A calorimetric and fluorescence study of batch cultures of Saccharomyces cerevisiae

Summary Batch growth of Saccharomyces cerevisiae was studied using a fluorescence probe as well as a microcalorimeter. The biphasic growth pattern previously demonstrable by microcalorimetry was also seen using the fluorescence probe. Acid production rate changes in the transition phase could be detected by pH measurements. Qualitative similarities between calorimetric and fluorescence measurements are discussed.     

Protein-ligand interaction. A calorimetric study of the interaction of oligosaccharides and hen ovalbumin glycopeptides with concanavalin A

A calorimetric study is reported concerning the interaction between concanavalin A (Con A) and some oligosaccharides and glycopeptides hydrolyzed from hen ovalbumin. The measurements were carried out in acetate buffer, pH 4.5, where, by far, the prevailing form of the protein is the dimeric one [Kalb, A.J., & Lustig, A. (1968) Biochim. Biophys. Acta 168, 366; Dani, M., Manca, F., & Rialdi, G. (1981) Biochim. Biophys. Acta 667, 108]. The calorimetric technique allows the direct determination of the binding enthalpy delta H, degrees B, the evaluation of the apparent association constant K'B, and then the evaluation of the apparent free energy and entropy, delta G degrees' B and delta S degrees' B. Three groups of data have been collected in the present study. The first one concerns the interaction between concanavalin A and some mono- and disaccharides [methyl alpha-glucopyranoside (alpha MGlup), methyl alpha-mannopyranoside (alpha MManp), D-maltose, D-trehalose, and D-cellobiose]. The analysis of the data indicates that in these cases there are small favorable entropic and enthalpic contributions to the affinity. The stoichiometry of the reaction is 2 mol of ligand/mol of Con A dimer, the sites resulting being equivalent and noninteracting. Melezitose, the only trisaccharide studied, shows a different behavior: its affinity for Con A is higher as compared to the other oligosaccharides containing alpha-glucosyl residues and closer to that of methyl alpha-mannopyranoside. However, the stoichiometry is different, namely, 1 mol of ligand/dimer of Con A.(ABSTRACT TRUNCATED AT 250 WORDS)     

A calorimetric study of subunit interaction in immunoglobulin G

The non covalent interaction of the heavy and light chains of immuno-globulin G has been studied in a batch calorimeter and shown to be exothermic. The enthalpy of association ranged from ?5.6 to ?112.5 kJ/mole of heavy-light chain pairs formed at 25° for subunits derived from eight myeloma proteins. The values for kappa and lambda chains were not significantly different. The enthalpy showed a marked temperature dependance giving changes in heat capacity near -9kJ/deg/mole between 25° and 35° suggesting the involvement of apolar bonds in the association. However the large negative enthalpy of association suggests the presence of additional types of bonding.     

Calmodulin-drug interaction. A fluorescence and flow dialysis study

Various Ca2+-antagonists and related compounds were probed for possible anti-calmodulin properties. Some of them efficiently inhibit calmodulin dependent activity (the plasma membrane Ca2+-ATPase and the cyclic nucleotide phosphodiesterase). The I50-values for the most potent inhibitors varied between 15 and 30 uM. Using fluorescence spectroscopy and flow dialysis methods the stoichiometry of the binding of some of the drugs to calmodulin has been investigated. The number of Ca2+-dependent high affinity binding sites has been studied on trypsin fragments of calmodulin. Compound 12-114 was bound with high affinity in a Ca2+-dependent way to both halves of calmodulin, compound 200-737 recognized one high affinity binding site only in the C-terminal half of the molecule, whereas compound 36-079 demanded the intact protein to be able to interact with high affinity in a Ca2+-dependent manner.     

A calorimetric study of the interaction of ATP with rabbit muscle phosphofructokinase.

The heat of interaction of ATP with phosphofructokinase from rabbit muscle was determined at 25 degrees C in 0.1 M potassium phosphate, pH 7.0 and 8.0. The limiting value of the enthalpy change at high ATP concentrations was found to be -11.5 kcal mol of enzyme polypeptide chains. Since phosphate and imidazole have very different heats of ionization (+0.8 and +7.5 kcal/mol, respectively), this suggests that the binding of at least two protons to the enzyme occurs concomitantly with the binding of ATP at the regulatory site.     

A study of interaction of 7-aminoactinomycin D with DNA by fluorescence correlation spectroscopy

The investigation of interaction of 7-aminoactinomycin D with DNA is hampered at nanomolar concentrations by low quantum yield of the dye fluorescence and high adsorbability of the dye. It was found that, instead of the increase in the fluorescence of 7-aminoactinomycin D after binding with DNA, a decrease in the fluorescence takes place, because 7-aminoactinomycin D complexed with DNA exists in two states: photochemically active and inactive. The presence of the photochemically active state of 7-aminoactinomycin D causes an apparent increase in the diffusion coefficient upon embedding of 7-aminoactinomycin D into DNA. The data obtained allow one to state that 7-aminoactinomycin D: (1) forms dimers at micromolar concentration, (2) has two specific photodestruction times, and (3) binds with DNA more effectively than actinomycin D. Polarization measurements made it possible to estimate numerically the adsorption of 7-aminoactinomycin D on the walls of the measuring cell.     

2.3 A crystal structure of tetanus neurotoxin light chain

TeNT is the causative agent of the neuroparalytic disease tetanus. A key component of TeNT is its light chain, a Zn(2+) endopeptidase that targets SNAREs. Recent structural studies of closely related BoNT endopeptidases indicate that substrate-binding exosites remote from a conserved active site are the primary determinants of substrate specificity. Here we report the 2.3 A X-ray crystal structure of TeNT-LC, determined by combined molecular replacement and MAD phasing. As expected, the overall structure of TeNT-LC is similar to the other known CNT light chain structures, including a conserved thermolysin-like core inserted between structurally distinct amino- and carboxy-terminal regions. Differences between TeNT-LC and the other CNT light chains are mainly limited to surface features such as unique electrostatic potential profiles. An analysis of surface residue conservation reveals a pattern of relatively high variability matching the path of substrate binding around BoNT/A, possibly serving to accommodate the variations in different SNARE targets of the CNT group.     

The active site structure of tetanus neurotoxin resolved by multiple scattering analysis in X-Ray absorption spectroscopy.

A detailed study of the x-ray absorption spectrum of tetanus neurotoxin in the K-edge EXAFS region of the zinc absorber is presented that allows the complete identification of the amino acid residues coordinated to the zinc active site. A very satisfactory interpretation of the experimental data can be given if multiple scattering contributions are included in the analysis. Comparing the absorption spectrum of tetanus neurotoxin to that of two other structurally similar zinc-endopeptidases, thermolysin and astacin, in which the zinc coordination mode is known from crystallographic data, we conclude that in tetanus neurotoxin, besides a water molecule, zinc is coordinated to two histidines and a tyrosine.     

Molecular structure of tetanus neurotoxin as revealed by Fourier transform infrared and circular dichroic spectroscopy

Secondary structure contents of tetanus neurotoxin have been estimated at neutral and acidic pH using circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy. An analysis of the far-ultraviolet CD spectra of the neurotoxin dissolved in 50 mM citrate-phosphate buffer (pH 7.0) revealed 20.0 +/- 2.1% alpha-helix, 50.5 +/- 2.1% beta-pleated sheets, no beta-turns, and 29.5% random coils, which is at considerable variance with results from an earlier detailed study of tetanus neurotoxin's secondary structures (J.P. Robinson, L.A. Holladay, J.H. Hash and D. Puett, J. Biol. Chem. 257 (1982) 407). However, the alpha-helix content estimated in this study is consistent with the earlier studies of Robinson et al. (J.P. Robinson, L.A. Holladay, J.B. Picklesimer and D. Puett, Mol. Cell. Biochem. 5 (1974) 147; J.P. Robinson, J.B. Picklesimer and D. Puett, J. Biol. Chem. 250 (1975) 7435) and with the study by Lazarovici et al. (P. Lazarovici, P. Yanai and E. Yavin, J. Biol. Chem. 262 (1986) 2645), although other secondary structural features do not agree with those of the previous studies. Secondary structure estimation from Fourier transform infrared spectra in both amide I and amide III frequency regions revealed 22-23% alpha-helix, 49-51% beta-pleated sheets and 27-28% random coils, indicating a good correlation with the secondary structure content estimated from CD analysis. Lowering of the pH of the neurotoxin to 5.5 or 4.0 did not result in any noticeable change in the overall secondary structures. However, there were significant pH-induced variations observed in the individual curve-fitted FT-IR bands in the amide III frequency region. For example, the 1302 cm-1 band (relative area, 4.2%) observed at pH 7.0 was shifted to 1297 cm-1 (relative area, 2.2%) at pH 5.5, and the relative area of the band at 1316-1317 cm-1 (alpha-helix) increased by approx. 40%. This study suggests that contrary to earlier reports, tetanus neurotoxin is a beta-pleated sheet dominated structure, and although lower pH does not change the overall contents of the secondary structures, significant conformational alterations are observed.     

Substrate recognition of VAMP-2 by botulinum neurotoxin B and tetanus neurotoxin

Botulinum neurotoxin (BoNT; serotypes A-G) and tetanus neurotoxin elicit flaccid and spastic paralysis, respectively. These neurotoxins are zinc proteases that cleave SNARE proteins to inhibit synaptic vesicle fusion to the plasma membrane. Although BoNT/B and tetanus neurotoxin (TeNT) cleave VAMP-2 at the same scissile bond, their mechanism(s) of VAMP-2 recognition is not clear. Mapping experiments showed that residues 60-87 of VAMP-2 were sufficient for efficient cleavage by BoNT/B and that residues 40-87 of VAMP-2 were sufficient for efficient TeNT cleavage. Alanine-scanning mutagenesis and kinetic analysis identified three regions within VAMP-2 that were recognized by BoNT/B and TeNT: residues adjacent to the site of scissile bond cleavage (cleavage region) and residues located within N-terminal and C-terminal regions relative to the cleavage region. Analysis of residues within the cleavage region showed that mutations at the P7, P4, P2, and P1' residues of VAMP-2 had the greatest inhibition of LC/B cleavage (> or =32-fold), whereas mutations at P7, P4, P1', and P2' residues of VAMP-2 had the greatest inhibition of LC/TeNT cleavage (> or =64-fold). Residues within the cleavage region influenced catalysis, whereas residues N-terminal and C-terminal to the cleavage region influenced binding affinity. Thus, BoNT/B and TeNT possess similar organization but have unique residues to recognize and cleave VAMP-2. These studies provide new insights into how the clostridial neurotoxins recognize their substrates.     

Affinity-purified tetanus neurotoxin interaction with synaptic membranes: properties of a protease-sensitive receptor component

The pharmacokinetic interaction of an affinity-purified 125I-labeled tetanotoxin fraction with guinea pig brain synaptosomal preparations was investigated. Binding of tetanotoxin was time- and temperature-dependent, was proportional to protein concentration, and was saturable at about 8 X 10(-9) M as estimated by a solid-surface binding assay. Binding was optimal at pH 6.5 under low ionic strength buffer and was almost entirely blocked by gangliosides or antitoxin. In analogy to intact nerve cells, binding of toxin to membranes resulted in a tight association operationally defined as sequestration. Binding and sequestration were abolished after membrane pretreatment with sialidase. The enzyme could not dissociate the membrane-bound toxin formed at 4 or 37 degrees C under low ionic strength conditions, which is in part compatible with internalization as defined in nerve cell cultures. In the latter system the toxin could be removed at 4 degrees C but not at 37 degrees C. Binding was significantly reduced upon pretreatment of guinea pig brain membranes by a variety of hydrolytic enzymes. Trypsin and chymotrypsin inhibited binding between 55% and 68% while bacterial protease abolished it by 91-95%. The effect was species-specific as it was not seen in rat or bovine synaptosomes. Collagenase and hyaluronidase had little or no inhibitory effect when applied to synaptosomes (27% and 9%) but inhibited binding to synaptic vesicles by 56% and 49%, respectively. Phospholipases A2 and C caused 42-43% inhibition of binding in vesicles and less than 22% in synaptosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Site-specific interaction of thrombin and inhibitors observed by fluorescence correlation spectroscopy.

We report on the application of fluorescence correlation spectroscopy (FCS) to observe the interaction between thrombin and thrombin inhibitors. Two site-specific fluorescent labels were used to distinguish between inhibitors directed to the active site, the exosite, or both binding sites of thrombin. For several well-known inhibitors of thrombin, the binding sites observed by FCS correspond to previous studies. The interaction of the recently discovered thrombin inhibitor ornithodorin from the tick Ornithodorus moubata with thrombin was investigated. It was found that this inhibitor, like hirudin and rhodniin, binds to both the active site and exosite of thrombin simultaneously. This study shows the feasibility of FCS as a sensitive and selective method for observing protein-ligand interactions. As an additional technique, simultaneous labeling with both fluorescent labels was successfully demonstrated.     

Reconstitution of tetanus neurotoxin from two antigenically active polypeptide fragments.

Whole tetanus toxin indistinguishable from the undissociated toxin (molecular weight 160,000), in terms of molecular weight, electrophoretic behavior, antigenicity, and toxicity, was reconstituted. This was achieved by removal of urea and dithiothreitol (DTT) by dialysis from a mixture of freshly purified fragment α (molecular weight 53,000) and fragment β (molecular weight 107,000) in buffer containing 2 M urea and 1 mM DTT. The highest yield (almost 100%) of reconstituted toxin was obtained when the two isolated fragments were mixed in a molar ratio of 1:1. No toxin was reconstituted from either one of two fragments alone.     

Protein-protein interaction analysis by C-terminally specific fluorescence labeling and fluorescence cross-correlation spectroscopy

Here, we describe novel puromycin derivatives conjugated with iminobiotin and a fluorescent dye that can be linked covalently to the C-terminus of full-length proteins during cell-free translation. The iminobiotin-labeled proteins can be highly purified by affinity purification with streptavidin beads. We confirmed that the purified fluorescence-labeled proteins are useful for quantitative protein-protein interaction analysis based on fluorescence cross-correlation spectroscopy (FCCS). The apparent dissociation constants of model protein pairs such as proto-oncogenes c-Fos/c-Jun and archetypes of the family of Ca2+-modulated calmodulin/related binding proteins were in accordance with the reported values. Further, detailed analysis of the interactions of the components of polycomb group complex, Bmi1, M33, Ring1A and RYBP, was successfully conducted by means of interaction assay for all combinatorial pairs. The results indicate that FCCS analysis with puromycin-based labeling and purification of proteins is effective and convenient for in vitro protein-protein interaction assay, and the method should contribute to a better understanding of protein functions by using the resource of available nucleotide sequences.     

Dissociation of tetanus neurotoxin into two polypeptide fragments

Analyses of neurotoxin protein of $\text{Clostridium tetani}$ by polyacrylamide gel electrophoresis showed that the toxin as purified from culture filtrates (“extracellular” toxin, molecular weight 160,000) could be dissociated into two polypeptide chains of molecular weight 53,000 (Fragment α) and 107,000 (Fragment β) by treatment with dithiothreitol and sodium dodecyl sulfate. The toxin as purified from bacterial extracts (“intracellular” toxin) was found to consist of a single 160,000 dalton polypeptide chain, which is undissociable by such treatment but, when pretreated with trypsin, becomes dissociable into two fragments apparently identical with α and β.     

Hydration and thermal denaturation of beta-lactoglobulin. A calorimetric study.

The thermal properties of the beta-lactoglobulin-water system were investigated by differential scanning calorimetry in the temperature range from -50 to 130 degrees C. Determination of the heat and temperature of fusion of the absorbed water allowed resolution of the water into four different states. The amounts of water in these states were different for samples before and after heat denaturation. In the case of denatured beta-lactoglobulin, a smaller amount of water with thermal properties different from ordinary water was observed and its total water binding capacity was lower. The thermal stability of beta-lactoglobulin in the water content range from 0 to 0.75 g/g showed a strong dependence on the degree of hydration. A correlation was observed between the changes in the thermal stability of the protein and the changes in the state of the absorbed water. The results are compared with those obtained from similar measurements of other globular proteins and of fibrillar proteins.     

Botulinum neurotoxin type A: Structure and interaction with the micellar concentration of SDS determined by FT-IR spectroscopy

Secondary structures of botulinum neurotoxin type A have been determined using Fourier transform infrared spectroscopy in the amide I and amide III frequency regions. Using Fourier self-deconvolution, second derivatization, and curve-fit analysis, the amide I frequency contour was resolved into Gaussian bands at 1678, 1654, 1644, and 1634 cm^–1. In the amide III frequency region, several small bands were resolved between 1320 and 1225 cm^–1. Assignments of the bands in both amide I and amide III frequency regions to various types of secondary structures and the estimation of spectral band strengths by integrating areas under each band suggested that the neurotoxin contains 29% -helix, 45–49% -sheets and 22–26% random coils. These values agreed very well with those determined earlier from CD spectra. The neurotoxin was treated with a micellar concentration of sodium dodecyl sulfate to simulate interaction between the protein and the amphipathic molecules. Sodium dodecyl sulfate micelles induced significant alterations both in the spectral band positions, and their strengths suggest refolding of the neurotoxin polypeptides. However, these changes were not entirely reversible, which could implicate the role of the altered structures in the function of the neurotoxin.     

Lipid diffusion in planar membranes investigated by fluorescence correlation spectroscopy

Investigation of lipid lateral mobility in biological membranes and their artificial models provides information on membrane dynamics and structure; methods based on optical microscopy are very convenient for such investigations. We focus on fluorescence correlation spectroscopy (FCS), explain its principles and review its state of the art versions such as 2-focus, Z-scan or scanning FCS, which overcome most artefacts of standard FCS (especially those resulting from the need for an external calibration) making it a reliable and versatile method. FCS is also compared to single particle tracking and fluorescence photobleaching recovery and the applicability and the limitations of the methods are briefly reviewed. We discuss several key questions of lateral mobility investigation in planar lipid membranes, namely the influence which membrane and aqueous phase composition (ionic strength and sugar content), choice of a fluorescent tracer molecule, frictional coupling between the two membrane leaflets and between membrane and solid support (in the case of supported membranes) or presence of membrane inhomogeneities has on the lateral mobility of lipids. The recent FCS studies addressing those questions are reviewed and possible explanations of eventual discrepancies are mentioned.