peptide with a sequence identical to that of the interdomain linker (L(387)LLDVIPLS(395)) stimulated its ATPase activity and induced widespread NMR chemical shift perturbations indicative of a binding interaction in the trans configuration.

Activation of phenylalanine hydroxylase by phenylalanine does not require binding in the active site.

Phenylalanine hydroxylase (PheH), a liver enzyme that catalyzes the hydroxylation of excess phenylalanine in the diet to tyrosine, is activated by phenylalanine. The lack of activity at low levels of phenylalanine has been attributed to the N-terminus of the protein’s regulatory domain acting as an inhibitory peptide by blocking substrate access to the active site. The location of the site at which phenylalanine binds to activate the enzyme is unknown, and both the active site in the catalytic domain and a separate site in the N-terminal regulatory domain have been proposed. Binding of catecholamines to the active-site iron was used to probe the accessibility of the active site. Removal of the regulatory domain increases the rate constants for association of several catecholamines with the wild-type enzyme by ∼2-fold. Binding of phenylalanine in the active site is effectively abolished by mutating the active-site residue Arg270 to lysine. The kcat/Kphe value is down 104 for the mutant enzyme, and the Km value for phenylalanine for the mutant enzyme is >0.5 M. Incubation of the R270K enzyme with phenylalanine also results in a 2-fold increase in the rate constants for catecholamine binding. The change in the tryptophan fluorescence emission spectrum seen in the wild-type enzyme upon activation by phenylalanine is also seen with the R270K mutant enzyme in the presence of phenylalanine. Both results establish that activation of PheH by phenylalanine does not require binding of the amino acid in the active site. This is consistent with a separate allosteric site, likely in the regulatory domain.

The specialized Hsp70 (HscA) interdomain linker binds to its nucleotide-binding domain and stimulates ATP hydrolysis in both cis and trans configurations.

Proteins from the isc operon of Escherichia coli constitute the machinery used to synthesize iron–sulfur (Fe–S) clusters for delivery to recipient apoproteins. Efficient and rapid [2Fe-2S] cluster transfer from the holo-scaffold protein IscU depends on ATP hydrolysis in the nucleotide-binding domain (NBD) of HscA, a specialized Hsp70-type molecular chaperone with low intrinsic ATPase activity (0.02 min−1 at 25 °C, henceforth reported in units of min–1). HscB, an Hsp40-type cochaperone, binds to HscA and stimulates ATP hydrolysis to promote cluster transfer, yet while the interactions between HscA and HscB have been investigated, the role of HscA’s interdomain linker in modulating ATPase activity has not been explored. To address this issue, we created three variants of the 40 kDa NBD of HscA: NBD alone (HscA386), NBD with a partial linker (HscA389), and NBD with the full linker (HscA395). We found that the rate of ATP hydrolysis of HscA395 (0.45 min–1) is nearly 15-fold higher than that of HscA386 (0.035 min–1), although their apparent affinities for ATP are equivalent. HscA395, which contains the full covalently linked linker peptide, exhibited intrinsic tryptophan fluorescence emission and basal thermostability that were higher than those of HscA386. Furthermore, HscA395 displayed narrower 1HN line widths in its two-dimensional 1H–15N TROSY-HSQC spectrum in comparison to HscA386, indicating that the peptide in the cis configuration binds to and stabilizes the structure of the NBD. The addition to HscA386 of a synthetic peptide with a sequence identical to that of the interdomain linker (L387LLDVIPLS395) stimulated its ATPase activity and induced widespread NMR chemical shift perturbations indicative of a binding interaction in the trans configuration.

Interaction of cyclic and linear Labaditin peptides with anionic and zwitterionic micelles

Conformational changes of the cyclic (Lo) peptide Labaditin (VWTVWGTIAG) and its linear analogue (L1) promoted by presence of anionic sodium dodecyl sulfate (SDS) and zwitterionic L-α-Lysophosphatidylcholine (LPC) micelles were investigated. Results from λmax blue-shift of tryptophan fluorescence emission combined with Stern–Volmer constants values and molecular dynamics (MD) simulations indicated that L1 interacts with SDS micelles to a higher extent than does Lo. Further, the MD simulation demonstrated that both Lo and L1 interact similarly with LPC micelles, being preferentially located at the micelle/water interface. The peptide–micelle interaction elicits conformational changes in the peptides. Lo undergoes limited modifications and presents unordered structure in both LPC and SDS micelles. On the other hand, L1 displays a random-coil structure in aqueous medium, pH 7.0, and it acquires a β-structure upon interaction with SDS and LPC, albeit with structural differences in each medium.

Purification and structural stability of white Spanish broom (Cytisus multiflorus) peroxidase

New plant peroxidase has been isolated to homogeneity from the white Spanish broom Cytisus multiflorus. The enzyme purification steps included homogenization, NH4SO4 precipitation, extraction of broom colored compounds and consecutive chromatography on Phenyl-Sepharose, HiTrap™ SP HP and Superdex-75 and 200. The novel peroxidase was characterized as having a molecular weight of 50±3kDa. Steady-state tryptophan fluorescence and far-UV circular dichroism (CD) studies, together with enzymatic assays, were carried out to monitor the structural stability of C. multiflorus peroxidase (CMP) at pH 7.0. Thus changes in far-UV CD corresponded to changes in the overall secondary structure of enzyme, while changes in intrinsic tryptophan fluorescence emission corresponded to changes in the tertiary structure of the enzyme. It is shown that the process of CMP denaturation can be interpreted with sufficient accuracy in terms of the simple kinetic scheme, N⟶kD, where k is a first-order kinetic constant that changes with temperature following the Arrhenius equation; N is the native state, and D is the denatured state. On the basis of this model, the parameters of the Arrhenius equation were calculated.

The broken ring: reduced aromaticity in Lys-Trp cations and high pH tautomer correlates with lower quantum yield and shorter lifetimes.

Several nonradiative processes compete with tryptophan fluorescence emission. The difficulty in spectral interpretation lies in associating specific molecular environmental features with these processes and thereby utilizing the fluorescence spectral data to identify the local environment of tryptophan. Here, spectroscopic and molecular modeling study of Lys-Trp dipeptide charged species shows that backbone-ring interactions are undistinguished. Instead, quantum mechanical ground state isosurfaces reveal variations in indole π electron distribution and density that parallel charge (as a function of pK1, pK2, and pKR) on the backbone and residues. A pattern of aromaticity-associated quantum yield and fluorescence lifetime changes emerges. Where quantum yield is high, isosurfaces have a charge distribution similar to the highest occupied molecular orbital (HOMO) of indole, which is the dominant fluorescent ground state of the 1La transition dipole moment. Where quantum yield is low, isosurface charge distribution over the ring is uneven, diminished, and even found off ring. At pH 13, the indole amine is deprotonated, and Lys-Trp quantum yield is extremely low due to tautomer structure that concentrates charge on the indole amine; the isosurface charge distribution bears scant resemblance to the indole HOMO. Such greatly diminished fluorescence has been observed for proteins where the indole nitrogen is hydrogen bonded, lending credence to the association of aromaticity changes with diminished quantum yield in proteins as well. Thus tryptophan ground state isosurfaces are an indicator of indole aromaticity, signaling the partition of excitation energy between radiative and nonradiative processes.

Influence of Maillard conjugation on structural characteristics and rheological properties of whey protein/dextran systems

It is well known that protein/polysaccharide conjugates obtained by Maillard reaction (MR) have good emulsifying properties. However, there is little information about the use of these conjugates in gel systems. Structural characteristics and rheological properties of conjugates obtained by MR of whey protein isolate (WPI) and dextrans (DX) of various molecular weight (MW: 6, 40 and 70 kDa) were studied. Conjugation was confirmed by electrophoresis; browning intensity was measured by absorbance at 420 nm; and conformational changes were studied by fluorescence emission of tryptophan (Trp) (λex = 280 nm). Rheological properties were determined by oscillatory rheometry with temperature ramp (25–90 °C). After each measure, a mechanical spectrum (at 25 °C) was obtained. The electrophoresis indicated the presence of WPI/DX conjugates in all systems. Browning intensity increased with decreasing MW of DX. Fluorescence emission of WPI incubated increased, but decreased in WPI/DX incubated systems. The gelation time (obtained by G′–G″ crossover) and G′ value at 25 °C increased in conjugate systems compared with WPI alone. Stability of gel structures were shown by frequency sweeps.

Glucose and xylose stimulation of a β-glucosidase from the thermophilic fungus Humicola insolens: A kinetic and biophysical study

β-Glucosidases activated by glucose and xylose are uncommon yet intriguing enzymes that may enhance cellulose saccharification efficiency, and are of interest for application in bioethanol production processes. The molecular mechanisms of activation are completely unknown, and the aim of this study was the kinetic and biophysical characterization of the stimulation of a β-glucosidase from Humicola insolens by glucose and xylose. The effects of the monosaccharides were concentration dependent, where in a stimulatory range (0.1–50 mmol L−1), the activity increased up to 2-fold; in a stimulatory-inhibitory range (50–450 mmol L−1 glucose or 50–730 mmol L−1 xylose), the enzyme continued to be stimulated, but the activity was lower than maximal. Above 450 mmol L−1 glucose or 730 mmol L−1 xylose, increasing inhibition occurred. Dynamic light scattering confirmed that the enzyme is monomeric (54 kDa) and kinetic, intrinsic tryptophan fluorescence emission and far ultraviolet circular dichroism analyses indicated that the enzyme possesses a catalytic site (CS) and a modulator binding site (MS). Glucose or xylose binding to the MS induces conformational changes that stimulate the catalytic activity at the CS. Glucose and xylose may compete with the substrate for the CS while the substrate competes with the monosaccharides for binding to the MS. The stimulation of the enzymatic activity by glucose and xylose, which compete for the same sites on the enzyme molecule, is not synergistic. These data reveal allosteric interactions between the MS and the CS in H. insolens β-glucosidase that result in fine modulation of the catalytic activity by the monosaccharides. A kinetic model was developed that accurately described the experimental data for enzyme stimulation by glucose and/or xylose. Understanding the regulatory mechanisms of the enzyme activity, with the aid of kinetic models, may be useful for the application of the enzyme in cellulose hydrolysis processes.