Raised calcium promotes α-synuclein aggregate formation.

Parkinson's and Parkinson's-plus diseases are associated with abnormal, aggregated forms of the protein, α-synuclein. We have investigated the effects of calcium on α-synuclein aggregation in vitro and in vivo. We treated monomeric α-synuclein with calcium in vitro and used fluorescence imaging, fluorescence correlation and scanning electron microscopy to investigate protein aggregation. Incubation of fluorescent-labelled monomeric α-synuclein (24h) at low concentration (10 µM) with calcium resulted in surface aggregates (1.5±0.7 µm(2)) detected by fluorescence microscopy saturating at a half-maximum calcium concentration of 80 µM, whilst incubations without calcium showed few protein aggregates. Scanning electron microscopy revealed that α-synuclein surface plaques (0.5-1 µm) form in the presence of calcium and comprise 10-20 nm globular particles. Incubation of α-synuclein at high concentration (75 µM; 6h) resulted in soluble oligomeric aggregates detected by fluorescence correlation spectroscopy in a calcium dependent process, saturating at a half maximum calcium concentration of 180 µM. In cell culture experiments, we used thapsigargin or calcium ionophore A23187 to induce transient increases of intracellular free calcium in human 1321N1 cells expressing an α-synuclein-GFP construct and observed calcium flux and α-synuclein aggregation by fluorescence microscopy. The cell culture data shows that a transient increase in intracellular free calcium significantly increased the proportion of cells bearing cytoplasmic α-synuclein aggregates 6 and 12h post-treatment (P, 0.01). Our data indicates that calcium accelerates α-synuclein aggregation on surfaces, in free solution and in cultured cells and suggests that surface adsorption may play an important role in the calcium-dependent aggregation mechanism.

Comparative evaluation of stable TAFIa variants: importance of alpha-helix 9 and beta-sheet 11 for TAFIa (in)stability.

BACKGROUND: Activated thrombin activatable fibrinolysis inhibitor (TAFIa) plays a pivotal role in fibrinolysis. TAFIa activity is regulated by a temperature-dependent instability. This instability has not only complicated the study of structure-function relationships of TAFIa but has also prevented the crystallization of TAFIa. Furthermore, the TAFIa instability has severely compromised the development of activity inhibiting monoclonal antibodies. Recently, we combined all known stabilizing mutations (i.e. S305C, T325I, T329I, H333Y and H335Q) resulting in a synergistic (one hundred and eightyfold) stabilization of TAFIa at 37 degrees C. All these residues are located in an amino acid region (AA297-335) consisting of alpha-helix 9 and beta-sheet 11. OBJECTIVES: To provide a comparative evaluation of the characteristics of a panel of stable TAFIa mutants and an energy-minimized model of the most stable TAFI variant. RESULTS: The catalytic efficiency for activation of TAFI by thrombin/thrombomodulin was higher for all TAFI mutants compared with TAFI-wild type (wt). Except for TAFI variants carrying T325I-T329I, S305C-T325I or S305C-T325I-T329I mutations, the catalytic efficiency for Hip-Arg hydrolysis by TAFIa was similar for the TAFI mutants compared with the wild type. All TAFIa variants were equally well inhibited by potato tuber carboxypeptidase inhibitor (PTCI) and showed a significantly increased antifibrinolytic potential in accordance with their increased stability. Based on the intrinsic fluorescence decay of TAFIa, two independent structural transitions were found to be associated with the loss of functional activity. CONCLUSIONS: Using molecular dynamic calculations on both TAFI-wt and TAFI-S305C-T325I-T329I-H333Y-H335Q models, we were able to identify the molecular interactions that contribute to the increased stability of the mutants.

In vitro study of the pulmonary translocation of nanoparticles: a preliminary study.

Recent studies indicate that inhaled ultrafine particles can pass into the circulation. To study this translocation in an in vitro model three types of pulmonary epithelial cells were examined. The integrity of the cell monolayer was verified by measuring the transepithelial electrical resistance (TEER) and passage of sodium fluorescein. TEER was too low in A549 cells. In these preliminary experiments, TEER values of 1007+/-300 and 348+/-62 Omega cm2 were reached for the Calu-3 cell line, using permeable membranes of 0.4 and 3 microm pore size, respectively. Growing primary rat type II pneumocytes on 0.4 microm pores, a TEER value of 241+/-90 Omega cm2 was reached on day 5; on 3 microm pores, no acceptable high TEER value was obtained. Translocation studies were done using 46 nm fluorescent polystyrene particles. When incubating polystyrene particles on membranes without a cellular monolayer, significant translocation was only observed using 3 microm pores: 67.5% and 52.7% for carboxyl- and amine-modified particles, respectively. Only the Calu-3 cell line was used in an initial experiment to investigate the translocation: on 0.4 microm pores no translocation was observed, on 3 microm pores approximately 6% translocation was observed both for carboxyl- and amine-modified particles.

Secondary structure analysis of tubulin and microtubules with Raman spectroscopy.

Raman spectroscopy is used to study the secondary structure of tubulin in the assembled and the dissociated states from the analysis of the amide-I band. Essentially two states are recognized: the GTP- and the GDP-bound state, differing in alpha-helix and antiparallel beta-sheet content. Microtubules give a spectrum which is very similar to the GDP-bound state. MAPs and temperature have minor effects, while increasing the pH up to 8 causes a reduction in alpha-helix content and a increase in antiparallel beta-sheet. The binding of demecolcine also induces structural changes which are similar to the GDP-bound state.

The immune response towards beta-adrenergic ligands and their receptors--VII. Equilibrium and kinetic binding studies of l-alprenolol to a monoclonal anti-alprenolol antibody.

Binding of the catecholamine beta-adrenergic antagonist, l-alprenolol, by the IgGl anti-alprenolol monoclonal antibody 37A4 was examined using the radioligand 3H-dihydroalprenolol as an extrinsic signal and the increase in antibody fluorescence upon l-alprenolol binding as intrinsic signal. Equilibrium binding studies based on both signals indicated that the binding process was exothermic with a positive entropy change. The difference in the affinity constants obtained by radioligand binding studies and by fluorescence analysis could be ascribed to the higher affinity of the hydrogenated tritiated l-dihydroalprenolol compared to the unsaturated l-alprenolol. The association rate constants determined by both signals were 10(4)-10(5)/M/sec and showed a high activation enthalpy (8-10 kcal/mol), thus excluding a diffusion controlled reaction. At low temp (7 degrees C), the fluorescence stopped-flow studies showed non-linear pseudo first order kinetics, indicating the existence of a fast pre-equilibrium of low affinity, followed by a conformational change leading to the tight binding of the ligand. The dissociation rate constants determined using both signals were very similar. Thus, the differences in affinity between the hydrogenated and non-saturated l-alprenolol could be ascribed to the association rate constants. Affinity constants and thermodynamic parameters calculated from the kinetic data were in close agreement with those determined by equilibrium binding. The mechanisms of ligand binding are discussed in terms of the interactions of idiotypes and anti-idiotypes in the anti-catecholamine immune response.

A step toward the prediction of the fluorescence lifetimes of tryptophan residues in proteins based on structural and spectral data.

A method is presented that allows the calculation of the lifetimes of tryptophan residues on the basis of spectral and structural data. It is applied to two different proteins. The calcium binding protein from the sarcoplasm of the muscles of the sand worm Nereis diversicolor (NSCP) changes its conformation upon binding of Ca2+ or Mg2+. NSCP contains three tryptophan residues at position 4, 57, and 170, respectively. The fluorescence lifetimes of W57 are investigated in a mutant in which W4 and W170 have been replaced. The time resolved fluorescence properties of W57 are linked to its different microconformations, which were determined by a molecular dynamics simulation map. Together with the determination of the radiative rate constant from the wavelength of maximum intensity of the decay associated spectra, it was possible to determine an exponential relation between the nonradiative rate constant and the distance between the indole CE3 atom and the carbonyl carbon of the peptide bond reflecting a mechanism of electron transfer as the main determinant of the value for the nonradiative rate constant. This result allows the calculation of the fluorescence lifetimes from the protein structure and the spectra. This method was further tested for the tryptophan of Ha-ras p21 (W32) and for W43 of the Tet repressor, which resulted in acceptable values for the predicted lifetimes.

Characterization of the hinges of the effector loop in the reaction pathway of the activation of ras-proteins. Kinetics of binding of beryllium trifluoride to V29G and I36G mutants of Ha-ras-p21.

This work experimentally confirms the pathway of activation of Ha-ras-p21, which was calculated by the method of Targeted Molecular Dynamics (TMD) (Díaz JF, Wroblowski B, Schlitter J, Engelborghs Y, 1997a, Proteins Struct Funct Genet 28:434-451). The process can be studied experimentally by analyzing the binding of BeF3- to the GDP complex of the active fluorescent mutant Y32W (Díaz JF, Sillen A, Engelborghs Y, 1997b, J Biol Chem 227:23138-23143). Two mutants, V29G and 136G, have been constructed at both sides of the effector loop of the active fluorescent mutant. This was done to check the proposed reaction pathway and to provide further insight into the mechanism of the activation of ras proteins. Both mutations accelerate the conformational isomerization with two orders of magnitude, demonstrating convincingly the role of these residues as hinges of the effector loop in one or more of the transitions of the conformational change. These results provide experimental support to the pathway calculated by TMD analysis.

Role of the switch II region in the conformational transition of activation of Ha-ras-p21.

The role of the switch II region in the conformational transition of activation of Ha-ras-p21 has been investigated by mutating residues predicted to act as hinges for the conformational transition of this loop (Ala59, Gly60, and Gly75) (Díaz JF, Wroblowski B, Schlitter J, Engelborghs Y, 1997, Proteins 28:434-451), as well as mutating the catalytic residue Gln61. The proposed mutations of the hinge residues decrease the rate of the conformational transition of activation as measured by the binding of BeF3- to the GDP-p21 complex. Also, the thermodynamic parameters of the binding reaction are altered by a factor between three and five, depending on the temperature. (Due to changes in activation and reaction enthalpies, partially compensated by entropy changes.) The control mutation Q61H in which only the catalytic residue is changed has only a limited effect on the kinetic rate constants of the conformational transition and on the thermodynamic parameters of the reaction. The fact that mutations of the hinge residues of the switch II region affect both the binding of the phosphate analog and the conformational transition of activation indicates that the switch II is implicated both in the early and the late states of the transition.

Structure of a rapidly formed intermediate in ribonuclease T1 folding.

Kinetic intermediates in protein folding are short-lived and therefore difficult to detect and to characterize. In the folding of polypeptide chains with incorrect isomers of Xaa-Pro peptide bonds the final rate-limiting transition to the native state is slow, since it is coupled to prolyl isomerization. Incorrect prolyl isomers thus act as effective traps for folding intermediates and allow their properties to be studied more easily. We employed this strategy to investigate the mechanism of slow folding of ribonuclease T1. In our experiments we use a mutant form of this protein with a single cis peptide bond at proline 39. During refolding, protein chains with an incorrect trans proline 39 can rapidly form extensive secondary structure. The CD signal in the amide region is regained within the dead-time of stopped-flow mixing (15 ms), indicating a fast formation of the single alpha-helix of ribonuclease T1. This step is correlated with partial formation of a hydrophobic core, because the fluorescence emission maximum of tryptophan 59 is shifted from 349 nm to 325 nm within less than a second. After about 20 s of refolding an intermediate is present that shows about 40% enzymatic activity compared to the completely refolded protein. In addition, the solvent accessibility of tryptophan 59 is drastically reduced in this intermediate and comparable to that of the native state as determined by acrylamide quenching of the tryptophan fluorescence. Activity and quenching measurements have long dead-times and therefore we do not know whether enzymatic activity and solvent accessibility also change in the time range of milliseconds. At this stage of folding at least part of the beta-sheet structure is already present, since it hosts the active site of the enzyme. The trans to cis isomerization of the tyrosine 38-proline 39 peptide bond in the intermediate and consequently the formation of native protein is very slow (tau = 6,500 s at pH 5.0 and 10 degrees C). It is accompanied by an additional increase in tryptophan fluorescence, by the development of the fine structure of the tryptophan emission spectrum, and by the regain of the full enzymatic activity. This indicates that the packing of the hydrophobic core, which involves both tryptophan 59 and proline 39, is optimized in this step. Apparently, refolding polypeptide chains with an incorrect prolyl isomer can very rapidly form partially folded intermediates with native-like properties.

Extending the capabilities of targeted molecular dynamics: simulation of a large conformational transition in plasminogen activator inhibitor 1.

Plasminogen activator inhibitor type 1 (PAI-1) is an inhibitor of plasminogen activators such as tissue-type plasminogen activator or urokinase-type plasminogen activator. For this molecule, different conformations are known. The inhibiting form that interacts with the proteinases is called the active form. The noninhibitory, noncleavable form is called the latent form. X-ray and modeling studies have revealed a large change in position of the reactive center loop (RCL), responsible for the interaction with the proteinases, that is inserted into a beta-sheet (s4A) in the latent form. The mechanism underlying this spontaneous conformational change (half-life = 2 h at 37 degrees C) is not known in detail. This investigation attempts to predict a transition path from the active to the latent structure at the atomic level, by using simulation techniques. Together with targeted molecular dynamics (TMD), a plausible assumption on a rigid body movement of the RCL was applied to define an initial guess for an intermediate. Different pathways were simulated, from the active to the intermediate, from the intermediate to the latent structure and vice versa under different conditions. Equilibrium simulations at different steps of the path also were performed. The results show that a continuous pathway from the active to the latent structure can be modeled. This study also shows that this approach may be applied in general to model large conformational changes in any kind of protein for which the initial and final three-dimensional structure is known.

Evidence that folliculo-stellate cells do not impede the permeability of intercellular spaces to molecular diffusion in three-dimensional aggregate cell cultures of rat anterior pituitary.

The permeability of intercellular spaces within the anterior pituitary (AP) and the influence of folliculo-stellate (FS) cells on compartmentalization within this tissue, has become a matter of debate. In reaggregated pituitary cell cultures as well as in the AP in situ the intercellular gaps and follicle-like structures remain accessible to molecular diffusion, whereas in some studies FS cells were reported to form tight epithelia that impede macromolecular transport through the spaces between the epithelial cells. In the present study the permeability of AP cell reaggregates was examined using fluorescent BSA as a tracer. Using confocal scanning laser microscopy a direct visualization of the permeation process was achieved. Quantitative estimation of the effective diffusion coefficient (Deff) for fluorescein-BSA within the aggregates was obtained using the fluorescence photobleaching recovery technique. Deff was 1.33 +/- 0.31 x 10(-7) cm2/sec (mean +/- SD) in aggregates from 14-day-old female rats and 2.45 +/- 0.55 x 10(-7) cm2/sec in aggregates from adult female rats. These values are about three times lower than in free solution. Calculation of the time-dependent concentration distribution inside the aggregate for a Deff = 2 x 10(-7) cm2/sec revealed that the concentration of the fluorescent tracer in the center of the aggregate reaches 90% of the concentration outside the aggregate after 0.5 min for aggregates with a radius of 50 microns and 6 min for aggregates with a radius of 150 microns. Aggregates enriched in FS cells, in which we previously showed a sustained inhibition of secretory responses to stimulatory and inhibitory agents as compared to total population aggregates, showed a diffusion coefficient (Deff = 1.85 +/- 0.77 x 10(-7) cm2/sec) which was not significantly different from that in the total population aggregates. The present study shows that AP cell aggregates are fully permeable to diffusing molecules within minutes and that in a three-dimensional tissue configuration FS cells, which were reported to tonically inhibit AP hormone release in response to various secretagogues, do not impede molecular diffusion to an extent which would account for sustained inhibition of hormone release.

Novel inhibitors of HIV-1 integration.

Human immunodeficiency virus (HIV) is the etiological agent of the acquired immune deficiency syndrome (AIDS). The current strategy for the treatment of HIV infection is called Highly Active Antiretroviral Therapy (HAART) and is based on cocktails of drugs that are currently approved by the Food and Drug Administration. These drugs include compounds that target the viral entry step and the enzymes reverse transcriptase or protease. The introduction of HAART has dramatically changed the landscape of HIV disease. Death from AIDS-related diseases has been reduced significantly since HAART came into use. Nevertheless it is not clear how long clinical benefit will last taking into account the emergence of multiple drug-resistant viral strains. Addition of new anti-HIV drugs targeting other steps of the viral replication cycle may increase the potency of inhibition and delay resistance development. HIV integrase is an essential enzyme in the HIV life cycle and is an attractive target for new drug development. Despite years of intensive research, only two classes of compounds that inhibit integration have been identified until now, namely the diketo acids and the pyranodipyrimidines. In this review we will point to new potential antiviral targets related to retroviral integration that are amenable to drug development. We will describe the pitfalls of currently used integrase assays and propose new strategies and technologies for the discovery of HIV integration inhibitors. Furthermore, we will describe the two classes of integrase inhibitors and discuss their antiviral activity, molecular mechanism of anti-HIV action and the selection of HIV resistance against these drugs.

Kinetic analysis of novel multisubstrate analogue inhibitors of thymidine phosphorylase.

A kinetic analysis was performed for the novel 1-(8-phosphonooctyl)-6-amino-5-bromouracil and 1-(8-phosphonooctyl)-7-deazaxanthine inhibitors of Escherichia coli thymidine (dThd) phosphorylase (TPase). The structure of the compounds was rationally designed based on the available crystal structure coordinates of bacterial TPase. These inhibitors reversibly inhibited TPase. Kinetic analysis revealed that the compounds inhibited TPase in a purely competitive or mixed fashion not only when dThd, but also when inorganic phosphate (Pi), was used as the variable substrate. In contrast, the free bases 6-amino-5-bromouracil and 7-deazaxanthine behaved as non-competitive inhibitors of the enzyme in the presence of variable Pi concentrations while being competitive or mixed with respect to thymine as the natural substrate. Our kinetic data thus revealed that the novel 1-(8-phosphonooctyl)pyrimidine/purine derivatives are able to function as multisubstrate inhibitors of TPase, interfering at two different sites (dThd(Thy)- and phosphate-binding site) of the enzyme. To our knowledge, the described compounds represent the first type of such multisubstrate analogue inhibitors of TPase; they should be considered as lead compounds for the development of mechanistically novel type of TPase inhibitors.

Temperature jump relaxation study of microtubule elongation in the presence of GTP/GDP mixtures.

GDP was added to microtubules at steady state. The amount of dissociation obtained was dependent on the GDP/GTP ratio and a method was developed to extrapolate to pure GDP conditions. From this extrapolation it was concluded that in the absence of GTP no elongation events occur. It was shown that at 35 degrees C nucleotide exchange is very fast, but at 25 degrees C, it is rate limiting for GDP-induced dissociation. Relaxation experiments, using temperature jumps before and after the addition of GDP, show that the nucleotide composition of the ends has to be taken into account. The model accepted so far cannot explain the observations. Several model mechanisms are described and their implications for equilibrium and relaxation data are analysed. All the acceptable models predict an increase in treadmilling efficiency at high GDP concentrations.

Differential hydrogen ion titrations of the histidine residues in Helix pomatia haemocyanin.

The properties of the histidine residues in Helix pomatia haemocyanin have been studied by differential hydrogen ion titrations. In oxy-and deoxyhaemocyanin 31 X 10(-5) histidine residues per g protein are titrated in contrast to 35 X 10(-5) residues in apohaemocyanin. The difference corresponds to a stoichiometry of one histidine residue per copper atom bound. Even in apohaemocyanin about 6 X 10(-5) histidine residues per g protein are not titrated in their normal pH region. In the presence of sufficient calcium to displace the dissociation completely out of the titration region, the titration curve of apohaemocyanin could be linarized according to the model of Linderstrom--Lang. In oxy-and deoxyhaemocyanin, however, a distinct deviation from linearity was found under the same conditions. In the absence of calcium the effect of the dissociation adds up to this deviation. The electrostatic interaction factors were determined for the protein at 0.1 M KC1 and for the dissociation products: halves and tenths at 1.0 M KC1. The electrostatic interaction factor for the wholes and the halves are much smaller than the values calculated from the Linderstrom--Lang equation, using the radius of the equivalent sphere either obtained from electron microscopy or from the partial specific volume. This probably due to solvent penetration. For the tenths at 1.0 M KC1, this effect is small.

A fluorescence study of tryptophan-histidine interactions in the peptide anantin and in solution.

Anantin is a heptadecapeptide in which the C-terminal peptide chain pierces the covalently cyclized peptide ring formed by an amide link between the alpha-NH2 end group and the beta-carboxyl group of Asp(8). It contains a tryptophan and a histidine at positions 5 and 12, respectively. Des-Phe(17)-anantin lacks the C-terminal phenylalanine. Fluorescence emission intensity as a function of pH follows the ionization of a single residue. The pKa amounts to 7.23 +/- 0.03 for anantin and is attributed to His(12). At pH 9 the quantum yield is 0.12 +/- 0.01 for anantin, whereas at pH 4.5 the quantum yield decreases more than two-fold (0.05 +/- 0.01). Practically identical parameters are observed for des-Phe(17)-anantin. This pH dependency reveals intramolecular quenching of the excited indole ring of Trp(5) by the imidazole of His(12), which results in a marked decrease of the tryptophan fluorescence at low pH. In a multifrequency phase fluorometric study the fluorescence lifetimes for both peptides at pH 4.5 and pH 9 are determined. At both, pH fluorescence decay is well described by a sum of two exponentials. For anantin at pH 4.5 the lifetimes are 0.72 +/- 0.07 ns and 1.67 +/- 0.07 ns. At pH 9 the lifetimes are 1.11 +/- 0.12 ns and 2.55 +/- 0.03 ns. In methanol we find two lifetimes for anantin: 0.68 +/- 0.01 ns and 2.57 +/- 0.01 ns. The lifetimes are found to be slightly dependent upon emission wavelength. For des-Phe(17)-anantin practically the same values are observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Acrylamide quenching of the fluorescence of glyceraldehyde-3-phosphate dehydrogenase: reversible and irreversible effects.

The acrylamide quenching of the tryptophan fluorescence of apo and holo glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was studied. In the case of apo-GAPDH, the steady state fluorescence quenching cannot be described by the classical Stern-Volmer equation: strong cooperative quenching is observed. In the presence of Pi and/or cofactor NAD+, an inaccessible fraction appears. Cooperative quenching is partially suppressed in the presence of Pi and fully absent in the presence of NAD+. The measurements of the fluorescence lifetimes of the holo-enzyme by phasefluorometry allow the resolution of two lifetimes. The long-lived component is quenched by acrylamide, the short-lived component is not. Quenching induces a red shift of the steady state emission peak. The quenching parameters from the lifetime measurements allow the quantitative description of the steady state fluorescence quenching data. In agreement with the observations of Orstan and Gafni (Photochemistry and Photobiology, (1990) 31, 725-731), we find that acrylamide causes a slow, irreversible loss of activity and a reduction of titratable thiol groups when it acts on the apo-enzyme. This inactivation is strongly reduced in the presence of NAD+. We show that this inactivation is also slowed down by the presence of Pi, and that it is accompanied by a loss of the NAD+ binding site. Blocking the thiol groups with 5,5'-dithio-bis-(2-nitrobenzoic acid) does not lead to a protection against the irreversible inactivation by acrylamide, showing that reactions other than thiol modifications are involved in the irreversible effect. A fraction of the inactivation can be reversed by treatment with mercapto-ethanol.

Fluorescence study of the conformational properties of recombinant tick anticoagulant peptide (Ornithodorus moubata) using multifrequency phase fluorometry.

Steady-state and multifrequency phase fluorometry were used to characterize the conformational state and conformational dynamics of recombinant tick anticoagulant peptide (Ornithodorus moubata) (TAP). The TAP contains two tryptophan residues at positions 11 and 37. The fluorescence emission varies sigmoidally as a function of pH with a pKa of 6.01 +/- 0.07. This pH dependency suggests that tryptophan fluorescence is quenched by His43 at low pH. This is confirmed by modification of the histidine with diethylpyrocarbonate. At pH 9 the fluorescence decay is well described by a sum of three exponentials (0.52, 1.9 and 5.4 ns), which decrease all three at pH 4 (0.25, 1.61 and 4.4 ns). From the reactivity of the fluorescence lifetimes toward N-bromosuccinimide and from the calculation of the accessibility we can attribute the long lifetime to Trp11, the short one to Trp37 and the middle one to both. The anisotropy decay was resolved into two components of 3.85 ns and 0.27 ns at pH 4 and 4.5 ns and 0.6 ns at pH 9. The long anisotropy decay time corresponds to the rotational correlation time of the protein, the short one to local mobility of the tryptophan residues.

Interaction between human alpha1-acid glycoprotein (orosomucoid) and 2-p-toluidinylnaphthalene-6-sulfonate.

The interaction between human alpha1-acid glycoprotein (orosomucoid) and the fluorescent probe, 2-p-toluidinylnaphthalene-6-sulfonate (TNS) has been studied. An association constant of 16.7 (+/- 3) x 10(3) M-1 was obtained for the complex at 20 degrees C with a stoichiometry of 1:1. From the effect of temperature on the binding process, the standard enthalpy change for the binding is calculated to be delta H0 = -18 +/- 3 kJ mol-1 and the standard entropy change delta S0 = 19 +/- 12 J K-1 mol-1. The tryptophan fluorescence of the protein can be described by a sum of three exponentials. Upon TNS binding, the average fluorescence lifetime of the protein in the complex changes much less than the fluorescence intensity. The bound TNS is therefore a very efficient acceptor for the protein fluorescence. The TNS bound to orosomucoid present two fluorescence lifetimes 11 and 4.3 ns. The possible origins of the two lifetimes are discussed.

Immobilizing and imaging microtubules by atomic force microscopy.

Microtubules isolated from pig brains have been immobilized on an inorganic substrate for use in AFM studies. The method employs 4-aminobutyldimethylmethoxysilane and glutaraldehyde to activate a silicon wafer for binding the biopolymer. The covalent bond ensures the positional stability of the tubules on the substrate, and allows reproducible scanning probe experiments. Microtubules have been imaged both by atomic force and scanning tunneling microscopy, yielding results very similar to electron microscopy. The average apparent height of the tubules is smaller than observed with transmission electron microscopy (25 nm) and is smaller in buffer solution (10 nm) than in air (15 nm). The biopolymer surface is softer under buffer than in air. The highest resolution was obtained with the tapping mode where surface features as small as 10 nm in X and Y have been resolved. Gold-coated tubules bound on silicon have been successfully imaged by STM, while images of uncertain origin were generated for tubules deposited on graphite. It is shown that artefacts imaged on a blank graphite surface can easily be confounded with collapsed tubules.