Lawrence J. Parkhurst

[17] Circular dichroism spectra of hemoglobins

Publisher Summary This chapter deals with the circular dichroism (CD) spectra of hemoglobins. The measurement of CD spectra as a tool to investigate the structural organization of protein molecules is of particular advantage for hemoglobins. These molecules contain, in addition to the protein moiety, the heme, with electronic transitions that are quite intense, diverse, and very sensitive both to the surrounding environment and to ligand binding. This situation offers three distinct regions of investigation, each containing information concerning a part of the structural organization of the hemoglobin molecule. Consequently, the optical activity of hemoglobin spectra depends on the relative positions of the different chromophores in the tridimensional organization of the molecule. Alterations of the relative positions in different conformational states are likely to cause alterations in the interactions from which the CD spectra of the different chromophores derive. This is the reason that CD is so sensitive to the conformational states of the molecule. The chapter also presents the informational content of CD spectra.

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.

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.

Rapid preparation of native alpha and beta chains of human hemoglobin

1. Current procedures for the isolation of native chains of hemoglobin employ two ion exchange columns for each chain and result in readily autoxidizable chains with measurable contamination by Hb and Hg. 2. In the new procedure, altered buffer conditions on the first column reduce Hb contamination from 2 to 5% to less than 1%, the limit of detectability. 3. The second column and lengthy washes with beta mercaptoethanol are replaced by incubation with DTT for 1 min for alpha chains and, for beta chains, three incubations with DTT and separations by gel-filtration. The residual Hg is less than 0.1%. 4. Oxidations in the previous procedure resulted in low yields and unreliable spectroscopic assessments of bound Hg. The new procedure resulted in a simple UV assay for Hg-free chains. 5. Hemoglobin reconstituted from these oxy-chains was identical to native Hb in oxygen binding equilibria and in the kinetics of CO binding following laser photolysis.

Use of dual wavelength spectrophotometry and continuous enzymatic depletion of oxygen for determination of the oxygen binding constants of hemoglobin

A small stopped-flow cuvette was built into a computer-controlled Cary 210 spectrophotometer. The enzymatic depletion of oxygen in solutions of hemoglobin and myoglobin was initiated by flowing the hemeproteins with the enzyme against a solution of the hemeproteins containing the appropriate substrate. The deoxygenation was homogeneous throughout the solution. Oxygen activity was calculated at each instant of time from the fractional saturation of Mb, determined from observations at the Hb/HbO2 isosbestic wavelength. Fractional saturation of Hb was determined from absorbances at the Mb/MbO2 isosbestic wavelength. The spectrophotometer cycled between these two wavelengths during the deoxygenation. The deoxygenation of HbO2 was largely complete in 20-25 min, whereas the deoxygenation of MbO2 was allowed to proceed for about 1 h. This procedure eliminates equilibration of Hb solutions with a gas phase and replaces oxygen electrode readings with spectrophotometric sensing by Mb, providing essentially instantaneous determinations of oxygen activity and hence 250-500 or more independent data points per run. The Mb and Hb data vectors require several manipulations to correct for small relative displacements in time and for small non-isosbestic effects. Detailed consideration of the enzyme kinetics allowed oxygen activities to be determined in regions where Mb is a poor sensor. Studies of HbO2 deoxygenation as a function of wavelength show that the determination of the four Adair constants requires in addition the determination of three spectroscopic parameters. Values of the apparent Adair constants, determined without these spectroscopic parameters, depend strongly on the monitoring wavelength.

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 (τ0), 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 τ0 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 Å. We present evidence for the direct interaction of dyes attached to the termini of various single‐stranded DNA sequences.

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.

Distance parameters derived from time-resolved Förster resonance energy transfer measurements and their use in structural interpretations of thermodynamic quantities associated with protein-DNA interactions.

Publisher Summary Parameters that define a distance distribution for Forster resonance energy transfer (FRET) donor and acceptor pairs can be extracted from time-resolved FRET measurements. The ultimate interest in extracting such parameters is in determining distances that are relatively static on a nonosecond time scale. In turn, these distances provide information about multiple conformations of macromolecules and about the kinetics and energetics that link such conformations. Not only the mean distance, but parameters from higher moments of the distribution in particular the width— provide the information about the flexibility of macromolecules. These distances and particularly the changes in distances can be determined to high precision allowing FRET to play a key role in providing highly precise distances in solution. This chapter reviews a few essentials of FRET with attention to experimental and computational methods and shows the relationship of distance parameters to macromolecular changes in the field of DNA and DNA–TATA-binding protein (TBP) interactions. These structural changes in turn are intimately linked to various thermodynamic properties. Parameters that characterize a distance distribution P(R) can be obtained from time-resolved FRET measurements. These measurements can involve various combinations of donor-detected FRET and acceptor-detected FRET constrained by steady state emission intensity differences between the donor and that of the donor in the presence of an acceptor. Highly precise average interdye distances R can ultimately lead to precise intra-molecular distances in solution. The width of the P(R) distribution, σ, preferably and more precisely after removal of the tether contributions, yields a measure of conformational equilibria and of conformational dynamics of the macromolecule to which the probes are attached. FRET measurements combined with equilibrium determinations and with rapid-mixing or relaxation kinetics provide structure-energy, entropy profiles of intermediates and transition states along the reaction coordinate.

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.

Detection of point mutations in DNA by fluorescence energy transfer

A method has been developed for the rapid and direct identification of a single point mutation in a DNA sequence using fluorescence resonance energy transfer (FRET). The probe was a 16-base oligomer with 58-bound x-rhodamine and 38-bound fluorescein (R*16*F); the two dyes acted as a donor/acceptor pair for FRET, resulting in a dramatic difference in the fluorescence emission of the R*16*F in a duplex structure (hybridized to a complementary strand) and as a single strand (melted). This difference was used to obtain the melting temperature (Tm), by spectroscopically following the transition from double to single strand, for the probe hybridized to three different strands: the 16-base complement, the 16-base complement containing a single base mismatch, and the 16-base complementary sequence in the phage DNA M13mp18(+). The Tms thus determined for the perfectly base-paired duplexes, with R*16*F hybridized to the 16-mer complement and to M13, differed by 2°C, whereas the Tm obtained for R*16*F hybridized to the mismatched 16-mer complement was 10°C lower than that for the perfect duplex. The sharpness of the transition and the ease of detection allow single base mismatches to be reliably detected in nano- and subnanomolar concentrations in less than 1 h following hybridization.