Correlation of tryptophan fluorescence spectral shifts and lifetimes arising directly from heterogeneous environment.

Tryptophan (Trp) fluorescence is potentially a powerful probe for studying the conformational ensembles of proteins in solution, as it is highly sensitive to the local electrostatic environment of the indole side chain. However, interpretation of the wavelength-dependent complex fluorescence decays of proteins has been stymied by controversy about two plausible origins of the typical multiple fluorescence lifetimes: multiple ground-state populations or excited-state relaxation. The latter naturally predicts the commonly observed wavelength-lifetime correlation between decay components, which associates short lifetimes with blue-shifted emission spectra and long lifetimes with red-shifted spectra. Here we show how multiple conformational populations also lead to the same strong wavelength-lifetime correlation in cyclic hexapeptides containing a single Trp residue. Fluorescence quenching in these peptides is due to electron transfer. Quantum mechanics-molecular mechanics simulations with 150-ps trajectories were used to calculate fluorescence wavelengths and lifetimes for the six canonical rotamers of seven hexapeptides in aqueous solution at room temperature. The simulations capture most of the unexpected diversity of the fluorescence properties of the seven peptides and reveal that rotamers having blue-shifted emission spectra, i.e., higher average energy, have an increased probability for quenching, i.e., shorter average lifetime, during large fluctuations in environment that bring the nonfluorescent charge transfer state and the fluorescing state into resonance. This general mechanism should also be operative in proteins that exhibit multiexponential fluorescence decays, where myriad other sources of conformational heterogeneity besides rotamers are possible.

Wound healing monitoring using near infrared fluorescent fibrinogen.

We demonstrate a method for imaging the wound healing process with near infrared fluorescent fibrinogen. Wound healing studies were performed on a rat punch biopsy model. Fibrinogen was conjugated with a near infrared fluorescent dye and injected into the tail vein. Fibrinogen is a useful protein for tracking wound healing because it is involved in fibrin clot formation and formation of new provisional matrix through transglutaminase's crosslinking activity. Strong fluorescence specific to the wound was observed and persisted for several days, indicating that the fibrinogen is converted to crosslinked fibrin. Administration of contrast agent simultaneously with wound creation led to primary labeling of the fibrin clot, indicating that the wound was in its early phase of healing. Administration on the following day showed labeling on the wound periphery, indicating location of formation of a new provisional matrix. This method may prove to be useful as a diagnostic for basic studies of the wound healing process, in drug development, or in clinical assessment of chronic wounds.

Dependence of tryptophan emission wavelength on conformation in cyclic hexapeptides.

The wavelength of maximum emission of tryptophan depends on the local electrostatic environment of the indole chromophore. The time-resolved emission spectra of seven rigid cyclic hexapeptides containing a single tryptophan residue were measured. The emission maxima of the three decay-associated spectra for the seven peptides ranged from 341 to 359 nm, suggesting that different tryptophan rotamers have different emission maxima even in the case of solvent-exposed tryptophans. This conclusion is supported by quantum mechanical/molecular dynamics simulations of the six canonical side chain rotamers of tryptophan in solvated hexapeptides. The calculated range of emission maxima for the tryptophan rotamers of the seven peptides is 344-365 nm. The precision of the wavelength calculations and the peptide, water, and charged side chain contributions to the spectral shifts are examined. The results indicate that the emission maxima of decay-associated spectra can aid in the assignment of fluorescence lifetimes to tryptophan rotamers.

Conformational effects on tryptophan fluorescence in cyclic hexapeptides.

The peptide bond quenches tryptophan fluorescence by excited-state electron transfer, which probably accounts for most of the variation in fluorescence intensity of peptides and proteins. A series of seven peptides was designed with a single tryptophan, identical amino acid composition, and peptide bond as the only known quenching group. The solution structure and side-chain chi(1) rotamer populations of the peptides were determined by one-dimensional and two-dimensional (1)H-NMR. All peptides have a single backbone conformation. The -, psi-angles and chi(1) rotamer populations of tryptophan vary with position in the sequence. The peptides have fluorescence emission maxima of 350-355 nm, quantum yields of 0.04-0.24, and triple exponential fluorescence decays with lifetimes of 4.4-6.6, 1.4-3.2, and 0.2-1.0 ns at 5 degrees C. Lifetimes were correlated with ground-state conformers in six peptides by assigning the major lifetime component to the major NMR-determined chi(1) rotamer. In five peptides the chi(1) = -60 degrees rotamer of tryptophan has lifetimes of 2.7-5.5 ns, depending on local backbone conformation. In one peptide the chi(1) = 180 degrees rotamer has a 0.5-ns lifetime. This series of small peptides vividly demonstrates the dominant role of peptide bond quenching in tryptophan fluorescence.

X-ray structures, photophysical characterization, and computational analysis of geometrically constrained copper(I)--phenanthroline complexes.

A series of three geometrically constrained C(2)-symmetric Cu(I) mono-phenanthroline complexes were characterized by X-ray structural analysis, and their photophysical properties were investigated by absorption and emission spectroscopy. Visible light excitation yielded metal-to-ligand charge-transfer (MLCT) excited states with luminescence lifetimes up to 155 ns. Ultrafast transient absorption spectroscopy provided further insights into the excited-state dynamics and suggests for all three complexes the formation of a phenanthroline radical anion. In agreement with electrochemical measurements, the data further indicate that coordinative rearrangements are involved in nonradiative deactivation of the excited states. According to time-dependent density functional theory calculations (B3LYP/6-31G), the major MLCT transitions are polarized along the C(2) axis of the complex and originate predominantly from the copper d(xz) orbital. The computational analysis identifies an excited-state manifold with a number of close-lying, potentially emissive triplet states and is in agreement with the multiexponential decay kinetics of the MLCT luminescence. The relationship between structural and photophysical data of the studied Cu(I) mono-phenanthroline complexes agrees well with current models describing the photophysics of the related Cu(I) bis-diimine complexes.

Endogenous tryptophan residues of cAPK regulatory subunit type IIbeta reveal local variations in environments and dynamics.

The amino terminal dimerization/docking domain and the two-tandem, carboxy-terminal cAMP-binding domains (A and B) of cAMP-dependent protein kinase regulatory (R) subunits are connected by a variable linker region. In addition to providing a docking site for the catalytic subunit, the linker region is a major source of sequence diversity between the R-subunit isoforms. The RIIbeta isoform uniquely contains two endogenous tryptophan residues, one at position 58 in the linker region and the other at position 243 in cAMP-binding domain A, which can act as intrinsic reporter groups of their dynamics and microenvironment. Two single-point mutations, W58F and W243F, allowed the local environment of each Trp to be probed using steady-state and time-resolved fluorescence techniques. We report that: (a) the tryptophan fluorescence of the wild-type protein largely reflects Trp243 emission; (2) cAMP selectively quenches Trp243 and thus acts as a cAMP sensor; (3) Trp58 resides in a highly solvated, unstructured, and mobile region of the protein; and (4) Trp243 resides in a stable, folded domain and is relatively buried and rigid within the domain. The use of endogenous Trp residues presents a non-perturbing method for studying R-subunit subdomain characteristics in addition to providing the first biophysical data on the RIIbeta linker region.

Two-step FRET as a structural tool.

The power of FRET to study molecular complexes is expanded by the use of two or more donor/acceptor pairs. A general theoretical framework for distance measurements in three-chromophore systems is presented. Three energy transfer schemes applicable to many diverse situations are considered: (I) two-step FRET relay with FRET between the first and second chromophores and between the second and third, (II) FRET from a single donor to two different acceptors, and (III) two-step FRET relay with FRET also between the first and third chromophores. Equations for the efficiencies involving multiple energy transfer steps are derived for both donor quenching and sensitized emission measurements. The theory is supported by experimental data on model systems of known structure using steady-state donor quenching, lifetime quenching, and sensitized emission. The distances measured in the three-chromophore systems agree with those in two-chromophore systems and molecular models. Finally, labeling requirements for diagnosis of the energy transfer scheme and subsequent distance measurements are discussed.