Giancarlo Baldini

Competitive binding of fatty acids and the fluorescent probe 1‐8‐anilinonaphthalene sulfonate to bovine β‐lactoglobulin

The use of spectroscopy in the study of fatty acids binding to bovine β‐lactoglobulin (BLG) appears to be a difficult task, as these acid compounds, assumed as the protein natural ligands, do not exhibit favorable optical response such as, for example, absorption or fluorescence. Therefore, the BLG fatty‐acid equilibrium has been tackled by exploiting the competition between fatty acids and ANS, a widely used fluorescent hydrophobic probe, whose binding sites on the protein have been characterized recently. Two lifetime decays of the ANS–BLG complex have been found; the longer one has been attributed to the internal binding site and the shorter one to the external site. At increasing fatty acids concentration, the fractional weight associated with ANS bound to the internal site drops, in agreement with a model describing the competition of the dye with fatty acids, whereas the external site occupancy appears to be unaffected by the fatty acids binding to BLG. This model is supported by docking studies. An estimate of the acid‐binding affinities for BLG has been obtained by implementing the fitting of the bound ANS intensities with a competitive binding model. A relevant dependence has been found upon the solution pH, in the range from 6 to 8, which correlates with the calyx accessibility modulated by the conformation of the EF loop. Fatty acids with longer aliphatic chains (palmitate and laurate) are found to display larger affinities for the protein and the interaction free energy nicely correlates with the number of contacts inside the protein calyx, in agreement with docking simulations.

Evidence of heterogeneous 1-anilinonaphthalene-8-sulfonate binding to beta-lactoglobulin from fluorescence spectroscopy

Steady-state and dynamic fluorescence titrations show that: (a) the complex between beta-lactoglobulin (BLG) and 1-anilinonaphthalene-8-sulfonate (ANS) displays a heterogeneous equilibrium with large changes in the binding strength vs. pH and ion concentration; and (b) the fluorescence response of bound ANS reveals two separate lifetimes that suggest two different sites (or binding modes). While steady-state fluorescence titrations yield effective values of the binding constant and of the bound ANS quantum efficiency, it is shown that, by combining steady-state fluorescence and lifetime decay of ANS, it is possible to give quantitative estimates of the association constants for each site. When heading from the acid (pH approximately 2) to the native state (pH approximately 6) the main result is a very large reduction of the effective binding constant. This and the results of titrations vs. ionic strength suggest that electrostatic interactions are a major contribution to ANS binding to BLG.

Two-photon thermal bleaching of single fluorescent molecules.

We have studied the fluorescence emission by two-photon excitation of four dyes widely used for bioimaging studies, rhodamine 6G, fluorescein, pyrene and indo-1 at the single molecule level. The single dye molecules, spread on a glass substrate by spin coating, show a constant fluorescence output until a sudden transition to a dark state very close to the background. The bleaching time that is found to vary in the series pyrene, indo-1, fluorescein and rhodamine 6G from the fastest to the slowest one respectively, has a Gaussian distribution indicating that the observed behavior is not due to photobleaching. Moreover, the bleaching time decreases with the glass substrate temperature reaching a vanishing nonmeasurable value for a limiting temperature whose value is found in the same series as for the bleaching time, from the lowest to the highest temperature respectively. The observed bleaching shows a clear correlation to the amount of absorbed power not reirradiated as fluorescence and to the complexity of the molecule. These observations are interpreted as thermal bleaching where the temperature increase is induced by the two-photon absorption of the single dyes as confirmed also by numerical simulations.

Conformation of interacting lysozyme by polarized and depolarized light scattering

The fluctuations of the polarized and depolarized light scattered by lysozyme solutions in acetate buffer and in 60% w/w glycerol–acetate mixtures have been studied by measuring the correlation function with a resolution of 12.5 ns. This result has been achieved by processing two replicas of the same scattered signal with two separate detectors and computing their cross correlation. The correlograms have been investigated at various temperatures and protein concentration at pH≃4.6 and buffer ionic strength ≃45 mM. The rotational relaxation times obtained from depolarized scattering have been found to lie in the range 150–400 μs, depending on the solution temperature, and no appreciable concentration dependence has been observed. On the other hand, the mutual translational diffusion coefficients derived from polarized scattering have been found to be strongly dependent on protein concentration. The main result is that the protein hydrodynamic radius, obtained by polarized photon correlation measurements is...

Measurement of the laser pulse width on the microscope objective plane by modulated autocorrelation method

We report on the construction details of a compact autocorrelator set‐up for the measurement of the width of infrared laser pulses at the focal plane of a microscope for two‐photon excitation fluorescence imaging. One of the novelties of the set‐up, which leads to an improved measurement accuracy, is the use of a modulation technique that is achieved by mounting one of the interferometer mirrors on a loudspeaker driven by a sinusoidal bias at low frequency. A non‐linear least‐square routine selects only that part of the fluorescence signal that is modulated at the same frequency as the loudspeaker bias. To further increase the accuracy, the laser pulse width is obtained from a series of measurements at different values of the modulation bias. The autocorrelator is a compact single bread‐board (10 × 20 cm); it is PC‐controlled both for the acquisition and the analysis of the data and can be coupled to different ports of the microscope. The increase in the pulse width measured for three different ports of the microscope is well accounted for by the group velocity dispersion and the glass thickness of the optics found along these paths.

Photon correlation spectroscopy of interacting and dissociating hemoglobin

Experimental and theoretical analysis of the effect of both intermolecular interactions and dissociation on the diffusional properties of carbon–monoxide bovine hemoglobin in solution are reported here. When performing accurate photon correlation spectroscopy measurements versus protein concentration, even on dilute solutions, one finds that the first cumulant diffusion coefficient of the macromolecule has a relevant dependence upon pH (5⩽pH⩽9.5), ionic strength (10–100 mM), and, particularly, on hemoglobin concentration (1⩽c⩽20 mg/ml). The results cannot be interpreted by considering the occurrence of either protein dissociation or intermolecular interactions only. As a consequence a simple theoretical expansion of the first cumulant diffusion coefficient, to first order in concentration, is derived here with the inclusion of protein interactions and dissociation. A fit procedure based on this expression leads to a good description of the dissociation and an accurate estimate of the native protein charge...

Porcine beta‐lactoglobulin chemical unfolding: Identification of a non‐native α‐helical intermediate

The chemical unfolding behavior of porcine beta‐lactoglobulin (PLG) has been followed at pH 2 and 6 in the presence of guanidinium hydrochloride. The PLG unfolding transition, monitored by tryptophan fluorescence, far and near UV circular dichroism and 1D‐NMR, can be described by a three‐state transition suggesting the presence of at least one intermediate state that appears to display an excess of non‐native α‐helical structures. The thermodynamic parameters, as determined through a global analysis fitting procedure, give estimates of the free energy differences of the transitions connecting the native, the intermediate and the unfolded state: ΔG  NI0 = 2.8 ± 0.7 kcal mol−1 (pH 2) and 4.2 ± 0.5 kcal mol−1 (pH 6) and ΔG  NU0 = 7.2 ± 0.6 kcal mol−1 (pH 2) and 6.9 ± 0.6 kcal mol−1 (pH 6). CD unfolding data of the bovine species (BLG) have been collected here under the same experimental conditions of PLG to allow a careful comparison of the two beta‐lactoglobulins. Intermediates with different characteristics have been identified for BLG and PLG, and their nature has been discussed on a structural analysis basis. The thermodynamic data reported here for PLG and BLG and the comparative analysis with data reported for equine beta lactoglobulin, show that homologous beta‐barrel proteins, belonging to the same family and displaying high sequence identity (52–64%) populate unfolding intermediates to different extents, even though a common tendency to the formation of non‐native alpha‐helical intermediates, can be envisaged. The present results provide a prerequisite foundation of knowledge for the design and interpretation of future folding kinetic studies. Proteins 2005.

ROTATIONAL DIFFUSION AND INTERNAL MOTIONS OF CIRCULAR DNA. I. POLARIZED PHOTON CORRELATION SPECTROSCOPY

The conformation and slow internal motions of a DNA plasmid molecule pUC18 (2687 base pairs) are studied by means of depolarized photon correlation spectroscopy. The autocorrelation functions are measured at different scattering angles between 6.7° and 90° and analyzed as heterodyne autocorrelation functions. One obtains both a relaxation time of ≂140 μs, which is interpreted as the tumbling rotational diffusion coefficient of the plasmid, and some faster component with relaxation time around 15 μs. The value of the rotational diffusion relaxation time is in good agreement with the results of Monte Carlo simulations, while the faster decay has a nonsingle exponential decay behavior. The results of measurements made in the depolarized scattering configuration are compared to those obtained from polarized configuration and critically discussed.

Probing protein aggregation by time-resolved fluorescence during β-lactoglobulin crystal growth

Abstract. We have used the fluorescence anisotropy (FA) decay of retinol bound to bovine β-lactoglobulin to monitor the time evolution of protein aggregation during the early stages of crystal growth. With this approach we have followed the formation of aggregates at different concentrations of ammonium sulfate, the precipitant used for crystallization. The average aggregation number is found to depend on precipitant concentration, and to be restricted to small numbers ranging from 2 to 5, also in the presence of visible growing crystals. The effect of particle distribution and of low probe-to-protein saturation on the FA response is also discussed in detail.

Novel efficient and stable heteroaromatic two-photon absorbing dyes

The synthesis and characterization of novel heteroaromatic-based two-photon absorption (TPA) dyes for bio-conjugation is described; the new isothiocyanate and maleimide derive from a class of novel efficient quadrupolar and octupolar/branched chromophores relying on the electronic effects of electron-poor and electron-rich simple heteroaromatic rings; the new systems exhibit very large TPA cross-sections, high chemical stability, and very low photobleaching.