Fluorescence resonance energy transfer studies on anthrax lethal toxin.

Anthrax lethal toxin is a binary bacterial toxin consisting of two proteins, protective antigen (PA) and lethal factor (LF), that self-assemble on receptor-bearing eukaryotic cells to form toxic, non-covalent complexes. PA(63), a proteolytically activated form of PA, spontaneously oligomerizes to form ring-shaped heptamers that bind LF and translocate it into the cell. Site-directed mutagenesis was used to substitute cysteine for each of three residues (N209, E614 and E733) at various levels on the lateral face of the PA(63) heptamer and for one residue (E126) on LF(N), the 30 kDa N-terminal PA binding domain of LF. Cysteine residues in PA were labeled with IAEDANS and that in LF(N) was labeled with Alexa 488 maleimide. The mutagenesis and labeling did not significantly affect function. Time-resolved fluorescence methods were used to study fluorescence resonance energy transfer (FRET) between the AEDANS and Alexa 488 probes after the complex assembled in solution. The results clearly indicate energy transfer between AEDANS labeled at residue N209C on PA and the Alexa 488-labeled LF(N), whereas transfer from residue E614C on PA was slight, and none was observed from residue E733C. These results support a model in which LF(N) binds near the top of the ring-shaped (PA(63))(7) heptamer.

Conformational dynamics and temperature dependence of photoinduced electron transfer within self-assembled coproporphyrin:cytochrome c complexes.

The focus of the present study is to better understand the complex factors influencing intermolecular electron transfer (ET) in biological molecules using a model system involving free-base coproporphyrin (COP) complexed with horse heart cytochrome c (Cc). Coproporphyrin exhibits bathochromic shifts in both the Soret and visible absorption bands in the presence of Cc and an absorption difference titration reveals a 1:1 complex with an association constant of 2.63 +/- 0.05 x 10(5) M(-1). At 20 degrees C, analysis of time-resolved fluorescence data reveals two lifetime components consisting of a discrete lifetime at 15.0 ns (free COP) and a Gaussian distribution of lifetimes centered at 2.8 ns (representing (1)COP --> Cc ET). Temperature-dependent, time-resolved fluorescence data demonstrate a shift in singlet lifetime as well as changes in the distribution width (associated with the complex). By fitting these data to semiclassical Marcus theory, the reorganizational energy (lambda) of the singlet state electron transfer was calculated to be 0.89 eV, consistent with values for other porphyrin/Cc intermolecular ET reactions. Using nanosecond transient absorption spectroscopy the temperature dependences of the forward and thermal back ET originating from triplet state were examined ((3)COP --> Cc ET). Fits of the temperature dependence of the rate constants to semiclassical Marcus theory gave lambda of 0.39 eV and 0.11 eV for the forward and back triplet ET, respectively (k(f) = (7.6 +/- 0.3) x 10(6) s(-1), k(b) = (2.4 +/- 0.3) x 10(5) s(-1)). The differing values of lambda for the forward and back triplet ET demonstrate that these ET reactions do not occur within a static complex. Comparing these results with previous studies of the uroporphyrin:Cc and tetrakis (4-carboxyphenyl)porphyrin:Cc complexes suggests that side-chain flexibility gives rise to the conformational distributions in the (1)COP --> Cc ET whereas differences in overall porphyrin charge regulates gating of the back ET reaction (reduced Cc --> COP(+)).

Fluorescence polarization: past, present and future.

Fluorescence polarization was first observed in 1920 and during the next few decades the theoretical foundations of the phenomenon were clearly established. In the last two decades of the 20(th) century, fluorescence polarization became one of the most prevalent methods used in clinical and biomedical sciences. In this article we review the history of fluorescence polarization, its theoretical foundations and some of the more important practical developments, which helped to popularize the method. We also discuss important, but often misunderstood, practical considerations including the wavelength dependence of the limiting polarization and the effect of energy transfer on polarization. The present state of fluorescence polarization, both in pure research as well as in the applied biosciences is also reviewed. Finally, we speculate on possible future developments in the field, such as the use of multi-photon techniques.

Fluorescence: basic concepts, practical aspects, and some anecdotes.

We hope that we have conveyed information of interest and value to present and future fluorescence practitioners. Those readers with a sustaining interest in this topic may wish to consult more comprehensive sources such as Molecular Fluorescence: Principles and Applications, an excellent text by Valeur, or Principles of Fluorescence Spectroscopy by Lakowicz. Many specialized fluorescence topics are covered in the series Topics in Fluorescence Spectroscopy (Volumes 1-6), and several volumes of Methods in Enzymology (e.g., Volumes 246 and 278) have dealt with issues in fluorescence spectroscopy. Proceedings from the International Conference on Methods and Applications of Fluorescence Spectroscopy, 1997 (MAFS 97) and MAFS 98 (in press) also present fluorescence work on many different topics in biological and chemical fields. The Molecular Probes Handbook and web site (www.probes.com) are also rich sources of useful information. Finally, any reader with a question or seeking advice on some topic related to fluorescence is welcome to e-mail D.M.J. at djameson@hawaii.edu.

The 2'-O- and 3'-O-Cy3-EDA-ATP(ADP) complexes with myosin subfragment-1 are spectroscopically distinct.

Ribose-modified highly-fluorescent sulfoindocyanine ATP and ADP analogs, 2'(3')-O-Cy3-EDA-AT(D)P, with kinetics similar to AT(D)P, enable myosin and actomyosin ATPase enzymology with single substrate molecules. Stopped-flow studies recording both fluorescence and anisotropy during binding to skeletal muscle myosin subfragment-1 (S1) and subsequent single-turnover decay of steady-state intermediates showed that on complex formation, 2'-O- isomer fluorescence quenched by 5%, anisotropy increased from 0.208 to 0.357, and then decayed with turnover rate k(cat) 0.07 s(-1); however, 3'-O- isomer fluorescence increased 77%, and anisotropy from 0.202 to 0.389, but k(cat) was 0.03 s(-1). Cy3-EDA-ADP.S1 complexes with vanadate (V(i)) were studied kinetically and by time-resolved fluorometry as stable analogs of the steady-state intermediates. Upon formation of the 3'-O-Cy3-EDA-ADP.S1.V(i) complex fluorescence doubled and anisotropy increased to 0.372; for the 2'-O- isomer, anisotropy increased to 0.343 but fluorescence only 6%. Average fluorescent lifetimes of 2'-O- and 3'-O-Cy3-EDA-ADP.S1.V(i) complexes, 0.9 and 1.85 ns, compare with approximately 0.7 ns for free analogs. Dynamic polarization shows rotational correlation times higher than 100 ns for both Cy3-EDA-ADP.S1.V(i) complexes, but the 2'-O-isomer only has also a 0.2-ns component. Thus, when bound, 3'-O-Cy3-EDA-ADP's fluorescence is twofold brighter with motion more restricted and turnover slower than the 2'-O-isomer; these data are relevant for applications of these analogs in single molecule studies.

Structural and biochemical characterization of a fluorogenic rhodamine-labeled malarial protease substrate.

Activation of the proenzyme form of the malarial protease PfSUB-1 involves the autocatalytic cleavage of an Asp-Asn bond within the internal sequence motif (215)LVSADNIDIS(224). A synthetic decapeptide based on this sequence but with the N- and C-terminal residues replaced by cysteines (Ac-CVSADNIDIC-OH) was labeled with 5- or 6-isomers of iodoacetamidotetramethylrhodamine (IATR). The doubly labeled peptides have low fluorescence because of ground-state, noncovalent dimerization of the rhodamines. Cleavage of either peptide by recombinant PfSUB-1 results in dissociation of the rhodamine dimers, which abolishes the self-quenching and consequently leads to an approximately 30-fold increase in the fluorescence. This spectroscopic signal provides a continuous assay of proteolysis, enabling quantitative kinetic measurements to be made, and has also enabled the development of a fluorescence-based assay suitable for use in high-throughput screens for inhibitors of PfSUB-1. The structure of the rhodamine dimer in the 6-IATR-labeled peptide was shown by NMR to be a face-to-face stacking of the xanthene rings. Time-resolved fluorescence measurements suggest that the doubly labeled peptides exist in an equilibrium consisting of rhodamines involved in dimers (closed forms) and rhodamines not involved in dimers (open forms). These data also indicate that the rhodamine dimers fluoresce and that the associated lifetimes are subnanosecond.

Discrete bathochromic shifts exhibited by fluorescein ligand bound to rabbit polyclonal anti-fluorescein Fab fragments.

Eleven individual hyperimmune rabbit polyclonal anti-fluorescein Fab fragment preparations were resolved into heterogeneous subfractions based on differential dissociation times from a specific adsorbent. Four Fab subfractions (i.e., 0.1-, 1.0-, 10-, and 100-day elutions) that differed in affinity were characterized and classified according to the extent of the bathochromic shift in the absorption properties of antibody-bound fluorescein ligand. Absorption maxima of bound fluorescein were shifted in all cases to two distinct narrow ranges, namely, 505 to 507 nm or 518 to 520 nm relative to 491 nm for free fluorescein. There was no direct correlation between the two spectral shift populations and antibody affinity, fluorescence polarization, fluorescence quenching, or fluorescence lifetimes of bound ligand. Fluorescence emission maxima varied with the bathochromic shift range. Bound fluorescein ligand, with absorption maxima of 505 to 507 nm and 518 to 520 nm showed fluorescence emission maxima of 519 to 520 nm and 535 nm, respectively. The two spectral shift ranges differed by approximately 14 to 15 nm and/or energies of approximately 1.5 kcal mol(-1) relative to each other and to the absorption maximum for free fluorescein. Spectral effects on the antibody-bound ligand were discussed relative to solvent-water studies and the atomic structure of a high-affinity liganded anti-fluorescein active site.