Franklyn G. Prendergast

Spectral and photophysical studies of benzo[c]xanthene dyes: Dual emission pH sensors

A series of fluorescent, long-wavelength, benzo[c]-xanthene dyes has been characterized for pH measurement in both excitation and emission ratio applications. The two general classes of these indicators are seminaphthofluoresceins (SNAFLs) and seminaphthorhodafluors (SNARFs) which are substituted at the 10-position with oxygen or nitrogen, respectively. These probes show separate emissions from the protonated and deprotonated forms of the fluorophores. The dyes may be excited at 488 or 514 nm with argon ion lasers. Most of the indicators have pKa values between 7.6 and 7.9. Detailed photophysical studies were conducted on the carboxy-SNAFL-1 system and excited-state prototropic reactions were compared to structurally related derivatives, such as the umbelliferones. Membrane permeant esters, such as diacetates and acetoxymethyl esters have also been prepared. The indicators are spectrally well resolved from calcium indicators such as fura-2 and indo-1 and should be suitable for simultaneous determination of pH and Ca2+ transients.

Interpretation of fluorescence decays in proteins using continuous lifetime distributions.

The decay of the tryptophanyl emission in proteins is often complex due to the sensitivity of the tryptophan excited state to its surroundings. The traditional analysis of the decay curve using exponential components is based on the identification of each component with a particular protein conformation. An alternative approach assumes that proteins can exhibit a large number of conformations and that, at room temperature, the interconversion rate between conformations can be of the same order of magnitude as the excited-state decay rate. Following this assumption, the analysis of the protein emission was performed using continuous distributions of lifetime values. The number of average protein conformations, the range of mobility around each conformation, and the rate of interconversion between conformations determines the characteristics of the lifetime distribution. The fluorescence decay from some single tryptophan proteins was measured using multifrequency phase fluorometry and analyzed using a sum of exponentials, unimodal and bimodal probability-density functions, and the analytical form for lifetime distribution obtained for a model in which the tryptophan residue can move in a single potential well. For ribonuclease T1 and neurotoxin variant 3, the sum of two exponentials and bimodal probability-density functions gave comparable results, whereas for phospholipase A2, the description of the decay required three exponentials or bimodal probability-density functions. Also the temperature dependence of the fluorescence decay was investigated. It was found that the lifetime distribution was broader and shifted toward longer lifetime values at lower temperature. The analysis of the decay of tryptophan in buffer and of some tryptophan derivatives gave single-exponential decays. The single-potential well lifetime distribution, which has only three adjustable parameters, gave good fits for all cases investigated, but in the case of phopholipase A2, the temperature dependence of the parameters that describe the single-potential well distribution indicated the inadequacy of this model at lower temperature, suggesting that multiple potential wells can describe better the decay for this protein.

Fluorescence lifetime distributions in proteins

The fluorescence lifetime value of tryptophan residues varies by more than a factor of 100 in different proteins and is determined by several factors, which include solvent exposure and interactions with other elements of the protein matrix. Because of the variety of different elements that can alter the lifetime value and the sensitivity to the particular environment of the tryptophan residue, it is likely that non-unique lifetime values result in protein systems. The emission decay of most proteins can be satisfactorily described only using several exponential components. Here it is proposed that continuous lifetime distributions can better represent the observed decay. An approach based on protein dynamics is presented, which provides fluorescence lifetime distribution functions for single tryptophan residue proteins. First, lifetime distributions for proteins interconverting between two conformations, each characterized by a different lifetime value, are derived. The evolution of the lifetime values as a function of the interconversion rate is studied. In this case lifetime distributions can be obtained from a distribution of rates of interconversion between the two conformations. Second, the existence of a continuum of energy substates within a given conformation was considered. The occupation of a particular energy substate at a given temperature is proportional to the Boltzmann factor. The density of energy states of the potential well depends upon the width of the well, which determines the degree of freedom the residue can move in the conformational space. Lifetime distributions can be obtained by association of each energy substate with a different lifetime value and assuming that the average conformation can change as the energy of the substate is increased. Finally, lifetime distributions for proteins interconverting between two conformations, each characterized by a quasi-continuum of energy substates, are presented. The origin of negative components of the lifetime distribution is also discussed. In the companion paper that will follow (Alcala, J. R., E. Gratton, and F. J.Prendergast, 1987, Biophys. J., in press) lifetime distributions obtained here are used to fit experimental data.

Resolvability of fluorescence lifetime distributions using phase fluorometry

The analysis of the fluorescence decay using discrete exponential components assumes that a small number of species is present. In the absence of a definite kinetic model or when a large number of species is present, the exponential analysis underestimates the uncertainty of the recovered lifetime values. A different approach to determine the lifetime of a population of molecules is the use of probability density functions and lifetime distributions. Fluorescence decay data from continuous distributions of exponentially decaying components were generated. Different magnitudes of error were added to the data to simulate experimental conditions. The resolvability of the distributional model was studied by fitting the simulated data to one and two exponentials. The maximum width of symmetric distributions (uniform, gaussian, and lorentzian), which cannot be distinguished from single and double exponential fits for statistical errors of 1 and 0.1%, were determined. The width limits are determined by the statistical error of the data. It is also shown that, in the frequency domain, the discrete exponential analysis does not uniformly weights all the components of a distribution. This systematic error is less important when probability and distribution functions are used to recover the decay. Finally, it is shown that real lifetime distributions can be proved using multimodal probability density functions. In the companion paper that follows we propose a physical approach, which provides lifetime distribution functions for the tryptophan decay in proteins. In the third companion paper (Alcala, J.R., E. Gratton, and F.J. Prendergast, 1987, Biophys. J., in press) we use the distribution functions obtained to fit data from the fluorescence decay of single tryptophan proteins.

Green-fluorescent protein mutants with altered fluorescence excitation spectra.

Using random mutagenesis and visual selection of fluorescent clones, we have isolated a T2031 and a E222G mutant of the Aequorea green‐fluorescent protein. Each mutant has one of the two fluorescence excitation bands of the wild type deleted and retains the other without a wavelength shift. This finding is consistent with each excitation band corresponding to a distinct spectroscopic state of the chromophore. Both mutations are single amino acid exchanges which in the linear sequence are located remotely from the chromophore but in the folded protein may be situated in its vicinity. We conclude that the mutations influence the fluorescence properties by changing the interactions between the chromophore and its protein environment.

A Conserved α-Helical Motif Mediates the Interaction of Sp1-Like Transcriptional Repressors with the Corepressor mSin3A

ABSTRACT Sp1-like proteins are defined by three highly homologous C2H2 zinc finger motifs that bind GC-rich sequences found in the promoters of a large number of genes essential for mammalian cell homeostasis. Here we report that TIEG2, a transforming growth factor β-inducible Sp1-like protein with antiproliferative functions, represses transcription through recruitment of the mSin3A-histone deacetylase complex. The interaction of TIEG2 with mSin3A is mediated by an alpha-helical repression motif (α-HRM) located within the repression domain (R1) of TIEG2. This α-HRM specifically associates with the second paired amphipathic helix (PAH2) domain of mSin3A. Mutations in the TIEG2 α-HRM domain that disrupt its helical structure abolish its ability to both bind mSin3A and repress transcription. Interestingly, the α-HRM is conserved in both the TIEG (TIEG1 and TIEG2) and BTEB (BTEB1, BTEB3, and BTEB4) subfamilies of Sp1-like proteins. The α-HRM from these proteins also mediates direct interaction with mSin3A and represses transcription. Surprisingly, we found that the α-HRM of the Sp1-like proteins characterized here exhibits structural and functional resemblance to the Sin3A-interacting domain previously described for the basic helix-loop-helix protein Mad1. Thus, our study defines a mechanism of transcriptional repression via the interactions of the α-HRM with the Sin3-histone deacetylase complex that is utilized by at least five Sp1-like transcriptional factors. More importantly, we demonstrate that a helical repression motif which mediates Sin3 interaction is not an exclusive structural and functional characteristic of the Mad1 subfamily but rather has a wider functional impact on transcriptional repression than previously demonstrated.

EUDOC: a computer program for identification of drug interaction sites in macromolecules and drug leads from chemical databases.

The completion of the Human Genome Project, the growing effort on proteomics, and the Structural Genomics Initiative have recently intensified the attention being paid to reliable computer docking programs able to identify molecules that can affect the function of a macromolecule through molecular complexation. We report herein an automated computer docking program, EUDOC, for prediction of ligand–receptor complexes from 3D receptor structures, including metalloproteins, and for identification of a subset enriched in drug leads from chemical databases. This program was evaluated from the standpoints of force field and sampling issues using 154 experimentally determined ligand–receptor complexes and four “real‐life” applications of the EUDOC program. The results provide evidence for the reliability and accuracy of the EUDOC program. In addition, key principles underlying molecular recognition, and the effects of structural water molecules in the active site and different atomic charge models on docking results are discussed.

Fluorescent rhodol derivatives: Versatile, photostable labels and tracers

A series of chemically reactive, fluorescent rhodol derivatives was prepared and evaluated. Reactive functional groups included activated esters, amines, haloacetamides, fixable hydrazide derivatives, acrylamides, and photoaffinity reagents. Depending on the choice of substituents, absorption maxima of the dyes varied from 490 to 550 nm with extinction coefficients that were generally greater than 50,000 M-1 cm-1 in aqueous solution and emission maxima from 520 to 580 nm. Most of the compounds investigated exhibited fluorescence lifetimes between 3 and 4 ns. Individual derivatives were suitable for excitation with the 488 and 514-nm lines of the argon ion laser and the 546-nm line of the mercury arc lamp and were compatible for use with standard fluorescein and rhodamine filter sets. The rhodol dyes were more photostable and less sensitive to pH changes in the physiological range than fluorescein derivatives. Some examples show absorption maxima at or near 514 nm, an excitation wavelength that is useful for multicolor fluorescence microscopy, flow cytometry, and DNA sequencing. Derivatives were also prepared that exhibit absorption and emission maxima similar to those of tetramethylrhodamine (TMR) analogs but with higher quantum yields in aqueous solution. A number of the dyes had higher solubilities in aqueous systems and were less quenched on conjugation to proteins than TMR derivatives. Appropriate substitution results in a wider range of solubilities in hydrophilic or lipophilic solvents than is easily accomplished with fluorescein or TMR derivatives. Conjugates of a number of the rhodol fluorophores were generally more photostable and less pH sensitive than fluorescein conjugates and more fluorescent than TMR conjugates.

Alterations in the structure, physicochemical properties, and pH of hepatocyte lysosomes in experimental iron overload.

While hemochromatosis is characterized by sequestration of iron-protein complexes in hepatocyte lysosomes, little is known about the effects of excess iron on these organelles. Therefore, we studied the effects of experimental iron overload on hepatocyte lysosomal structure, physicochemical properties, and function in rats fed carbonyl iron. A sixfold increase (P less than 0.0001) in hepatic iron and a fivefold increase in lysosomal iron (P less than 0.01) was observed after iron loading; as a result, hepatocyte lysosomes became enlarged and misshapen. These lysosomes displayed increased (P less than 0.0001) fragility; moreover, the fluidity of lysosomal membranes isolated from livers of iron-loaded rats was decreased (P less than 0.0003) as measured by fluorescence polarization. Malondialdehyde, an end product of lipid peroxidation, was increased by 73% (P less than 0.008) in lysosomal membranes isolated from livers of iron-overloaded rats. While amounts of several individual fatty acids in isolated lysosomal membranes were altered after iron overload, cholesterol/phospholipid ratios, lipid/protein ratios, double-bond index, and total saturated and unsaturated fatty acids remained unchanged. The pH of lysosomes in hepatocytes isolated from livers of iron-loaded rats and measured by digitized video microscopy was increased (control, 4.70 +/- 0.05; iron overload, 5.21 +/- 0.10; P less than 0.01). Our results demonstrate that experimental iron overload causes marked alterations in hepatocyte lysosomal morphology, an increase in lysosomal membrane fragility, a decrease in lysosomal membrane fluidity, and an increase in intralysosomal pH. Iron-catalyzed lipid peroxidation is likely the mechanism of these structural, physicochemical, and functional disturbances.

Time-resolved fluorescence techniques: methods and applications in biology

Abstract Modern time-resolved fluorescence spectroscopy affords detection and accurate quantification of fluorescence intensity decays as short as a few picoseconds. The method allows for measurements of dynamic events in the picosecond-nanosecond time frame. It is therefore especially useful for the study of macromolecular structure and dynamics, in particular for evaluating predictions from molecular dynamics simulations.