No abstract

Xanthene dyes are known to form dimers with spectral characteristics that have been interpreted in terms of exciton theory. A unique aspect of H-type dimers is the fluorescence quenching that accompanies their formation. Using the principles of exciton theory as a guide, a series of protease substrates was synthesized with a xanthene dye on each side of the cleavage site. To bring the attached dyes into spatial proximity to form a dimer, the molecular design included structure determinant regions in the amino acid sequence. In addition, chromophores were chosen such that changes in absorption spectra indicative of exciton splitting were anticipated. Cleavage of the peptides by a protease resulted in disruption of the dimers and indeed significant absorption spectral changes were observed. Furthermore, substrate cleavage was accompanied by at least an order of magnitude increase in fluorescence intensity. This has allowed determination of intracellular elastase activity using a fluorescence microscope equipped with standard optics.The molecular exciton represents a delocalized electronic excitation in a system of identical molecular units (1). Based on a resonance dipole-dipole interaction mechanism, exciton theory predicts and explains the spectroscopic characteristics in systems of interacting fluorophores. Although the original definition of excitons was introduced for molecular crystals (2), the same formalism can also be applied to explain spectra of other molecular aggregates such as monomolecular lamellar systems (3) as well as dye dimers (4, 5). The latter are usually classified as either H-or J-type aggregates depending on the spatial arrangement of the fluorophores and the resulting spectral characteristics (6). Xanthene dyes are known to form H-type dimers, the characteristics of which are a blue shift in the absorption spectrum and the loss of fluorescence (7-9).If one were to design a functional molecular structure that fits the exciton model, one could have a reporter molecule with a unique self-contained analytical tool. We now report the design of a new class of profluorescent protease substrates. First, polypeptides containing amino acid sequences of naturally occurring protease inhibitors for protease recognition were synthesized; second, synthesis was followed by derivatization with a fluorophore on each side of the cleavage site. The size and geometry of the doubly labeled polypeptides are favorable for the formation of H-type dimers. Cleavage of such a polypeptide by a protease results in disruption of the H-type dimer and appearance of fluorescence.MATERIALS AND METHODS Materials. N"-9-Fluorenylmethoxycarbonyl (Fmoc) amino acids were purchased from Calbiochem-Novabiochem. 2-Chlorotrityl resin was obtained from Peptides International. The coupling reagent benzotriazol-1-yl-oxy-tris-pyrrolidinophosphonium hexafluorophosphate (PyBOP) was bought from Advanced ChemTech. Solvents such as HPLC grade dichloromethane, methanol, and acetonitrile were from Fisher Scientific. Other reagents such...

We report an experimental study of protein relaxation dynamics on the picosecond and nanosecond time scales. The protein equilibrium state is perturbed by the redistribution of electric charge density over the side chain of a tryptophan residue. Electronic excitation of the residue induces the charge shift and triggers a relaxation process, the dynamics of which is reflected in tryptophan fluorescence emission. In the case of homogeneous emission, the relaxation dynamics can be extracted from a time-dependent red shift in the emission spectrum. In the case of heterogeneous emission, the spectral shift may not represent relaxation dynamics. A criterion for distinguishing between homogeneous and heterogeneous fluorescence emission is suggested here. Emission from the mutants E21W and F3W of IIA Glc is found to be free from permanent or long-lived heterogeneity. In E21W, the only tryptophan residue is in a rigid globule, whereas in F3W it is on a flexible tail. The relaxation dynamics reported by the tryptophan residue in F3W is much faster than that in E21W. Addition of glycerol to the solvent slows down the relaxation dynamics for both tryptophan residues.

The B1 domain of Streptococcal protein G (GB1) is a small, thermostable protein containing a single tryptophan residue. We recorded time-resolved fluorescence of the wild-type GB1 and its 5-fluorotryptophan (5FTrp) variant at more than 30 emission wavelengths between 300 and 470 nm. The time-resolved emission spectra reveal no signs of heterogeneity, but show a time-dependent red shift characteristic of microscopic dielectric relaxation. This is true for both 5FTrp and unmodified Trp in GB1. The time-dependent red shifts in the fluorescence of 5FTrp and unmodified Trp are essentially identical, confirming that the shift is caused by the relaxation of the protein matrix rather than by the fluorophore itself. The total amplitude (but not the rate) of the time-dependent red shift depends on the fluorophore, specifically, on the magnitude of the vector difference between its excited state and ground state electric dipole moments; for 5FTrp this is estimated to be about 88% of that for the unmodified Trp. The decay of the excited state fluorophore population is not monoexponential for either fluorophore; however, the deviation from the monoexponential decay law is larger in the case of unmodified Trp. The relaxation dynamics of GB1 was found to be considerably faster than that of other proteins studied previously, consistent with the small size, tightly packed core, and high thermodynamic stability of GB1.