The Tryptophan Fluorescence of Tetracarbidium conophorum Agglutinin II and a Solution-Based Assay for the Binding of a Biantennary Glycopeptide

The plant lectin Tetracarbidium conophorum agglutinin II binds to glycoproteins and glycopeptides in a structurally specific manner [Animashaun et al., (1994) Glycoconjugate J. 11, 299–303]. We have characterized the steady-state and time-resolved fluorescence of the tryptophan residues of this lectin. The fluorescence (λ_ex = 295 nm, λ_em = 350 nm) decay is complex and can be described by four decay times with the following values: τ₁ = 7.4nsec, α₁ = 0.22; τ₂ = 2.9 nsec, α₂ = 0.25; τ₃ = l.0 nsec, α₃ = 0.34; τ₄ = 0.2 nsec, α₄ = 0.18. The addition of a biantennary glycopeptide $\begin{array}{*{20}c} {Gal\beta (1 \to 4)GlcNAc\beta (1 \to 2)Man\alpha (1 \to 6)\neg } \\ {Man\beta (1 \to 4)GlcNAC\beta (1 \to 4)GlcAc\beta (1 \to )\begin{array}{*{20}c} {Glu - Nh_2 } \\ | \\ {Asn} \\ | \\ {COOH} \\ \end{array} } \\ {Gal\beta (1 \to 4)GlcNAc\beta (1 \to 2)Man\alpha (1 \to 3)} \\ \end{array} $ to the lectin results in a quench and an 8 nm blue shift of the emission spectrum. The effect is saturable, and is described by an association constant of 1.8×10⁵ M^?1. The tryptophan fluorescence of Tetracarbidium conophorum agglutinin II may therefore be utilized to characterize thermodynamically the binding interactions between this lectin and complex glycoprotein.     

Ionic Charge, Hydrophobicity and Tryptophan Fluorescence of the Folate Binding Protein Isolated from Cow's Milk

A high-affinity folate binding protein was isolated and purified from cow's milk by a combination of cation exchange chromatography and methotrexate affinity chromatography. Chromatofocusing studies revealed that the protein possessed isoelectric points in the pH-interval 8–7. Polymers of the protein prevailing at pH values close to the isoelectric points seemed to be more hydrophobic than monomers present at pH 5.0 as evidenced by hydrophobic interaction chromatography and turbidity (absorbance at 340 nm) in aqueous buffer solutions (pH 5–8). Ligand binding seemed to induce a conformation change that decreased the hydrophobicity of the protein. In addition, Ligand binding quenched the tryptophan fluorescence of folate binding protein suggesting that tryptophan is present at the binding site and/or ligand binding induces a conformation change that affects tryptophan environment in the protein. There was a noticeable discordance between the ability of individual folate analogues to compete with folate for binding and the quenching effect.     

克木毒蛋白三种酶活性与色氨酸残基的关系

对一种新的核糖体失活蛋白──克木毒蛋白的研究表明,该分子中仅含一个色氨酸.此色氨酸与克木毒蛋白具有的三种酶活性有明显不同的关系.     

Intrinsic Fluorescence in Endoglucanase and Cellobiohydrolase from Trichoderma pseudokiningii S-38: Effects of pH, Quenching Agents, and Ligand Binding

To gain further insight into the difference in substrate specificity between endoglucanase and cellobiohydrolase, the intrinsic fluorescence properties of cellobiohydrolase I (CBH I) and endoglucanase I (EG I) from Trichoderma pseudokiningii S-38 were investigated. The results for the spectral characteristics, ligand binding and fluorescence quenching suggest that the fluorescence of two enzymes comes from tryptophan residues, and that tryptophan residue(s) may be involved in the function of the two enzymes. The results also suggest that the binding tryptophan in EG I may be more exposed to solvent than that in CBH I. This interpretation is supported by the observations that the effects of pH upon the fluorescence of EG I are greater than that of CBH I; spectral shifts are different in EG I and CBH I under various conditions, and fluorescence lifetime changes caused by cellobiose binding are larger for EG I than for CBH I.     

Binding Studies of Bile Acids using the Native Fluorescence of the Tryptophan Residue Of Bax Protein

Ursodeoxycholic acid (UDCA) and its taurine-conjugate, tauroursodeoxycholic acid (TUDCA), play a unique role in modulating the apoptotic threshold in cells. The mechanism is thought to involve, in part, inhibition of translocation for Bax from the cytosol to mitochondria. Here, we attempted to use the native fluorescence of the tryptophan residues of Bax to determine whether bile acids bind directly to recombinant Bax protein. The results showed that UDCA had no effect on the tryptophan fluorescence of Bax. Similarly, there was no evidence of direct binding between Bax protein and the more hydrophobic bile acid, deoxycholic acid (DCA). In contrast, the fluorescence change detected for Bax solution titrated against TUDCA in dimethylsulfoxide was greater than that observed with solvent alone. In conclusion, data from fluorescence spectroscopy does not support a direct interaction of UDCA or DCA with Bax protein, whereas it suggests that there may be some potential interaction with TUDCA.     

DNA与蛋白质结合的荧光测定

构建了插入λ阻抑蛋白（Ｒｅｐ）的操纵基因（ＯＲ）和ＢｇｌⅡ识别位点的ＰＢＲ３２２重组质粒。阻抑蛋白与该质粒的相互作用可用ＢｇｌⅡ对它的水解作用引起的ＥＢ荧光变化来研究。在Ｅ．ｃｏｌｉ中表达的Ｒｅｐ表现了与该重组质粒结合的活力。核苷酸序列具精确二重对称性的ＯＲ（ＯＲｃｏｎｓ）对Ｒｅｐ的亲和力比天然的ＯＲ１小。     

Experimental indication for the existence of multiple Trp rotamers in von Willebrand Factor A3 domain

The first step in both normal haemostasis and arterial thrombosis is the interaction between collagen, von Willebrand factor (vWF), and glycoprotein Ib. The A3 domain of vWF forms the principal binding site for collagen type I and type III. Inhibition of the vWF-collagen interaction by an anti-human vWF monoclonal antibody (MoAb) 82D6A3 can be a potential way to prevent arterial thrombosis. Identification of the epitope of MoAb 82D6A3 showed recently that the consensus sequence SPWR obtained by phage display could adopt the conformation of the discontinuous epitope. Modelling showed that Trp982 in the vWF had to obtain a more solvent accessible conformation. We performed a detailed fluorescence study of Trp982 in the vWF A3. Using the method described by Hellings et al. (Biophys J 2003;85:1894-1902), we were able to identify two different low-energy Trp982 rotamers and to link them with their experimentally derived fluorescence lifetimes. Fluorescence anisotropy showed no interconversion in the nanosecond timescale between the two different rotameric states. With these experiments, we gather strong indications for the existence of an exposed rotamer conformation and a rotamer that corresponds to the one observed in the X-ray structure. These results strongly support the modeling work (Vanhoorelbeke et al., J Biol Chem 2003;278:37815-37821).     

An unusual red-edge excitation and time-dependent Stokes shift in the single tryptophan mutant protein DD-carboxypeptidase from Streptomyces: the role of dynamics and tryptophan rotamers

The fluorescence emission of the single tryptophan (W233) of the mutant protein DD-carboxypeptidase from streptomyces is characterized by a red-edge excitation shift (REES), i.e., the phenomenon that the wavelength of maximum emission depends on the excitation wavelength. This phenomenon is an indication for a strongly reduced dynamic environment of the single tryptophan, which has a very low accessibility to the solvent. The REES shows, however, an unusual temperature and time dependence. This, together with the fluorescence lifetime analysis, showing three resolvable lifetimes, can be explained by the presence of three rotameric states that can be identified using the Dead-End Elimination method. The three individual lifetimes increase with increasing emission wavelength, indicating the presence of restricted protein dynamics within the rotameric states. This is confirmed by time-resolved anisotropy measurements that show dynamics within the rotamers but not among the rotamers. The global picture is that of a protein with a single buried tryptophan showing strongly restricted dynamics within three distinct rotameric states with different emission spectra and an anisotropic environment.     

Fluorescence quenching method for the determination of carbazochrome sodium sulfonate with aromatic amino acids

In Britton‐Robinson (BR) buffer medium (pH 3.3), carbazochrome sodium sulfonate (CSS) can react with some aromatic amino acids such as tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe) to form a 1:1 complex by electrostatic attraction, aromatic stacking interaction and Van der Waals' force, resulting in fluorescence quenching of these amino acids. Maximum quenching wavelengths were located at 352 nm (CSS‐Trp system), 303 nm (CSS‐Tyr system) and 284 nm (CSS‐Phe system), respectively. The fluorescence quenching value (ΔF) was proportional to the concentration of CSS in a certain range. The fluorescence quenching method for the determination of CSS showed high sensitivity, with detection limits of 31.3 ng/mL (CSS‐Trp system), 44.6 ng/mL (CSS‐Tyr system) and 315.0 ng/mL (CSS‐Phe system), respectively. The optimum conditions of the reaction conditions and the effect of coexisting substances were investigated and results showed that the method had good selectivity. The method was successfully applied for the rapid determination of CSS in blood and urine samples. Based on the bimolecular quenching constant K_q, the effect of temperature and Stern‐Volmer plots, this study showed that quenching of fluorescence of amino acids by CSS was a static quenching process. Copyright © 2012 John Wiley & Sons, Ltd.     

A New Assay Based on Fluorescence Resonance Energy Transfer to Determine the Binding Affinity of Bcl-xL Inhibitors

We developed a new assay of Bcl-x_L inhibitors based on fluorescence resonance energy transfer that occurs between an AEDANS-labeled Bak-BH3 peptide and three tryptophans in the BH1 and BH2 domains of Bcl-x_L. The method can tolerate up to 5% DMSO, and it was validated with several Bcl-x_L inhibitors. It can be adapted to screen for compounds targeting other Bcl-2 family proteins.     

Binding of isolectins from red kidney bean (Phaseolus vulgaris) to purified rat brush-border membranes

Ingestion of red kidney bean phytohemagglutinin causes impaired growth and intestinal malabsorption, and facilitates bacterial colonization in the small intestine of weanling rats. We have studied interactions of the highly purified phytohemagglutinin erythroagglutinating (E4) and mitogenic (L4) isolectins with microvillous membrane vesicles prepared from rat small intestines. E4 and L4 were radioiodinated with ¹²⁵I by the chloramine-T technique. E4 and L4 isolectins both bound to microvillous membrane vesicles. Binding was saturable and reversible. Each mg of membrane protein bound 744±86 μg E4 and 213±21 μg L4. The apparent K_a for E4 and L4 binding was 2.5·10⁻⁶ and 13.0·10⁻⁶ M⁻¹, respectively. Binding of each ¹²⁵I-labelled isolectin was abolished by 100-fold excess of unlabelled isolectin. In each case binding also was inhibited by appropriate oligosaccharide inhibitors, indicating that isolectin-microvillous membrane interactions were mediated by carbohydrate recognition. Patterns of saccharide inhibition of isolectin binding were different for E4 and L4. Competitive binding experiments demonstrated mutual noncompetitive inhibition of E4 and L4 binding consistent with steric hindrance. Therefore, E4 and L4 each bound to its own set of receptors. Based on the known saccharide specificities of E4 and L4, these data indicate that there are differences in expression of complex asparagine-linked biantennary and tri- or tetraantennary oligosaccharides at the microvillous surface. The data also provide the possibility that direct interactions of one or more phytohemagglutinin isolectins with intestinal mucosa in vivo may contribute to the antinutritional effects associated with ingestion of crude red kidney beans.     

Binding of the Activating Ion Co(II) to myo-Inositol Monophosphatase Monitored by Fluorescence and Phosphorescence Spectroscopy

Two extrinsic probes, pyrene-maleimide and eosin-maleimide, were used to label specific SH groups of the enzyme myo-inositol monophosphatase. The fluorescence of pyrene-monophosphatase is enhanced upon addition of the activating metal ions Co(II) and Mg(II). Co(II) ions bind with a dissociation constant of 4 M, whereas the apparent activation constant K _a is 0.4 mM. Energy transfer measurements demonstrated that the pyrene chromophore, covalently linked to Cys-218, is within 9 Å of the metal ion Tb(III) coordinated to the metal-binding site. The phosphorescence emitted by eosin covalently linked to the protein is quenched by the addition of the activating cations Co(II) and Mg(II). Phosphorescence titrations conducted under anaerobic conditions were used to determine a dissociation constant of approximately 3 M for the binding of Co(II) ions. The results are consistent with the hypothesis that two activating ions per monomeric subunit participate in the catalytic mechanism. The affinity of the tightly bound ion is at least 100-fold greater than the affinity of the weakly bound ion.     

Excited‐state lifetime studies of the three tryptophan residues in the N‐lobe of human serum transferrin

The energy transfer from the three Trp residues at positions 8, 128, and 264 within the human serum transferrin (hTF) N-lobe to the ligand to metal charge transfer band has been investigated by monitoring changes in Trp fluorescence emission and lifetimes. The fluorescence emission from hTF N-lobe is dominated by Trp264, as revealed by an 82% decrease in the quantum yield when this Trp residue is absent. Fluorescence lifetimes were determined by multifrequency phase fluorometry of mutants containing one or two Trp residues. Decays of these samples are best described by two or three discrete lifetimes or by a unimodal Lorentzian distribution. The discrete lifetimes and the center of the lifetime distribution for samples containing Trp128 and Trp264 are affected by iron. The distribution width narrows on iron removal and is consistent with a decrease in dynamic mobility of the dominant fluorophore, Trp264. Both the quantum yield and the lifetimes are lower when iron is present, however, not proportionally. The greater effect of iron on quantum yields is indicative of nonexcited state quenching, i.e., static quenching. The results of these experiments provide quantitative data strongly suggesting that Förster resonance energy transfer is not the sole source of Trp quenching in the N-lobe of hTF.     

Intact Glycopeptide Analysis of Influenza A/H1N1/09 Neuraminidase Revealing the Effects of Host and Glycosite Location on Site‐Specific Glycan Structures

Influenza H1N1 virus has posed a serious threat to human health. The glycosylation of neuraminidase (NA) could affect the infectivity and virulence of the influenza virus, but detailed site‐specific glycosylation information of NA is still missing. In this study, intact glycopeptide analysis is performed on an influenza NA (A/H1N1/California/2009) that is expressed in human 293T and insect Hi‐5 cells. The data indicate that three of four potential N‐linked glycosylation sites are glycosylated, including one partial glycosylation site from both cell lines. The NA expressed in human cells has more complex glycans than that of insect cells, suggesting the importance of selecting an appropriate expression system for the production of functional glycoproteins. Different types of glycans are identified from different glycosites of NA expressed in human cells, which implies the site‐dependence of glycosylation on NA. This study provides valuable information for the research of influenza virus as well as the functions of viral protein glycosylation.     

Identification of N-acetylglucosamine binding residues in Griffonia simplicifolia lectin II

Primary structure and crystallographic data of several legume lectins were used to predict the involvement in carbohydrate binding of six amino acid residues (Asp⁸⁸, Glu¹⁰⁸, Tyr¹³⁴, Asn¹³⁶, Leu²²⁶ and Gln²²⁷) in Griffonia simplicifolia lectin II (GS-II). The functional involvement of these residues was evaluated by assessing GlcNAc binding of modified forms of GS-II in which these residues were eliminated in truncated peptides or systematically substituted with other amino acids by site-specific mutations. Mutations at (Asp⁸⁸, Tyr¹³⁴ or Asn¹³⁶ eliminated GlcNAc binding activity by GS-II, while those at Glut¹⁰⁸, Leu²²⁶ or Gln²²⁷ did not alter the activity. The former three amino acids were functionally essential for carbohydrate binding by GS-II presumably through hydrogen bonding to and hydrophobic interactions with GlcNAc. Although an Asp or Gly substitution for Tyr¹³⁴ eliminated GlcNAc affinity, substitution with Phe did not appreciably affect binding. Despite the fact that mutations to Leu²²⁶ and Gln²²⁷ did not alter carbohydrate binding, a truncated form of GS-II lacking these residues no longer exhibited carbohydrate binding affinity.     

双分子荧光互补技术及其在蛋白质相互作用研究中的应用

双分子荧光互补(bimolecularfluorescencecomplementation,BiFC)分析技术,是由Hu等在2002年最先报道的一种直观、快速地判断目标蛋白在活细胞中的定位和相互作用的新技术.该技术巧妙地将荧光蛋白分子的两个互补片段分别与目标蛋白融合表达,如果荧光蛋白活性恢复则表明两目标蛋白发生了相互作用.其后发展出的多色荧光互补技术(multicolorBiFC),不仅能同时检测到多种蛋白质复合体的形成,还能够对不同蛋白质间产生相互作用的强弱进行比较.目前,该技术已用于转录因子,G蛋白βγ亚基的二聚体形式,不同蛋白质间产生相互作用强弱的比较以及蛋白质泛素化等方面的研究工作上.     

The fluorescence intensities ratio is not a reliable parameter for evaluation of protein unfolding transitions

Monitoring the fluorescence of proteins, particularly the fluorescence of intrinsic tryptophan residues, is a popular method often used in the analysis of unfolding transitions (induced by temperature, chemical denaturant, and pH) in proteins. The tryptophan fluorescence provides several suitable parameters, such as steady‐state fluorescence intensity, apparent quantum yield, mean fluorescence lifetime, position of emission maximum that are often utilized for the observation of the conformational/unfolding transitions of proteins. In addition, the fluorescence intensities ratio at different wavelengths (usually at 330 nm and 350 nm) is becoming an increasingly popular parameter for the evaluation of thermal transitions. We show that, under certain conditions, the use of this parameter for the analysis of unfolding transitions leads to the incorrect determination of thermodynamic parameters characterizing unfolding transitions in proteins (e.g., melting temperature) and, hence, can compromise the hit identification during high‐throughput drug screening campaigns.     

Multiple Column Synthesis of a Library of T-Cell Stimulating Tn-Antigenic Glycopeptide Analogues for the Molecular Characterization of T-Cell–Glycan Specificity

A series of peptides and glycopeptides derived by amino acid and glycosyl amino acid scans through the self peptide from CBA/J mouse haemoglobin Hb (67–76), VITAFNEGLK, was synthesized by multiple column peptide synthesis (MCPS). Investigation of glycopeptide binding to the mouse major histocompatibility class II molecule E^k showed that glycans in position 72 did not interfere with the binding to E^k. Immunization experiments revealed that glycopeptides with the glycan in position 72 were immunogenic. Therefore a series of N-linked and O-linked glycopeptides with the glycan attached in the position 72 either to serine, threonine or asparagine was synthesized by MCPS. The glycan structure was furthermore varied with respect to monosacc haride component, size of oligosaccharide, anomer configuration and stereoche mistry of essential hydroxyl groups in order to investigate the specificity of the interaction with the T-cell receptor. Easy synthesis of ready to use Ser and Thr building blocks corresponding to mucin core 1, the T_n-antigen and its β-anomer were developed using trichloroacetimidates as glycosyl donors and reduction with in situ acetylation of the azide containing glycosylation products. Synthesis of an α-linked GlcNAc-Thr building block was achieved by glycosylation of Fmoc-Thr-OPfp with 2-azido-2-deoxy-3,4,6-tri-O-acetyl-D - glycopyranosyl trichloroacetimidate as a glycosyl donor. Other building blocks were obtained by previously described procedures.     

Gating-related Structural Dynamics of the MgtE Magnesium Channel in Membrane-Mimetics Utilizing Site-Directed Tryptophan Fluorescence

Magnesium is the most abundant divalent cation present in the cell, and an abnormal Mg²⁺ homeostasis is associated with several diseases in humans. However, among ion channels, the mechanisms of intracellular regulation and transport of Mg²⁺ are poorly understood. MgtE is a homodimeric Mg²⁺-selective channel and is negatively regulated by high intracellular Mg²⁺ concentration where the cytoplasmic domain of MgtE acts as a Mg²⁺ sensor. Most of the previous biophysical studies on MgtE have been carried out in detergent micelles and the information regarding gating-related structural dynamics of MgtE in physiologically-relevant membrane environment is scarce. In this work, we monitored the changes in gating-related structural dynamics, hydration dynamics and conformational heterogeneity of MgtE in micelles and membranes using the intrinsic site-directed Trp fluorescence. For this purpose, we have engineered six single-Trp mutants in the functional Trp-less background of MgtE to obtain site-specific information on the gating-related structural dynamics of MgtE in membrane-mimetic systems. Our results indicate that Mg²⁺-induced gating might involve the possibility of a ‘conformational wave’ from the cytosolic N-domain to transmembrane domain of MgtE. Although MgtE is responsive to Mg²⁺-induced gating in both micelles and membranes, the organization and dynamics of MgtE is substantially altered in physiologically important phospholipid membranes compared to micelles. This is accompanied by significant changes in hydration dynamics and conformational heterogeneity. Overall, our results highlight the importance of lipid-protein interactions and are relevant for understanding gating mechanism of magnesium channels in general, and MgtE in particular.