Studies on tryptophan residues of Abrus agglutinin Stopped-flow kinetics of modification and fluorescence-quenching studies

The presence of two essential tryptophan residues/molecule was implicated in the binding site of Abrus agglutinin [Patanjali, Swamy, Anantharam, Khan & Surolia (1984) Biochem. J. 217, 773-781]. A detailed study of the stopped-flow kinetics of the oxidation of tryptophan residues revealed three classes of tryptophan residues in the native protein. A discrete reorganization of tryptophan residues into two phases was observed upon ligand binding. The heterogeneity of tryptophan exposure was substantiated by quenching studies with acrylamide, succinimide and Cs+. Our study revealed the microenvironment of tryptophan residues to be hydrophobic, and also the presence of acidic amino acid residues in the vicinity of surface-localized tryptophan residues.

Saccharide binding to three Gal/GalNAc specific lectins: Fluorescence, spectroscopic and stopped-flow kinetic studies

Fluorescence and stopped-flow spectrophotometric studies on three plant lectins fromPsophocarpus tetragonolobus (winged bean),Glycine max (soybean) andArtocarpus integrifolia (jack fruit) have been studied usingN-dansylgalactosamine as a fluorescent ligand. The best monosaccharide for the winged bean agglutinin I (WBA I) and soybean (SBA) is Me-agrGalNAc and for jack fruit agglutinin (JFA) is Me-agrGal. Examination of the percentage enhancement and association constants (1.51×106, 6.56×106 and 4.17×105 M–1 for SBA, WBA I and JFA, respectively) suggests that the combining regions of the lectins SBA and WBA I are apolar whereas that of JFA is polar. Thermodynamic parameters obtained for the binding of several monosaccharides to these lectins are enthalpically favourable. The binding of monosaccharides to these lectins suggests that the-OH groups at C-1, C-2, C-4 and C-6 in thed-galactose configuration are important loci for interaction with these lectins. An important finding is that the JFA binds specifically to Galß1-3GaINAc with much higher affinity than the other disaccharides which are structurally and topographically similar.The results of stopped-flow spectrometry on the binding ofN-dansylgalactosamine to these lectins are consistent with a bimolecular single step mechanism. The association rate constants (2.4×105, 1.3×104, and 11.7×105 M–1 sec–1 for SBA, WBA I and JFA, respectively) obtained are several orders of magnitude slower than the ones expected for diffusion controlled reactions. The dissociation rate constants (0.2, 3.2×10–2, 83.3 sec–1 for SBA, WBA I and JFA, respectively) obtained for the dissociation ofN-dansylgalactosamine from its lectin complex are slowest for SBA and WBA I when compared with any other lectin-ligand dissociation process.

Milk lactate determination with a rotating bioreactor based on an electron transfer mediated by osmium complexes incorporating a continuous-flow/stopped-flow system

The high sensitivity that can be attained using a bienzymatic system and mediated by the redox polymer [Os(bpy)₂ClPyCH₂NHpoly(allylamine)] (Os-PAA), has been verified by on-line interfacing of a rotating bioreactor and continuous-flow/stopped-flow/continuous-flow processing. When the hydrogen peroxide formed by LO_x layer reaches the inner layer, the electronic flow between the immobilized peroxidase and the electrode surface produces a current, proportional to lactate concentration. The determination of lactate was possible with a limit of detection of 5 nmol l⁻¹ in the processing of as many as 30 samples per hour. This arrangement allows working in undiluted milk samples with a good stability and reproducibility. Horseradish peroxidase [EC 1.11.1.7] and Os-PAA were covalently immobilized on the glassy carbon electrode surface (upper cell body), lactate oxidase [EC 1.1.3.x] was immobilized on a disk that can be rotated.

Denaturant-induced movement of the transition state of protein folding revealed by high-pressure stopped-flow measurements

The small all-β protein tendamistat folds and unfolds with two-state kinetics. We determined the volume changes associated with the folding process by performing kinetic and equilibrium measurements at variable pressure between 0.1 and 100 MPa (1 to 1,000 bar). GdmCl-induced equilibrium unfolding transitions reveal that the volume of the native state is increased by 41.4 ± 2.0 cm3/mol relative to the unfolded state. This value is virtually independent of denaturant concentration. The use of a high-pressure stopped-flow instrument enabled us to measure the activation volumes for the refolding (ΔVf0‡) and unfolding reaction (ΔVu0‡) over a broad range of GdmCl concentrations. The volume of the transition state is 60% native-like (ΔVf0‡ = 25.0 ± 1.2 cm3/mol) in the absence of denaturant, indicating partial solvent accessibility of the core residues. The volume of the transition state increases linearly with denaturant concentration and exceeds the volume of the native state above 6 M GdmCl. This result argues for a largely desolvated transition state with packing deficiencies at high denaturant concentrations and shows that the structure of the transition state depends strongly on the experimental conditions.

Least activation path for protein folding: investigation of staphylococcal nuclease folding by stopped-flow circular dichroism.

Is the pathway of protein folding determined by the relative stability of folding intermediates, or by the relative height of the activation barriers leading to these intermediates? This is a fundamental question for resolving the Levinthal paradox, which stated that protein folding by a random search mechanism would require a time too long to be plausible. To answer this question, we have studied the guanidinium chloride (GdmCl)-induced folding/unfolding of staphylococcal nuclease [(SNase, formerly EC 3.1.4.7; now called microbial nuclease or endonuclease, EC 3.1.31.1] by stopped-flow circular dichroism (CD) and differential scanning microcalorimetry (DSC). The data show that while the equilibrium transition is a quasi-two-state process, kinetics in the 2-ms to 500-s time range are triphasic. Data support the sequential mechanism for SNase folding: U3 <--> U2 <--> U1 <--> N0, where U1, U2, and U3 are substates of the unfolded protein and N0 is the native state. Analysis of the relative population of the U1, U2, and U3 species in 2.0 M GdmCl gives delta-G values for the U3 --> U2 reaction of +0.1 kcal/mol and for the U2 --> U1 reaction of -0.49 kcal/mol. The delta-G value for the U1 --> N0 reaction is calculated to be -4.5 kcal/mol from DSC data. The activation energy, enthalpy, and entropy for each kinetic step are also determined. These results allow us to make the following four conclusions. (i) Although the U1, U2, and U3 states are nearly isoenergetic, no random walk occurs among them during the folding. The pathway of folding is unique and sequential. In other words, the relative stability of the folding intermediates does not dictate the folding pathway. Instead, the folding is a descent toward the global free-energy minimum of the native state via the least activation path in the vast energy landscape. Barrier avoidance leads the way, and barrier height limits the rate. Thus, the Levinthal paradox is not applicable to the protein-folding problem. (ii) The main folding reaction (U1 --> N0), in which the peptide chain acquires most of its free energy (via van der Waals' contacts, hydrogen bonding, and electrostatic interactions), is a highly concerted process. These energy-acquiring events take place in a single kinetic phase. (iii) U1 appears to be a compact unfolded species; the rate of conversion of U2 to U1 depends on the viscosity of solution. (iv) All four relaxation times reported here depend on GdmCl concentrations: it is likely that none involve the cis/trans isomerization of prolines. Finally, a mechanism is presented in which formation of sheet-like chain conformations and a hydrophobic condensation event precede the main-chain folding reaction.

Stopped-flow NMR spectroscopy: real-time unfolding studies of 6-19F-tryptophan-labeled Escherichia coli dihydrofolate reductase.

Escherichia coli dihydrofolate reductase (DHFR; EC 1.5.1.3) contains five tryptophan residues that have been replaced with 6-19F-tryptophan. The 19F NMR assignments are known in the native, unliganded form and the unfolded form. We have used these assignments with stopped-flow 19F NMR spectroscopy to investigate the behavior of specific regions of the protein in real time during urea-induced unfolding. The NMR data show that within 1.5 sec most of the intensities of the native 19F resonances of the protein are lost but only a fraction (approximately 20%) of the intensities of the unfolded resonances appears. We postulate that the early disappearance of the native resonances indicates that most of the protein rapidly forms an intermediate in which the side chains have considerable mobility. Stopped-flow far-UV circular dichroism measurements indicate that this intermediate retains native-like secondary structure. Eighty percent of the intensities of the NMR resonances assigned to the individual tryptophans in the unfolded state appear with similar rate constants (k approximately 0.14 sec-1), consistent with the major phase of unfolding observed by stopped-flow circular dichroism (representing 80% of total amplitude). These data imply that after formation of the intermediate, which appears to represent an expanded structural form, all regions of the protein unfold at the same rate. Stopped-flow measurements of the fluorescence and circular dichroism changes associated with the urea-induced unfolding show a fast phase (half-time of about 1 sec) representing 20% of the total amplitude in addition to the slow phase mentioned above. The NMR data show that approximately 20% of the total intensity for each of the unfolded tryptophan resonances is present at 1.5 sec, indicating that these two phases may represent the complete unfolding of the two different populations of the native protein.

Two Components of Actin-based Retrograde Flow in Sea Urchin Coelomocytes

Sea urchin coelomocytes represent an excellent experimental model system for studying retrograde flow. Their extreme flatness allows for excellent microscopic visualization. Their discoid shape provides a radially symmetric geometry, which simplifies analysis of the flow pattern. Finally, the nonmotile nature of the cells allows for the retrograde flow to be analyzed in the absence of cell translocation. In this study we have begun an analysis of the retrograde flow mechanism by characterizing its kinetic and structural properties. The supramolecular organization of actin and myosin II was investigated using light and electron microscopic methods. Light microscopic immunolocalization was performed with anti-actin and anti-sea urchin egg myosin II antibodies, whereas transmission electron microscopy was performed on platinum replicas of critical point-dried and rotary-shadowed cytoskeletons. Coelomocytes contain a dense cortical actin network, which feeds into an extensive array of radial bundles in the interior. These actin bundles terminate in a perinuclear region, which contains a ring of myosin II bipolar minifilaments. Retrograde flow was arrested either by interfering with actin polymerization or by inhibiting myosin II function, but the pathway by which the flow was blocked was different for the two kinds of inhibitory treatments. Inhibition of actin polymerization with cytochalasin D caused the actin cytoskeleton to separate from the cell margin and undergo a finite retrograde retraction. In contrast, inhibition of myosin II function either with the wide-spectrum protein kinase inhibitor staurosporine or the myosin light chain kinase–specific inhibitor KT5926 stopped flow in the cell center, whereas normal retrograde flow continued at the cell periphery. These differential results suggest that the mechanism of retrograde flow has two, spatially segregated components. We propose a “push–pull” mechanism in which actin polymerization drives flow at the cell periphery, whereas myosin II provides the tension on the actin cytoskeleton necessary for flow in the cell interior.

Delivery of nitric oxide for analysis of the function of cytochrome c'

On delivery of nitric oxide (NO) to protein samples (e.g., cytochrome c'), for spectroscopic experiments it is important to avoid exposure to oxygen and to remove contaminants from the NO gas. We describe a number of techniques for steady-state UV/Vis spectrophotometry and pre-steady-state stopped-flow spectrophotometry analysis of cytochrome c'.

The carbon dioxide hydration activity of the sulfonamide-resistant carbonic anhydrase from the liver of male rat: pH independence of the steady-state kinetics

The steady-state kinetic constants for the catalysis of CO2 hydration by the sulfonamide-resistant and testosterone-induced carbonic anhydrase from the liver of the male rat has been determined by stopped-flow spectrophotometry. The turnover number was 2.6 ± 0.6 × 103 s− at 25 °C, and was invariant with pH ranging from 6.2 to 8.2 within experimental error. The Km at 25 °C was 5 ± 1 mImage , and was also pH independent. These data are in quantitative agreement with earlier findings of pH-independent CO2 hydration activity for the mammalian skeletal muscle carbonic anhydrase isozyme III. The turnover numbers for higher-activity isozymes I and II are strongly pH dependent in this pH range. Thus, the kinetic status of the male rat liver enzyme is that of carbonic anhydrase III. This finding is consistent with preliminary structural and immunologic data from other laboratories.

Thermodynamic and kinetic studies on saccharide binding to soya-bean agglutinin.

The fluorescence of N-dansylgalactosamine [N-(5-dimethylaminonaphthalene-1-sulphonyl)galactosamine] was enhanced 11-fold with a 25 nm blue-shift in the emission maximum upon binding to soya-bean agglutinin (SBA). This change was used to determine the association constants and thermodynamic parameters for this interaction. The association constant of 1.51 X 10(6) M-1 at 20 degrees C indicated a very strong binding, which is mainly due to a relatively small entropy value, as revealed by the thermodynamic parameters: delta G = -34.7 kJ X mol-1, delta H = -37.9 kJ X mol-1 and delta S = -10.9 J X mol-1 X K-1. The specific binding of this sugar to SBA shows that the lectin can accommodate a large hydrophobic substituent on the C-2 of galactose. Binding of non-fluorescent ligands, studied by monitoring the fluorescence changes when they are added to a mixture of SBA and N-dansylgalactosamine, indicates that a hydrophobic substituent at the anomeric position increases the affinity of the interaction. The C-6 hydroxy group also stabilizes the binding considerably. Kinetics of binding of N-dansylgalactosamine to SBA studied by stopped-flow spectrofluorimetry are consistent with a single-step mechanism and yielded k+1 = 2.4 X 10(5) M-1 X s-1 and k-1 = 0.2 s-1 at 20 degrees C. The activation parameters indicate an enthalpicly controlled association process.

Thermodynamic and kinetic analysis of carbohydrate binding to the basic lectin from winged bean (Psophocarpus tetragonolobus)

A basic lectin (pI approximately 10.0) was purified to homogeneity from the seeds of winged bean (Psophocarpus tetragonolobus) by affinity chromatography on Sepharose 6-aminocaproyl-D-galactosamine. The lectin agglutinated trypsinized rabbit erythrocytes and had a relative molecular mass of 58,000 consisting of two subunits of Mr 29,000. The lectin binds to N-dansylgalactosamine, leading to a 15-fold increase in dansyl fluorescence with a concomitant 25-nm blue shift in the emission maximum. The lectin has two binding sites/dimer for this sugar and an association constant of 4.17 X 10(5) M-1 at 25 degrees C. The strong binding to N-dansylgalactosamine is due to a relatively positive entropic contribution as revealed by the thermodynamic parameters: delta H = -33.62 kJ mol-1 and delta S0 = -5.24 J mol-1 K-1. Binding of this sugar to the lectin shows that it can accommodate a large hydrophobic substituent on the C-2 carbon of D-galactose. Studies with other sugars indicate that a hydrophobic substituent in alpha- conformation at the anomeric position increases the affinity of binding. The C-4 and C-6 hydroxyl groups are critical for sugar binding to this lectin. Lectin difference absorption spectra in the presence of N-acetylgalactosamine indicate perturbation of tryptophan residues on sugar binding. The results of stopped flow kinetics with N- dansylgalactosamine and the lectin are consistent with a simple one- step mechanism for which k+1 = 1.33 X 10(4) M-1 s-1 and k-1 = 3.2 X 10(- 2) s-1 at 25 degrees C. This k-1 is slower than any reported for a lectin-monosaccharide complex so far. The activation parameters indicate an enthalpically controlled association process.

Studies on a chitooligosaccharide-specific lectin from Coccinia indica. Thermodynamics and kinetics of umbelliferyl glycoside binding.

Coccinia indica agglutinin (CIA) is a chitooligosaccharide-specific lectin with two binding sites/homodimer of M(r) 32,000. Quenching studies implied tryptophan involvement in binding activity, which was confirmed by chemical modification experiments (A. R. Sanadi and A. Surolia, submitted for publication). Binding of 4-methylumbelliferyl chitooligosaccharides has been carried out to study their binding by CIA. Reversal experiments confirm the validity of the data previously obtained (A. R. Sanadi and A. Surolia, submitted for publication) from intrinsic fluorescence studies. Surprisingly, unlike wheat germ agglutinin, there is no consistent thermodynamic effect of the chromophoric label on binding activities as compared with the native sugars. From the changes in the optical properties of the chromophoric group upon binding to CIA, it has been possible to confirm that the tryptophan located in the binding site is closest to the fourth subsite. Thermodynamic analysis shows that the binding of the labeled tetrasaccharide is very strongly entropically driven, with the terminal, nonreducing sugar residue protruding from the binding pocket. The results of stopped-flow kinetic studies on the binding of the chromophoric trisaccharide by CIA show that the mechanism of binding is a one-step process.