Mary D. Barkley

Time-resolved fluorescence of the single tryptophan of Bacillus stearothermophilus phosphofructokinase

The fluorescence of the single tryptophan in Bacillus stearothermophilus phosphofructokinase was characterized by steady-state and time-resolved techniques. The enzyme is a tetramer of identical subunits, which undergo a concerted allosteric transition. Time-resolved emission spectral data were fitted to discrete and distributed lifetime models. The fluorescence decay is a double exponential with lifetimes of 1.6 and 4.4 ns and relative amplitudes of 40 and 60%. The emission spectra of both components are identical with maxima at 327 nm. The quantum yield is 0.31 +/- 0.01. The shorter lifetime is independent of temperature; the longer lifetime has weak temperature dependence with activation energy of 1 kcal/mol. The fluorescence intensity and decay are the same in H2O and D2O solutions, indicating that the indole ring is not accessible to bulk aqueous solution. The fluorescence is not quenched significantly by iodide, but it is quenched by acrylamide with bimolecular rate constant of 5 x 10(8) M-1 s-1. Static and dynamic light scattering measurements show that the enzyme is a tetramer in solution with hydrodynamic radius of 40 A. Steady-state and time-resolved fluorescence anisotropies indicate that the tryptophan is immobile. The allosteric transition has little effect on the fluorescence properties. The fluorescence results are related to the x-ray structure.

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.

Solution structural dynamics of HIV-1 reverse transcriptase heterodimer.

Crystal structures and simulations suggest that conformational changes are critical for the function of HIV-1 reverse transcriptase. The enzyme is an asymmetric heterodimer of two subunits, p66 and p51. The two subunits have the same N-terminal sequence, with the p51 subunit lacking the C-terminal RNase H domain. We used hydrogen exchange mass spectrometry to probe the structural dynamics of RT. H/D exchange revealed that the fingers and palm subdomains of both subunits form the stable core of the heterodimer. In the crystal structure, the tertiary fold of the p51 subunit is more compact than that of the polymerase domain of the p66 subunit, yet both subunits show similar flexibility. The p66 subunit contains the polymerase and RNase H catalytic sites. H/D exchange indicated that the RNase H domain of p66 is very flexible. The beta-sheet beta12-beta13-beta14 lies at the base of the thumb subdomain of p66 and contains highly conserved residues involved in template/primer binding and NNRTI binding. Using the unique ability of hydrogen exchange mass spectrometry to resolve slowly interconverting species, we found that beta-sheet beta12-beta13-beta14 undergoes slow cooperative unfolding with a t(1/2) of <20 s. The H/D exchange results are discussed in relation to existing structural, simulation, and sequence information.

Efavirenz binding to HIV-1 reverse transcriptase monomers and dimers.

Efavirenz (EFV) is a nonnucleoside reverse transcriptase inhibitor (NNRTI) of HIV-1 reverse transcriptase (RT) used for the treatment of AIDS. RT is a heterodimer composed of p66 and p51 subunits; p51 is produced from p66 by C-terminal truncation by HIV protease. The monomers can form p66/p66 and p51/p51 homodimers as well as the p66/p51 heterodimer. Dimerization and efavirenz binding are coupled processes. In the crystal structure of the p66/p51-EFV complex, the drug is bound to the p66 subunit. The binding of efavirenz to wild-type and dimerization-defective RT proteins was studied by equilibrium dialysis, tryptophan fluorescence, and native gel electrophoresis. A 1:1 binding stoichiometry was determined for both monomers and homodimers. Equilibrium dissociation constants are approximately 2.5 microM for both p66- and p51-EFV complexes, 250 nM for the p66/p66-EFV complex, and 7 nM for the p51/p51-EFV complex. An equilibrium dissociation constant of 92 nM for the p66/p51-EFV complex was calculated from the thermodynamic linkage between dimerization and inhibitor binding. Binding and unbinding kinetics monitored by fluorescence were slow. Progress curve analyses revealed a one-step, direct binding mechanism with association rate constants k(1) of approximately 13.5 M(-1) s(-1) for monomers and heterodimer and dissociation rate constants k(-1) of approximately 9 x 10(-5) s(-1) for monomers. A conformational selection mechanism is proposed to account for the slow association rate. These results show that efavirenz is a slow, tight-binding inhibitor capable of binding all forms of RT and suggest that the NNRTI binding site in monomers and dimers is similar.

Monte Carlo convolution method for simulation and analysis of fluorescence decay data

A Monte Carlo convolution method for simulating time‐correlated single photon counting data is presented. The random convolution automatically produces the Poisson statistics of the real experiment. The new simulation technique offers realistic treatment of various aspects of the single photon counting experiment, including pulse pileup. The random convolution is also incorporated in a data analysis technique using a reference fluorophore. Illustrative examples comparing the Monte Carlo and conventional simulation methods are given and the conceptual differences are discussed.

Kinetics of Association and Dissociation of HIV-1 Reverse Transcriptase Subunits †

The biologically active form of HIV-1 reverse transcriptase (RT) is the p66/p51 heterodimer. The process of maturation of the heterodimer from precursor proteins is poorly understood. Previous studies indicated that association of p66 and p51 is very slow. Three techniques, a pre-steady-state activity assay, intrinsic tryptophan fluorescence, and a FRET assay, were used to monitor the dimerization kinetics of RT. Kinetic experiments were conducted with purified p66 and p51 proteins in aqueous buffer. All three techniques gave essentially the same results. The dissociation kinetics of p66/p51 were first-order with rate constants (k(diss)) of approximately 4 x 10(-6) s(-1) (t(1/2) = 48 h). The association kinetics of p66 and p51 were concentration-dependent with second-order rate constants (k(ass)) of approximately 1.7 M(-1) s(-1) for the simple bimolecular association reaction. The implications of slow dimerization of p66/p51 for the maturation process are discussed. A reaction-controlled model invoking conformational selection is proposed to explain the slow protein-protein association kinetics.

Efavirenz Binding Site in HIV-1 Reverse Transcriptase Monomers

Efavirenz (EFV) is a potent nonnucleoside reverse transcriptase inhibitor (NNRTI) used in the treatment of AIDS. NNRTIs bind in a hydrophobic pocket located in the p66 subunit of reverse transcriptase (RT), which is not present in crystal structures of RT without an inhibitor. Recent studies showed that monomeric forms of the p66 and p51 subunits bind efavirenz with micromolar affinity. The effect of efavirenz on the solution conformations of p66 and p51 monomers was studied by hydrogen-deuterium exchange mass spectrometry (HXMS) and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). HXMS data reveal that five peptides, four of which contain efavirenz contact residues seen in the crystal structure of the RT-EFV complex, exhibit a reduced level of exchange in monomer-EFV complexes. Moreover, peptide 232-246 undergoes slow cooperative unfolding-refolding in the bound monomers, but at a rate much slower than that observed in the p66 subunit of the RT heterodimer [Seckler, J. M., Howard, K. J., Barkley, M. D., and Wintrode, P. L. (2009) Biochemistry 48, 7646-7655]. These results suggest that the efavirenz binding site on p66 and p51 monomers is similar to the NNRTI binding pocket in the p66 subunit of RT. Nanoelectrospray ionization FT-ICR mass spectra indicate that the intact monomers each have (at least) two different conformations. In the presence of efavirenz, the mass spectra change significantly and suggest that p51 adopts a single, more compact conformation, whereas p66 undergoes facile, electrospray-induced cleavage. The population shift is consistent with a selected-fit binding mechanism.

Ion effects on the lac repressor-operator interaction.

The effects of ions on the binding of lac repressor protein and operator DNA have been studied using the membrane filter technique. The association and dissociation rate constants were measured, and the equilibrium association constants calculated, as a function of monovalent and divalent cation concentrations, anions, and pH. The salt dependence of the binding parameters is interpreted in light of recent theoretical analyses based on Mannings counterion condensation model.

Nonradiative Processes in Constrained Trps and Model Compounds

Constrained derivatives and model compounds were used to elucidate the nonradiative decay pathways of Trp. Fluorescence quenching by electron transfer from the excited indole to the amide backbone was studied in 7 cyclic hexapeptides.

Time-resolved fluorescence studies of tomaymycin bonding to synthetic DNAs

Tomaymycin reacts covalently with guanine in the DNA minor groove, exhibiting considerable specificity for the flanking bases. The sequence dependence of tomaymycin bonding to DNA was investigated in synthetic DNA oligomers and polymers. The maximum extent of bonding to DNA is greater for homopurine and natural DNA sequences than for alternating purine-pyrimidine sequences. Saturation of DNA with tomaymycin has little effect on the melting temperature in the absence of unbound drug. Fluorescence lifetimes were measured for DNA adducts at seven of the ten unique trinucleotide bonding sites. Most of the adducts had two fluorescence lifetimes, representing two of the four possible binding modes. The lifetimes cluster around 2-3 ns and 5-7 ns; the longer lifetime is the major component for most bonding sites. The two lifetime classes were assigned to R and S diastereomeric adducts by comparison with previous NMR results for oligomer adducts. The lifetime difference between binding modes is interpreted in terms of an anomeric effect on the excited-state proton transfer reaction that quenches tomaymycin fluorescence. Bonding kinetics of polymer adducts were monitored by fluorescence lifetime measurements. Rates of adduct formation vary by two orders of magnitude with poly(dA-dG).poly(dC-dT), reacting the fastest at 4 x 10(-2) M-1 s-1. The sequence specificity of tomaymycin is discussed in light of these findings and other reports in the literature.