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.

A small molecule directly inhibits the p53 transactivation domain from binding to replication protein A.

Replication protein A (RPA), essential for DNA replication, repair and DNA damage signalling, possesses six ssDNA-binding domains (DBDs), including DBD-F on the N-terminus of the largest subunit, RPA70. This domain functions as a binding site for p53 and other DNA damage and repair proteins that contain amphipathic alpha helical domains. Here, we demonstrate direct binding of both ssDNA and the transactivation domain 2 of p53 (p53TAD2) to DBD-F, as well as DBD-F-directed dsDNA strand separation by RPA, all of which are inhibited by fumaropimaric acid (FPA). FPA binds directly to RPA, resulting in a conformational shift as determined through quenching of intrinsic tryptophan fluorescence in full length RPA. Structural analogues of FPA provide insight on chemical properties that are required for inhibition. Finally, we confirm the inability of RPA possessing R41E and R43E mutations to bind to p53, destabilize dsDNA and quench tryptophan fluorescence by FPA, suggesting that protein binding, DNA modulation and inhibitor binding all occur within the same site on DBD-F. The disruption of p53-RPA interactions by FPA may disturb the regulatory functions of p53 and RPA, thereby inhibiting cellular pathways that control the cell cycle and maintain the integrity of the human genome.

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.

Changes in DNA bending and flexing due to tethered cations detected by fluorescence resonance energy transfer.

Local DNA deformation arises from an interplay among sequence-related base stacking, intrastrand phosphate repulsion, and counterion and water distribution, which is further complicated by the approach and binding of a protein. The role of electrostatics in this complex chemistry was investigated using tethered cationic groups that mimic proximate side chains. A DNA duplex was modified with one or two centrally located deoxyuracils substituted at the 5-position with either a flexible 3-aminopropyl group or a rigid 3-aminopropyn-1-yl group. End-to-end helical distances and duplex flexibility were obtained from measurements of the time-resolved Förster resonance energy transfer between 5'- and 3'-linked dye pairs. A novel analysis utilized the first and second moments of the G(t) function, which encompasses only the energy transfer process. Duplex flexibility is altered by the presence of even a single positive charge. In contrast, the mean 5'-3' distance is significantly altered by the introduction of two adjacently tethered cations into the double helix but not by a single cation: two adjacent aminopropyl groups decrease the 5'-3' distance while neighboring aminopropynyl groups lengthen the helix.

Native human TATA-binding protein simultaneously binds and bends promoter DNA without a slow isomerization step or TFIIB requirement.

The association of TATA-binding protein (TBP) with promoter DNA is central to the initiation and regulation of eukaryotic protein synthesis. Our laboratory has previously conducted detailed investigations of this interaction using yeast TBP and seven consensus and variant TATA sequences. We have now investigated this key interaction using human TBP and the TATA sequence from the adenovirus major late promoter (AdMLP). Recombinant native human protein was used together with fluorescently labeled DNA, allowing real time data acquisition in solution. We find that the wild-type hTBP-DNAAdMLP reaction is characterized by high affinity (Kd < or = 5 nm), simultaneous binding and DNA bending, and rapid formation of a stable human TBP-DNA complex having DNA bent approximately 100 degrees. These data allow, for the first time, a direct comparison of the reactions of the full-length, native human and yeast TBPs with a consensus promoter, studied under identical conditions. The general reaction characteristics are similar for the human and yeast proteins, although the details differ and the hTBPwt-induced bend is more severe. This directly measured hTBPwt-DNAAdMLP interaction differs fundamentally from a recently published hTBPwt-DNAAdMLP model characterized by low affinity (microM) binding and an unstable complex requiring either a 30-min isomerization or TFIIB to achieve DNA bending. Possible sources of these significant differences are discussed.

Reflections on apparent DNA bending by charge variants of bZIP proteins.

Basic-leucine zipper (bZIP) proteins have been studied intensely as transcription factors. It has been proposed that the bZIP domain might modulate transcription activation through the induction of conformational changes in the DNA binding site. We have been interested in using bZIP peptides as convenient models with which to study the role of asymmetric phosphate neutralization in DNA bending. DNA bending experiments have yielded discordant results for bZIP peptides studied by electrophoretic- vs solution-based assays. We review the history of DNA bending assays involving bZIP peptides and introduce the reader to examples of discordant results. Our recent published experiments designed to clarify this field of study will then be reviewed. The engineering of protein fusions has established that electrophoretic phasing assays are relatively insensitive to precise protein structure/conformation and instead appear to report DNA bending, as influenced by protein charge. New applications of time-resolved fluorescence resonance energy transfer (FRET) have allowed for the first time corroboration of electrophoretic phasing assays with solution-based FRET measurements. We report that two conventional DNA bending assays that rely on DNA ligation cannot be applied to analysis of the bZIP peptides we studied due to ligation inhibition.

DNA bending by bZIP charge variants: a unified study using electrophoretic phasing and fluorescence resonance energy transfer.

The role of asymmetric charge neutralization as a primary determinant of protein-induced DNA helical bending remains controversial. Electrophoretic phasing experiments have been conducted previously for peptides derived from the yeast basic leucine zipper (bZIP) transcription factor GCN4 bound to AP-1 sites in duplex DNA. Mutations altering the electrostatic character of amino acids close to the DNA backbone result in phase-dependent gel mobility changes, interpreted as evidence of DNA bending. However, alternate interpretations are possible. The effect of electrostatic interactions on DNA conformation has now been investigated further, using purified peptides having indistinguishable AP-1 DNA affinity. Two independent techniques have been employed: electrophoretic phasing and fluorescence resonance energy transfer (FRET). The phasing results imply DNA bending by bZIP charge variants, consistent with earlier findings. FRET studies yield the mean 5' end to 3' end distance of AP-1 DNA when free or bound to neutral or charged bZIP peptides. These distances were reduced in the charged variant complexes relative to those in the free duplex and the wild-type complex. Bending of the DNA helical axis is shown by molecular modeling to be the simplest interpretation of these results. The electrophoretic phasing and FRET results thus offer two mutually supportive lines of evidence for induced bending of the DNA helical axis due to asymmetric changes in charge density caused by the electrostatic character of the amino acids residing near the DNA backbone.

Comparison of TATA-binding protein recognition of a variant and consensus DNA promoters.

Assembly of transcription pre-initiation complexes proceeds from the initial complex formed between "TATA" bearing promoter DNA and the TATA-binding protein (TBP). Our laboratory has been investigating the relationships among TATA sequence, TBP center dot TATA solution structure, recognition mechanisms, and transcription efficiency. TBP center dot TATA interactions have been modeled by global analysis of detailed kinetic and thermodynamic data obtained using fluorimetric and fluorometric techniques in conjunction with fluorescence resonance energy transfer. We have reported recently that TBP recognition of two consensus promoters, adenovirus major late (AdMLP: TATAAAAG) and E4 (TATATATA), is well described by a linear two-intermediate mechanism with simultaneous DNA binding and bending. Similar DNA geometries and high transcription efficiencies characterize these TBP x TATA complexes. Here we show that, in contrast to the consensus sequences, TBP recognition of a variant sequence (C7: TATAAACG) is described by a three-step model with two branching pathways. One pathway proceeds through an intermediate having severely bent DNA, reminiscent of the consensus interactions, with the other branch yielding a unique conformer with shallowly bent DNA. The resulting TBP x C7 complex has a dramatically different solution conformation than for TBP x DNA(CONSENSUS) and is correlated with diminished relative transcription activity. The temperature dependence of the TBP x C7 helical bend is postulated to derive from population shifts between the conformers with slightly and severely bent DNA.