Conformation and activity ofPhaseolus coccineus var. rubronanus lectin

The conformation of native and denaturedPhaseolus coccineus var. rubronanus lectin was studied by circular dichroism (CD) and correlated to the hemagglutinating activity. The far-UV CD spectrum at 25°C showed a broad, negative band around 223 nm and a positive one at 196 nm. CD data analysis of the lectin indicated a β-sheet-rich protein. At high temperatures, the spectrum was blue-shifted with increasing magnitude; these changes correlated well with the loss of the activity. The conformation of lectin betweenpH 2 and 10 remained essentially unchanged. AtpH 13 the CD spectrum resembled that of unordered form with a negative band near 200 nm and the activity was completely lost. The denatured lectin in 6 M guanidine hydrochloride would be renatured upon diluting the denaturant to 0.75 M; the changes in CD spectrum again correlated well with the loss of the activity. The effect of sodium dodecyl sulfate on the lectin was drastic; it sharply increased thea-helix at the expense of the β-sheet and reduced the activity; the changes reached a plateau above 20 mM surfactant.     

Effect of amino acid residue and oligosaccharide chain chemical modifications on spectral and hemagglutinating activity of Millettia dielsiana Harms. ex Diels. lectin

The effects of modifying the carbohydrate chain and amino acids on the conformation and activity of Millettia dielsiana Harms. ex Diels. lectin (MDL) were studied by hemagglutination, fluorescence and circular dichroism analysis. The modification of tryptophan residues led to a compete loss of hemagglutinating activity; however, the addition of mannose was able to prevent this loss of activity. The results indicate that two tryptophan residues are involved in the carbohydrate-binding site. Modifications of the carboxyl group residues produced an 80% loss of activity, but the presence of mannose protected against the modification. The results suggest that the carboxyl groups of aspartic and glutamic acids are involved in the carbohydrate-binding site of the lectin. However, oxidation of the carbohydrate chain and modification of the histidine and arginine residues did not affect the hemagglutinating activity of MDL. Fluorescence studies of MDL indicate that tryptophan residues are present in a relatively hydrophobic region, and the binding of mannose to MDL could quench tryptophan fluorescence without any change in λmax. The circular dichroism spectrum showed that all of these modifications affected the conformation of the MDL molecule to different extents, except the modification of arginine residues. Fluorescence quenching showed that acrylamide and iodoacetic acids are able to quench 77% and 98% of the fluorescence of tryptophan in MDL, respectively. However, KI produced a barely perceptible effect on the fluorescence of MDL, even when the concentration of I^- was 0.15M. This demonstrates that most of tryptophan residues are located in relatively hydrophobic or negatively charged areas near the surface of the MDL molecule.     

Conformation of bilirubin oxidase in native and denatured states

The conformation of bilirubin oxidase (EC 1.3.3.5) fromMyrothecium verrucaria was studied by circular dichroism (CD). The far-UV CD spectrum showed a single minimum at 215 nm and a maximum near 198 nm, suggesting the dominance of-sheets. There was another negative band at 187 nm that is absent from the spectra of model-helix or-sheet. CD analysis by the method of Changet al. agreed well with the estimates based on the Chou and Fasman sequence-predictive method, but the Provencher-Glöckner method of CD analysis agreed well with the sequence-predictive method of Garnieret al. AtpH 12 the 215- and 187-nm bands completely disappeared and the protein was denatured. This denaturation was accompanied by the appearance of a large positive band at 250 nm, probably due to ionization of tyrosine residues. In 20 mM sodium dodecyl sulfate the magnitude of the 215-nm band increased, but the spectrum transformed to that of partial helices after heating at 100°C. In 6 M guanidine hydrochloride the far-UV CD spectrum was monotonic and became more negative at the lower wavelength limit (near 212 nm), suggesting that the secondary structure of the protein was disrupted. However, the near-UV CD spectrum retained residual aromatic bands even after heating at 100°C. Thus, our denaturation studies suggest that bilirubin oxidase has a rigid tertiary structure.     

Purification and characterization of a mannose-binding lectin from the rhizomes of Aspidistra elatior Blume with antiproliferative activity

A lectin with a novel N-terminal amino acid sequence was purified from the rhizomes of Aspidistra elatior Blume by ammonium sulphate precipitation, ion exchange chromatography on diethylaminoethyl-Sepharose and carboxymethyl-Sepharose and gel filtration chromatography on Sephacryl S-100. The A. elatior Blume lectin （AEL） is a heterotetramer with a molecular mass of 56 kDa and composed of two homodimers consisting of two different polypeptides of 13.5 kDa and 14.5 kDa held together by noncovalent interactions. Hapten inhibition assay indicated that hemagglutinating activity of AEL towards rabbit erythrocytes could be inhibited by D-mannose, mannan, thyroglobulin and ovomucoid. The lectin was stable up to 70 ℃ , and showed maximum activity in a narrow pH range of 7.0-8.0. Chemical modification and spectrum analysis indicated that tryptophan, arginine, cysteine and carboxyl group residues were essential for its hemagglutinating activity. However, they might not be present in the active center, except some carboxyl group residues. AEL also showed significant in vitro antiproliferative activity towards Bre-04 （66%）, Lu-04 （60%） and HepG2 （56%） of human cancer cell lines.     

Effect of Heat and pH Denaturation on the Structure and Conformation of Recombinant Human Hepatic Stimulator Substance

Hepatic stimulator substance (HSS) is a novel liver-specific growth-promoting factor. Although HSS has been successfully crystallized, several properties of this protein have yet to be determined. This study shows that recombinant human HSS (rhHSS) is a dimer with a molecular mass of 31 kDa, The protein is weakly acidic and has an isoelectric point (pI) of 4.50. rhHSS was able to protect hepatoma cells from H₂O₂-induced apoptosis and to stimulate cell growth. The recombinant protein was thermostable up to 80°C and resistant to changes in pH, as determined by synchronous fluorescence and far-UV circular dichroism (CD). Within the range of pH 4.0–10.0, rhHSS assumed a folded conformation identical to the secondary structure of the original, native protein and a native-like far-UV CD spectrum. Denatured rhHSS could be partly reconstituted with respect to its structure, but not its activity. Thus, rhHSS is a structurally stable protein insensitive to thermal and acid–alkaline denaturation.     

Conformation and orientation of penetratin in phospholipid membranes.

The binding, conformation and orientation of a hydrophilic vector peptide penetratin in lipid membranes and its state of self-association in solution were examined using circular dichroism (CD), analytical ultracentrifugation and fluorescence spectroscopy. In aqueous solution, penetratin exhibited a low helicity and sedimented as a monomer in the concentration range approximately 50-500 microM. The partitioning of penetratin into phospholipid vesicles was determined using tryptophan fluorescence anisotropy titrations. The apparent penetratin affinity for 20% phosphatidylserine/80% egg phosphatidylcholine vesicles was inversely related to the total peptide concentration implying repulsive peptide-peptide interactions on the lipid surface. The circular dichroism spectra of the peptide when bound to unaligned 20% phosphatidylserine/80% egg phosphatidylcholine vesicles and aligned hydrated phospholipid multilayers were attributed to the presence of both alpha-helical and beta-turn structures. The orientation of the secondary structural elements was determined using oriented circular dichroism spectroscopy. From the known circular dichroism tensor components of the alpha-helix, it can be concluded that the orientation of the helical structures is predominantly perpendicular to the membrane surface, while that of the beta-type carbonyls is parallel to the membrane surface. On the basis of our observations, we propose a novel model for penetratin translocation.     

Conformational study reveals amino acid residues essential for hemagglutinating and anti-proliferative activities of Clematis montana lectin

Clematis montana lectin （CML）, a novel mannose-binding lectin purified from C. montana Buch.-Ham stem （Ranunculaceae）, has been proved to have hemagglutinat- ing activity in rabbit erythrocytes and apoptosis-inducing activity in tumor cells. However, the biochemical properties of CML have not revealed and its structural information still needs to be elucidated. In this study, it was found that CML possessed quite good thermostability and alkaline re- sistance, and its hemagglutinating activity was bivalent metal cation dependent. In addition, hemagglutination test and fluorescence spectroscopy proved that GuHCI, urea, and sodium dodecyl sulfate could change the conformation of CML and further caused the loss of hemagglutination activity. Moreover, the changes of fluorescence spectrum indicated that the tryptophan （Trp） microenvironment conversion might be related to the conformation and bioactivities of CML. In addition, it was also found that Trp residues, arginine （Arg） residues, and sulfhydryl were important for the hemagglutinating activity of CML, but only Trp was proved to be crucial for the CML con- formation. Furthermore, the Trp, Arg, and sulfhydryl- modified CML exhibited 97.17%, 76.99%, and 49.64% loss of its anti-proliferative activity, respectively, which was consistent with the alterations of its hemagglutinating activity. Given these findings, Trp residues on the surface of CML are essential for the active center to form sub- strate-accessible conformation and suitable environment for carbohydrate binding.     

Possible mechanism for the inhibition of lectin-erythrocyte interaction in presence of endogenous lectin receptor

The presence of hydrophobic sites in the lectin-I molecule was indicated by hydrophobic probes like 1-anilinonapthalene-8-sulfonic acid (ANS), 2-p-toluidinyl napthalene-6-sulfonic acid (TNS), N-phenyl-1-napthylamine (NA) and rose bengal (RB). This was further confirmed by amino acid modifications in the hydrophobic region of the lectin-I molecule. The binding of ANS, TNS, NA and RB to lectin-I was affected in the presence of NaCl. The involvement of hydrophobic interactions in rice-bean lectin-I-endogenous lectin receptor (ELR) complex were indicated by alterations in the circular dichroism and fluorescence emission spectra. The percentage of -conformation (55–63%) of lectin-I was decreased by addition of ELR. ELR on reacting with lectin-I reduced the fluorescence emissions of the hydrophobic probes while fluorescence emission of ANS, TNS, NA and RB were greatly enhanced in presence of lectin-I alone. N-aceyl-galactosamine did not change the fluorescence emissions of any of the hydrophobic probes in presence or in absence of lectin-I. This demonstrates that carbohydrate and hydrophobic sites may be different and non-interacting. It is proposed that the ELR in reacting with lectin-I, induced conformational changes in the lectin-I molecule and thereby affected its erythroagglutinating activity with human blood group A erythrocytes.     

Domain Structure and Conformation of a Cellobiohydrolase from Trichoderma pseudokiningii S-38

A cellobiohydrolase (CBH) with a molecular mass of 66 kD was purified from Trichoderma pseudokiningii S-38. Papain digestion produced a 59- to 60-kD core domain with 54% of intact activity on crystalline cellulose and with full activity against soluble substrates. Digestion products also included two small peptides with molecular mass of about 3–4 kD, which are heavily glycosylated and difficult to purify; the mixed peptides displayed the capacity to disorganize the cellulose fiber. The sequencing results indicated that the intact enzyme had a blocked N-terminal and there was a 10-amino-acid sequence in the N-terminal of the core protein of Ser-Gly-Thr-Ala-Val-Thr-Cys-Leu-Ala-Asp. Fluoresence and circular dichroism properties indicated that the core protein has an independent conformation and is conformationally similar to intact enzyme, suggesting that the spectroscopic properties of the intact enzyme come from the core protein.     

Photosynthetic rate and lectin activity of soybean leaves after inoculation with rhizobia together with homologous lectin

Nodule bacteria (Bradyrhizobium japonicum) of various activities were preincubated with homologous lectin and then used for inoculating soybean (Glycine max (L.) Merrill) seeds. The effect of this inoculation on the photosynthetic rate, lectin activity in leaves, and plant development at different supply of mineral nitrogen was investigated under the conditions of pot experiments. There was a positive relationship between the photosynthetic rate and the lectin activity of proteins isolated from soybean leaves. Under the conditions of effective symbiosis, activation of functioning of the symbiotic apparatus by preincubation of the rhizobia with lectin exerted an additional stimulating effect on the photosynthetic rate. It is suggested that a relationship between the effectiveness of legume-rhizobium symbiosis and the lectin activity in leaves is mediated by the regulation of photosynthesis through a demand for assimilates in the source-sink system of soybean plants.     

Effects of denaturation and amino acid modification on fluorescence spectrum and hemagglutinating activity of Hericium erinaceum Lectin

In a broad sense, lectins are proteins or glycoproteins ofnon-immune origin that bind specifically to carbohydrates[1]. But most lectins are usually multivalent, which meansthey have more than one carbohydrate-binding site in onemolecule, a property that enables them to agglutinate eryth-rocytes and other cells [2,3]. Some lectins exhibit blood-group specificity [4] and can be used in blood grouping;some agglutinate transformed cells better than the normalones [5]. Therefore, clinical research…     

Conformational changes of recombinant human granulocyte-colony stimulating factor induced bypH and guanidine hydrochloride

Fluorescence and circular dichroism were used to follow thepH-dependent conformational changes of granulocyte colony stimulating factor (G-CSF). Tryptophan fluorescence of the spectra monitored at 344 nm, or after deconvolution of the emission spectra, at 345 nm, showed a decrease in intensity on going frompH 7 to 4, with a midtransitionpH of 5.8. On the other hand, tyrosine fluorescence measured either by the ratio of intensity at 308 nm to that at 344 nm, or by the fluorescence intensity at 303 nm after deconvolution of the spectra, increased in intensity as thepH was changed from 6 to 2.5, with a midtransitionpH of 4.5. Near UV circular dichroic spectra also showed changes betweenpH 7.5 and 4.5, which correlated with the transition monitored by the tryptophan fluorescence. The guanidine hydrochloride-induced conformational changes of G-CSF at fivepH values from 2.5 to 7.5 were also studied. Circular dichroic and fluorescence spectra revealed minor conformational changes by the addition of 1 or 2 M guanidine HCl at allpH values examined, while the major conformational transition occurred between 2 and 4 M guanidine hydrochloride. The secondary structure of the protein was most stable betweenpH 3.3 and 4.5. The guanidine HCl-induced denaturation of G-CSF involved more than a two-state transition, with detectable intermediate(s) present, and the structure of the intermediate(s) appeared to depend on thepH used. These results are consistent with thepH dependence of the structure described above, and demonstrate the complex conformational properties of G-CSF.     

Conformational stability of porcine serum transferrin.

The conformation of porcine serum ferric transferrin (Tf) and its stability against denaturation were studied by circular dichroism. Tf was estimated to have 19-24% alpha-helix and 50-55% beta-sheet based on the methods of Chang et al. (Chang, C.T., Wu, C.-S.C., & Yang, J.T., 1978, Anal. Biochem. 91, 13-31) and Provencher and Glöckner (Provencher, S.W. & Glöckner, J., 1981, Biochemistry 20, 33-37). Removal of the bound ferric ions (apo-Tf) did not alter the overall conformation, but there were subtle changes in local conformation based on its near-UV CD spectrum. The Tfs were stable between pH 3.5 and 11. Denaturation by guanidine hydrochloride (Gu-HCl) showed two transitions at 1.6 and 3.4 M denaturant. The process of denaturation by acid and base was reversible, whereas that by Gu-HCl was partially reversible. The irreversible thermal unfolding of Tfs began at temperatures above 60 degrees C and was not complete even at 80 degrees C. The bound irons (based on absorbance at 460 nm) were completely released at pH < 4 or in Gu-HCl solution above 1.7 M, when the protein began to unfold, but they remained intact in neutral solution even at 85 degrees C. The NH2- and COOH-terminal halves of the Tf molecule obtained by limited trypsin digestion had CD spectra similar to the spectrum of native Tf, and the COOH-terminal fragment had more stable secondary structure than the NH2-terminal fragment.     

Optimization of the enzyme-linked lectin assay for enhanced glycoprotein and glycoconjugate analysis

Lectins are proteins capable of recognizing and binding to specific oligosaccharide structures found on glycoproteins and other biomolecules. As such, they have utility for glycoanalytical applications. One common difficulty encountered in the application of these proteins, particularly in multiwell plate assay formats known as enzyme-linked lectin assays (ELLAs), is finding appropriate blocking solutions to prevent nonspecific binding with plate surfaces. Many commonly used blocking agents contain carbohydrates and generate significant background signals in ELLAs, limiting the utility of the assays. In this study, we examined the suitability of a range of blocking reagents, including protein-based, synthetic, and commercially available carbohydrate-free blocking reagents, for ELLA applications. Each blocking reagent was assessed against a panel of 19 commercially available biotinylated lectins exhibiting diverse structures and carbohydrate specificities. We identified the synthetic polymer polyvinyl alcohol (PVA) as the best global blocking agent for performing ELLAs. We ultimately present an ELLA methodology facilitating broad spectrum lectin analysis of glycoconjugates and extending the utility of ELLAs.     

Ligand binding and thermodynamic stability of a multidomain protein, calmodulin

Chemical and thermal denaturation of calmodulin has been monitored spectroscopically to determine the stability for the intact protein and its two isolated domains as a function of binding of Ca2+ or Mg2+. The reversible urea unfolding of either isolated apo-domain follows a two-state mechanism with relatively low deltaG(o)20 values of approximately 2.7 (N-domain) and approximately 1.9 kcal/mol (C-domain). The apo-C-domain is significantly unfolded at normal temperatures (20-25 degrees C). The greater affinity of the C-domain for Ca2+ causes it to be more stable than the N-domain at [Ca2+] > or = 0.3 mM. By contrast, Mg2+ causes a greater stabilization of the N- rather than the C-domain, consistent with measured Mg2+ affinities. For the intact protein (+/-Ca2+), the bimodal denaturation profiles can be analyzed to give two deltaG(o)20 values, which differ significantly from those of the isolated domains, with one domain being less stable and one domain more stable. The observed stability of the domains is strongly dependent on solution conditions such as ionic strength, as well as specific effects due to metal ion binding. In the intact protein, different folding intermediates are observed, depending on the ionic composition. The results illustrate that a protein of low intrinsic stability is liable to major perturbation of its unfolding properties by environmental conditions and liganding processes and, by extension, mutation. Hence, the observed stability of an isolated domain may differ significantly from the stability of the same structure in a multidomain protein. These results address questions involved in manipulating the stability of a protein or its domains by site directed mutagenesis and protein engineering.     

Testing the role of chain connectivity on the stability and structure of dihydrofolate reductase from E. coli: fragment complementation and circular permutation reveal stable, alternatively folded forms

The effects of chain cleavage and circular permutation on the structure, stability, and activity of dihydrofolate reductase (DHFR) from Escherichia coli were investigated by various spectroscopic and biochemical methods. Cleavage of the backbone after position 86 resulted in two fragments, (1--86) and (87--159) each of which are poorly structured and enzymatically inactive. When combined in a 1 : 1 molar ratio, however, the fragments formed a high-affinity (K(a) = 2.6 x 10(7) M(-1)) complex that displays a weakly cooperative urea-induced unfolding transition at micromolar concentrations. The retention of about 15% of the enzymatic activity of full-length DHFR is surprising, considering that the secondary structure in the complex is substantially reduced from its wild-type counterpart. In contrast, a circularly permuted form with its N-terminus at position 86 has similar overall stability to full-length DHFR, about 50% of its activity, substantial secondary structure, altered side-chain packing in the adenosine binding domain, and unfolds via an equilibrium intermediate not observed in the wild-type protein. After addition of ligand or the tight-binding inhibitor methotrexate, both the fragment complex and the circular permutant adopt more native-like secondary and tertiary structures. These results show that changes in the backbone connectivity can produce alternatively folded forms and highlight the importance of protein-ligand interactions in stabilizing the active site architecture of DHFR.     

A glucose/mannose binding lectin from litchi (Litchi chinensis) seeds: Biochemical and biophysical characterizations

BackgroundLectins are highly important biomolecules to study several biological processes. A novel α-D-glucose/mannose specific lectin was isolated from the seeds of litchi fruits (Litchi chinensis) and its various biophysical and biochemical properties were studied.MethodsPurification was done by successive Sephadex G 100 and Con A-Sepharose 4B affinity chromatography. SDS-PAGE, Surface Plasmon Resonance (SPR), steady state absorbance, fluorescence, time-correlated single-photon counting, circular dichroism and antibiofilm activity by measuring total protein estimation and azocasein degradation assay have been performed.ResultsThe purified lectin is a homodimer of molecular mass ~ 54 kDa. The amount of lectin required for hemagglutination of normal human O erythrocytes was 6.72 µg/ml. Among the saccharides tested, Man-α-(1,6)-Man was found to be the most potent inhibitor (0.01 mM) determined by hemagglutination inhibition assay. Steady state and time resolved fluorescence measurements revealed that litchi lectin formed ground state complex with maltose (K_a=4.9 (±0.2)×10⁴ M^?1), which indicated static quenching (Stern-Volmer (SV) constant K_sv=4.6 (±0.2)×10⁴ M^?1). CD measurements demonstrated that litchi lectin showed no overall conformational change during the binding process with maltose. The lectin showed antibiofilm activity against Pseudomonus aeruginosa.ConclusionsA novel homodimeric lectin has been purified from the seeds of litchi fruits (Litchi chinensis) having specificity for α-d-glucose/mannose. The thermodynamics and conformational aspects of its interaction with maltose have been studied in detail. The antibiofilm activity of this lectin towards Pseudomonus aeruginosa has been explored.General significanceThe newly identified litchi lectin is highly specific for α-d-glucose/mannose with an important antibiofilm activity towards Pseudomonus aeruginosa.     

A putative carbohydrate-binding domain of the lactosebindingCytisus sessilifolius anti-H(O) lectin has a similar amino acid sequence to that of thel-fucose-bindingUlex europaeus anti-H(O) lectin

The complete amino acid sequence of a lactose-bindingCytisus sessilifolius anti-H(O) lectin II (CSA-II) was determined using a protein sequencer. After digestion of CSA-II with endoproteinase Lys-C or Asp-N, the resulting peptides were purified by reversed-phase high performance liquid chromatography (HPLC) and then subjected to sequence analysis. Comparison of the complete amino acid sequence of CSA-II with the sequences of other leguminous seed lectins revealed regions of extensive homology. The amino acid sequence of a putative carbohydrate-binding domain of CSA-II was found to be similar to those of several anti-H(O) leguminous lectins, especially to that of thel-fucose-bindingUlex europaeus lectin I (UEA-I).Abbreviations BPA Bauhinia purpurea lectin - Con A concanavalin A - CMA-I Cytisus multiflorus lectin I - CMA-II Cytisus multiflorus lectin II - CSA-I Cytisus sessilifolius lectin I - CSA-II Cytisus sessilifolius lectin II - CSII Cytisus scoparius lectin II - ECorL Erythrina corallodendron lectin - GSIV Griffonia simplicifolia lectin IV - HPLC high performance liquid chromatography - LAA-I Laburnum alpinum lectin I - LAA-II Laburnum alpinum lectin II - LOL Lathyrus ochrus lectin - LTA Lotus tetragonolobus lectin - MAH Maackia amurensis haemagglutinin - PSA Pisum sativum lectin - SDS sodium dodecyl sulfate - TFA trifluoroacetic acid - UEA-I Ulex europaeus lectin I - UEA-II Ulex europaeus lectin II - VFA Vicia faba lectin     

Purification of a lectin from Eugenia uniflora L. seeds and its potential antibacterial activity

Aims: The aim of this work was to analyse the antimicrobial properties of a purified lectin from Eugenia uniflora L. seeds. Methods and Results: The E. uniflora lectin (EuniSL) was isolated from the seed extract and purified by ion‐exchange chromatography in DEAE‐Sephadex with a purification factor of 11·68. The purified lectin showed a single band on denaturing electrophoresis, with a molecular mass of 67 kDa. EuniSL agglutinated rabbit and human erythrocytes with a higher specificity for rabbit erythrocytes. The haemagglutination was not inhibited by the tested carbohydrates but glycoproteins exerted a strong inhibitory action. The lectin proved to be thermo resistant with the highest stability at pH 6·5 and divalent ions did not affect its activity. EuniSL demonstrated a remarkable nonselective antibacterial activity. EuniSL strongly inhibited the growth of Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella sp. with a minimum inhibitory concentration (MIC) of 1·5 μg ml^?1, and moderately inhibited the growth of Bacillus subtilis, Streptococcus sp. and Escherichia coli with a MIC of 16·5 μg ml^?1. Conclusions: EuniSL was found to be effective against bacteria. Significance and Impact of the Study: The strong antibacterial activity of the studied lectin indicates a high potential for clinical microbiology and therapeutic applications.