A FLIM Microscopy Based on Acceptor-Detected Förster Resonance Energy Transfer.

Time-resolved donor-detected Förster resonance energy transfer (trDDFRET) allows the observation of molecular interactions of dye-labeled biomolecules in the ∼10-100 Å region. However, we can observe longer-range interactions when using time-resolved acceptor-detected FRET (trADFRET), since the signal/noise ratio can be improved when observing the acceptor emission. Therefore, we propose a new methodology based on trADFRET to construct a new fluorescence lifetime microscopy (FLIM-trADFRET) technique to observe biological machinery in the range of 100-300 Å in vivo, the last frontier in biomolecular medicine. The integrated trADFRET signal is extracted in such a way that noise is canceled, and more photons are collected, even though trADFRET and trDDFRET have the same rate of transfer. To assess our new methodology, proof of concept was demonstrated with a set of well-defined DNA scaffolds.

Dual-Channel Stopped-Flow Apparatus for Simultaneous Fluorescence, Anisotropy, and FRET Kinetic Data Acquisition for Binary and Ternary Biological Complexes.

The Stopped-Flow apparatus (SF) tracks molecular events by mixing the reactants in sub-millisecond regimes. The reaction of intrinsically or extrinsically labeled biomolecules can be monitored by recording the fluorescence, F(t), anisotropy, r(t), polarization, p(t), or FRET, F(t)FRET, traces at nanomolar concentrations. These kinetic measurements are critical to elucidate reaction mechanisms, structural information, and even thermodynamics. In a single detector SF, or L-configuration, the r(t), p(t), and F(t) traces are acquired by switching the orientation of the emission polarizer to collect the IVV and IVH signals however it requires two-shot experiments. In a two-detector SF, or T-configuration, these traces are collected in a single-shot experiment, but it increases the apparatus' complexity and price. Herein, we present a single-detector dual-channel SF to obtain the F(t) and r(t) traces simultaneously, in which a photo-elastic modulator oscillates by 90° the excitation light plane at a 50 kHz frequency, and the emission signal is processed by a set of electronic filters that split it into the r(t) and F(t) analog signals that are digitized and stored into separated spreadsheets by a custom-tailored instrument control software. We evaluated the association kinetics of binary and ternary biological complexes acquired with our dual-channel SF and the traditional methods; such as a single polarizer at the magic angle to acquire F(t), a set of polarizers to track F(t), and r(t), and by energy transfer quenching, F(t)FRET. Our dual-channel SF economized labeled material and yielded rate constants in excellent agreement with the traditional methods.

Detailed characterization of the solution kinetics and thermodynamics of biotin, biocytin and HABA binding to avidin and streptavidin.

The high affinity (KD ~ 10-15 M) of biotin for avidin and streptavidin is the essential component in a multitude of bioassays with many experiments using biotin modifications to invoke coupling. Equilibration times suggested for these assays assume that the association rate constant (kon) is approximately diffusion limited (109 M-1s-1) but recent single molecule and surface binding studies indicate that they are slower than expected (105 to 107 M-1s-1). In this study, we asked whether these reactions in solution are diffusion controlled, which reaction model and thermodynamic cycle describes the complex formation, and if there are any functional differences between avidin and streptavidin. We have studied the biotin association by two stopped-flow methodologies using labeled and unlabeled probes: I) fluorescent probes attached to biotin and biocytin; and II) unlabeled biotin and HABA, 2-(4'-hydroxyazobenzene)-benzoic acid. Both native avidin and streptavidin are homo-tetrameric and the association data show no cooperativity between the binding sites. The kon values of streptavidin are faster than avidin but slower than expected for a diffusion limited reaction in both complexes. Moreover, the Arrhenius plots of the kon values revealed strong temperature dependence with large activation energies (6-15 kcal/mol) that do not correspond to a diffusion limited process (3-4 kcal/mol). Accordingly, we propose a simple reaction model with a single transition state for non-immobilized reactants whose forward thermodynamic parameters complete the thermodynamic cycle, in agreement with previously reported studies. Our new understanding and description of the kinetics, thermodynamics, and spectroscopic parameters for these complexes will help to improve purification efficiencies, molecule detection, and drug screening assays or find new applications.

Stability of the Plasmodium falciparum AMA1-RON2 Complex Is Governed by the Domain II (DII) Loop.

Plasmodium falciparum is an obligate intracellular protozoan parasite that employs a highly sophisticated mechanism to access the protective environment of the host cells. Key to this mechanism is the formation of an electron dense ring at the parasite-host cell interface called the Moving Junction (MJ) through which the parasite invades. The MJ incorporates two key parasite components: the surface protein Apical Membrane Antigen 1 (AMA1) and its receptor, the Rhoptry Neck Protein (RON) complex, the latter one being targeted to the host cell membrane during invasion. Crystal structures of AMA1 have shown that a partially mobile loop, termed the DII loop, forms part of a deep groove in domain I and overlaps with the RON2 binding site. To investigate the mechanism by which the DII loop influences RON2 binding, we measured the kinetics of association and dissociation and binding equilibria of a PfRON2sp1 peptide with both PfAMA1 and an engineered form of PfAMA1 where the flexible region of the DII loop was replaced by a short Gly-Ser linker (ΔDII-PfAMA1). The reactions were tracked by fluorescence anisotropy as a function of temperature and concentration and globally fitted to acquire the rate constants and corresponding thermodynamic profiles. Our results indicate that both PfAMA1 constructs bound to the PfRON2sp1 peptide with the formation of one intermediate in a sequential reversible reaction: A↔B↔C. Consistent with Isothermal Titration Calorimetry measurements, final complex formation was enthalpically driven and slightly entropically unfavorable. Importantly, our experimental data shows that the DII loop lengthened the complex half-life time by 18-fold (900 s and 48 s at 25°C for Pf and ΔDII-Pf complex, respectively). The longer half-life of the Pf complex appeared to be driven by a slower dissociation process. These data highlight a new influential role for the DII loop in kinetically locking the functional binary complex to enable host cell invasion.

Computational and biophysical approaches to protein-protein interaction inhibition of Plasmodium falciparum AMA1/RON2 complex.

Invasion of the red blood cell by Plasmodium falciparum parasites requires formation of an electron dense circumferential ring called the Moving Junction (MJ). The MJ is anchored by a high affinity complex of two parasite proteins: Apical Membrane Antigen 1 (PfAMA1) displayed on the surface of the parasite and Rhoptry Neck Protein 2 that is discharged from the parasite and imbedded in the membrane of the host cell. Structural studies of PfAMA1 revealed a conserved hydrophobic groove localized to the apical surface that coordinates RON2 and invasion inhibitory peptides. In the present work, we employed computational and biophysical methods to identify competitive P. falciparum AMA1-RON2 inhibitors with the goal of exploring the 'druggability' of this attractive antimalarial target. A virtual screen followed by molecular docking with the PfAMA1 crystal structure was performed using an eight million compound collection that included commercial molecules, the ChEMBL malaria library and approved drugs. The consensus approach resulted in the selection of inhibitor candidates. We also developed a fluorescence anisotropy assay using a modified inhibitory peptide to experimentally validate the ability of the selected compounds to inhibit the AMA1-RON2 interaction. Among those, we identified one compound that displayed significant inhibition. This study offers interesting clues to improve the throughput and reliability of screening for new drug leads.

Spectroscopic properties of fluorescein and rhodamine dyes attached to DNA.

We report the spectroscopic properties of fluorescein, x-rhodamine, tetramethyl-rhodamine, attached to single strand, duplex DNA, and to the digestion products by DNAse I. The properties reported include: molar absorptivity, quantum yield, absorbance and fluorescence spectra, fluorescence lifetime, intrinsic lifetime (tau0), static quenching (S) and the Förster critical distances (R0) between fluorescein and x-rhodamine or tetramethyl-rhodamine (acceptors). These spectroscopic properties depend strongly on the local dye environment. Fluorescein was studied: (1) attached to biotin (BF), (2) BF bound to avidin; and attached to two positions in DNA. X-rhodamine and tetramethyl-rhodamine were studied as free dyes and attached at the 5'-end of DNA. We propose a general method to determine the molar absorptivity and tau0 of a dye attached to DNA based on the reaction of a biotinylated and dye-labeled oligomer with standardized avidin. The molar absorptivity of a second dye attached to a DNA duplex can be obtained by comparing spectra of doubly and singly labeled sequences. S, arising from dye-DNA interactions can then be determined. R0 for free and attached dyes showed differences from 1.1 to 4.2 A. We present evidence for the direct interaction of dyes attached to the termini of various single-stranded DNA sequences.

The TATA-binding protein core domain in solution variably bends TATA sequences via a three-step binding mechanism.

Studies of the binding and bending of the AdMLP TATA sequence (TATAAAAG) by the core domain of yeast TBP allow quantitation of the roles of the N-terminal domains of yeast and human TBP. All three proteins bind DNA via a three-step mechanism with no evidence for an initially bound but unbent DNA. The large enthalpy and entropy of activation for the first step in yTBP binding can now be assigned to movement of the NTD from the DNA binding pocket and not to energetics of DNA bending. The energetic patterns for hTBP and cTBP suggest that the 158-amino acid NTD in hTBP does not initially occupy the DNA binding pocket. Despite the appearance of similar energetics for hTBP and cTBP, order of magnitude differences in rate constants lead to differing populations of intermediates during DNA binding. We find that the NTDs destabilize the three bound forms of DNA for both yTBP and hTBP. For all three proteins, the DNA bend angle (theta) depends on the TATA sequence, with theta for cTBP and hTBP being greater than that for yTBP. For all three proteins, theta for the G6 variant (TATAAGAG) varies with temperature and increases in the presence of osmolyte to be similar to that of AdMLP. Crystallographic studies of cTBP binding to a number of variants had shown no dependence of DNA bending on sequence. The results reported here reveal a clear structural difference for the bound DNA in solution versus the crystal; we attribute the difference to the presence of osmolytes in the crystals.

TATA-binding protein recognition and bending of a consensus promoter are protein species dependent.

The structure and behavior of full-length human TBP binding the adenovirus major late promoter (AdMLP) have been characterized using biophysical methods. The human protein induces a 97 degrees bend in DNA AdMLP. The high-resolution functional data provide a quantitative energetic and kinetic description of the partial reaction sequence as native human TBP binds rapidly to a consensus promoter with high affinity. The reaction proceeds with successive formation of three bound species, all having strongly bent DNA, with the concurrence of binding and bending demonstrated by both fluorescence and anisotropy stopped flow. These results establish the protein species dependence of the TBP-DNA AdMLP structure and recognition mechanism. Additionally, the strong correlation between the DNA bend angle and transcription efficiency demonstrated previously for yeast TBP is shown to extend to human TBP. The heterologous NH 2-terminal domains are the apparent source of the species-specific differences. Together with previous studies the present work establishes that TBP wt-DNA TATA function and structure depend both on the TATA box sequence and on the TBP species.