Solution structure of a mutant of transcription factor 1: implications for enhanced DNA binding.

An NMR solution structure of a mutant of the homodimer protein transcription factor 1, TF1-G15/I32 (22 kDa), has been solved to atomic resolution, with 23 final structures that converge to an r.m. s.d. of 0.78 A. The overall shape of TF1-G15/I32 remains similar to that of the wild-type protein and other type II DNA-binding proteins. Each monomer has two N-terminal alpha-helices separated by a short loop, followed by a three-stranded beta-sheet, whose extension between the second and third beta-strands forms an antiparallel beta-ribbon arm, leading to a C-terminal third alpha-helix that is severely kinked in the middle. Close examination of the structure of TF1-G15/I32 reveals why it is more stable and binds DNA more tightly than does its wild-type counterpart. The dimeric core, consisting of the N-terminal helices and the beta-sheets, is more tightly packed, and this might be responsible for its increased thermal stability. The DNA-binding domain, composed of the top face of the beta-sheet, the beta-ribbon arms and the C-terminal helices, is little changed from wild-type TF1. Rather, the enhancement in DNA affinity must be due almost exclusively to the creation of an additional DNA-binding site at the side of the dimer by changes affecting helices 1 and 2: helix 2 of TF1-G15/I32 is one residue longer than helix 2 of the wild-type protein, bends inward, and is both translationally and rotationally displaced relative to helix 1. This rearrangement creates a longer, narrower fissure between the V-shaped N-terminal helices and exposes additional positively charged surface at each side of the dimer.

Mechanisms for the enhanced thermal stability of a mutant of transcription factor 1 as explained by (1)H, (15)N and (13)C NMR chemical shifts and secondary structure analysis.

A variant of the bacteriophage SPO1-encoded transcription factor 1 (TF1) with two site-specific mutations (E15G and T32I) was shown to be more thermally stable and bind DNA more tightly compared to the wild-type protein. In order to understand the biochemical mechanisms underlying these properties, we are engaged in determining the solution structures of this mutant alone and in complex with DNA using nuclear magnetic resonance (NMR) spectroscopy. The first phase of this project is reported here, as we have completed most of the backbone and sidechain sequential NMR assignments of the mutant protein, TF1-G15/I32. Insights derived from the (1)H, (15)N and (13)C chemical shifts and from the secondary structure analysis provide us with an explanation for the noted increase in thermal stability of TF1-G15/I32. Compared to the structure of the wild-type protein, the beta-sheet and the C-terminal helix remain largely unaffected whereas the mutations cause great changes in the first two helices and their enclosed loop. Specifically, we have found that the second helix is extended by one residue at its N-terminus and rotated in a way that allows Ala-37 to interact with Tyr-94 of the C-terminal helix. The loop has been found to become more rigid as a result of hydrophobic interactions between the flanking second and first helices and also between the second helix and the loop itself. Furthermore, the T32I mutation allows tighter packing between the second helix and the beta-sheet. Collectively, these changes contribute to a more tightly associated dimer and hence, to a greater thermal stability.

NMR-derived solution structure of a 17mer hydroxymethyluracil-containing DNA.

Incorporation of 5-(hydroxymethyl)-2'-deoxyuridine into DNA in place of thymine by SPO1, a Bacillus subtilis bacteriophage, allows the viral DNA to bind selectively to transcription factor 1. We have synthesized a TF1-binding site: d(5'-ACCHACHCHHHGHAGGT-3')-d(5'-ACCHACAAAGAGHAGGT-3') and studied this molecule using NMR spectroscopy. The chemical shifts of exchangeable and non-exchangeable protons were sequentially assigned. Absence of corresponding NOEs in the imino-imino region suggested that the end base pairs did not form Watson-Crick hydrogen bond. Restrained molecular dynamics calculation yielded a family of B-DNA structures whose r.m.s.d. was 0.66 A (all atoms) for the internal 15 bp. The helical twist was 38.5 degrees per step. The base pairs were situated directly on the helix axis (X-displacement = -0.2 A). All sugars exhibited C2'-endo puckering with P = 167.3 degrees and upsilon(max)= 38.2 degrees. The OH groups of all hmU bases resided on the 3' side of the base plane and may affect the base orientation relative to the sugar plane as the average chi value for all hmU was 4 degrees more positive than that of other nucleosides (258 degrees versus 254 degrees ). Positive roll angles (rho) and small flanking twists (omega) at hmU suggested that the two hmU-A base pair steps open toward the minor grooves.

Specificity of hydroxylmethyluracil-containing DNA for transcription factor 1: structural insights.

The genomic materials from some Bacillus subtilis bacteriophages are found to contain 5-(hydroxymethyl)-2'-deoxyuridine in place of thymine. Phage-encoded proteins such as transcription factor 1 specifically and preferentially bind to the minor grooves of these hmU-containing DNA but not to thymine-containing DNA. Data from electrophoretic mobility shift assays suggest that the inherent, localized flexibility of hmU-DNA, which is sequence-specific, is responsible for its discriminative binding. We discuss here, from the NMR-derived structural point of view, how differential DNA flexibility can contribute to specific binding of TF1 to hmU-DNA.

Secondary structure of T4 gene 33 protein. Fourier transform infrared and circular dichroic spectroscopic studies.

The secondary structure of bacteriophage T4 gene 33 protein (gp33) has been quantitatively examined by using Fourier transform infrared (FT-IR) and circular dichroism (CD) spectroscopy. Resolution enhancement techniques, including Fourier deconvolution and derivative spectroscopy were used to quantitate the spectral information from the amide I bands. The relative areas of these component bands indicate 21% alpha-helix, 25% beta-sheet, 34% turn, 12% random coil and 8% other undefined structures in gp33. An analysis of the CD spectrum of gp33 at the same pH and temperature revealed 19% alpha-helix, 25% beta-sheet, 13% turn and 43% random coil structures. The possible reasons for the discrepancies in estimates of the contributions to the secondary structure from turns and random coils are discussed.

Nuclear magnetic resonance-based model of a TF1/HmU-DNA complex.

Transcription factor 1 (TF1), a type II DNA-binding protein encoded by the Bacillus subtilis bacteriophage SPO1, has the capacity for sequence-selective DNA binding and a preference for 5-hydroxymethyl-2'-deoxyuridine (HmU)-containing DNA. In NMR studies of the TF1/HmU-DNA complex, intermolecular NOEs indicate that the flexible beta-ribbon and C-terminal alpha-helix are involved in the DNA-binding site of TF1, placing it in the beta-sheet category of DNA-binding proteins proposed to bind by wrapping two beta-ribbon "arms" around the DNA. Intermolecular and intramolecular NOEs were used to generate an energy-minimized model of the protein-DNA complex in which both DNA bending and protein structure changes are evident.

Structure of the Bacillus subtilis phage SPO1-encoded type II DNA-binding protein TF1 in solution.

The solution structure of a type II DNA-binding protein, the bacteriophage SPO1-encoded transcription factor 1 (TF1), was determined using NMR spectroscopy. Selective 2H-labeling, 13C-labeling and isotopic heterodimers were used to distinguish contacts between and within monomers of the dimeric protein. A total of 1914 distance and dihedral angle constraints derived from NMR experiments were used in structure calculations using restrained molecular dynamics and simulated annealing protocols. The ensemble of 30 calculated structures has a root-mean-square deviation (r.m.s.d.) of 0.9 A, about the average structure for the backbone atoms, and 1.2 A for all heavy-atoms of the dimeric core (helices 1 and 2) and the beta-sheets. A severe helix distortion at residues 92-93 in the middle of helix 3 is associated with r.m.s.d. of approximately 1.5 A for the helix 3 backbone. Deviations of approximately 5 A or larger are noted for the very flexible beta-ribbon arms that constitute part of a proposed DNA-binding region. A structural model of TF1 has been calculated based on the previously reported crystal structure of the homologous HU protein and this model was used as the starting structure for calculations. A comparison between the calculated average solution structure of TF1 and a solution structure of HU indicates a similarity in the dimeric core (excluding the nine amino acid residue tail) with pairwise deviations of 2 to 3 A. The largest deviations between the average structure and the HU solution structure were found in the beta-ribbon arms, as expected. A 4 A deviation is found at residue 15 of TF1 which is in a loop connecting two helical segments; it has been reported that substitution of Glu15 by Gly increases the thermostability of TF1. The homology between TF1 and other proteins of this family leads us to anticipate similar tertiary structures.

1H NMR studies of the 5-(hydroxymethyl)-2'-deoxyuridine containing TF1 binding site.

The pyrimidine base 5-(hydroxymethyl)-2'-deoxyuridine (HmU) is a common nucleotide in SPO1 phage DNA. Numerous transcriptional proteins bind HmU-containing DNA preferentially implicating a regulatory function of HmU. We have investigated the conformation and dynamics of d-(5'-CHmUCHmUACACGHmUGHmUAGAG-OH-3')2 (HmU-DNA). This oligonucleotide mimics the consensus sequence of Transcription Factor 1 (TF1). The HmU-DNA was compared to the thymine-containing oligonucleotide. NOESY and DQF COSY spectroscopy provided resonance assignments of nonexchangeable and exchangeable protons, intranucleotide, internucleotide and intrastrand proton-proton distances, and dihedral angle constraints. Methylene protons of the hydroxymethyl group are nonequivalent protons and the hydroxymethyl group is not freely rotating. The hydroxymethyl group adopts a specific orientation with the OH group oriented on the 3' side of the plane of the base. Analysis of imino proton resonances and NOEs indicates additional end base pair fraying and a temperature-induced transition to a conformation in which the internal HmU-A base pairs are disrupted or have reduced lifetimes. Orientation of the hydroxymethyl group indicates the presence of internucleotide intrastrand hydrogen bonding between the HmU12C5 hydroxyl group and A13. All sugars in both DNAs show a C2'endo conformation (typical of B-DNA).

Multidimensional NMR spectroscopy of DNA-binding proteins: structure and function of a transcription factor.

The solution structure of a type II DNA-binding protein (DBPII), transcription factor 1 (TF1), has been determined using NMR spectroscopy. A multidimensional, heteronuclear strategy was employed to overcome assignment ambiguities due to resonance overlap and broadened crosspeaks. This approach involved the use of selectively deuteriated, 13C- and 15N-labeled samples and 'isotopic heterodimers' to distinguish between intra- and intermonomeric NOEs. A comparison with the crystal structure and NMR analysis of the E. coli HU protein suggests that other homologous proteins in this family will possess similar tertiary structures. This NMR strategy is applicable to the study of other proteins and their biomolecular complexes.

Structural characterization of d(CAACCCGTTG) and d(CAACGGGTTG) mini-hairpin loops by heteronuclear NMR: the effects of purines versus pyrimidines in DNA hairpins.

The DNA decamers, d(CAACCCGTTG) and d(CAACGGGTTG) were studied in solution by proton and heteronuclear NMR. Under appropriate conditions of pH, temperature, salt concentration and DNA concentration, both decamers form hairpin conformations with similar stabilities [Avizonis and Kearns (1995) Biopolymers, 35, 187-200]. Both decamers adopt mini-hairpin loops, where the first and last four nucleotides are involved in Watson-Crick hydrogen bonding and the central two nucleotides, CC or GG respectively, form the loop. Through the use of proton-proton, proton-phosphorus and natural abundance proton-carbon NMR experiments, backbone torsion angles (beta, gamma and epsilon), sugar puckers and interproton distances were measured. The nucleotides forming the loops of these decamers were found to stack upon one another in an L1 type of loop conformation. Both show gamma tr and unusual beta torsion angles in the loop-closing nucleotide G7, as expected for mini-hairpin loop formation. Our results indicate that the beta and epsilon torsion angles of the fifth and sixth nucleotides that form the loop and the loop-closing nucleotide G7 are not in the standard trans conformation as found in B-DNA. Although the loop structures calculated from NMR-derived constraints are not well defined, the stacking of the bases in the two different hairpins is different. This difference in the base stacking of the loop may provide an explanation as to why the cytosine-containing hairpin is thermodynamically more stable than the guanine-containing hairpin.

Kinetic and thermodynamic characterization of DNA duplex-hairpin interconversion for two DNA decamers: d(CAACGGGTTG) and d(CAACCCGTTG).

The duplex-hairpin interconversion of two DNA decamers, d(CAACGGGTTG) and d(CAACCCGTTG), has been characterized thermodynamically and kinetically by using uv-melting and nmr relaxation methods. Separately, each decamer shows slow exchange between hairpin and duplex conformations. The hairpin conformations have melting points of 47 and 50 degrees C, respectively, and exhibit similar thermodynamic stabilities. The enthalpies of duplex formation, measured by nmr, were found to be very similar (delta HDH = 26 +/- 3 kcal/mole) for both decamers at low salt concentrations (< 50 mM NaCl). However, as the salt concentration was increased the behavior of delta HDH and kinetics is significantly different for each decamer. The d(CAACGGGTTG) decamer forms a duplex containing two central G.G mismatches at high salt and DNA concentration. Based upon the measurement of high interconversion activation energies and a decrease in hairpin formation rate with increasing salt, the interconversion between hairpin and duplex was concluded to proceed by complete strand dissociation. In contrast, the d(CAACCCGTTG) decamer was determined to form a duplex with two centrally located C.C mismatches at pH values less than 6.2, consistent with the formation of a hemiprotonated C+.C mismatch. At pH values greater than 6.4, the hairpin-duplex equilibrium is almost completely shifted toward the hairpin conformation at DNA concentrations of 0.5-7.0 mM and salt concentrations of 10-100 mM. The interconversion of duplex and hairpin conformations was ascertained by means of both kinetic and thermodynamic measurements to proceed by a slightly different mechanism than its complementary decamer. Although the interconversion proceeds by complete strand separation as suggested by high duplex-hairpin interconversion activation enthalpies, the increasing hairpin formation rate with increasing ionic strength as well as the delta HDH dependence on salt indicate that an intermediate internally bulged duplex (no C+.C formation) is stabilized by increasing ionic strength. These data support an interconversion mechanism where an intermediate internally bulged duplex may be the rate limiting step before strand separation.

Proton and nitrogen NMR sequence-specific assignments and secondary structure determination of the Bacillus subtilis SPO1-encoded transcription factor 1.

Sequence-specific 1H and 15N NMR1 assignments are reported for the transcription factor 1 (TF1), a 22-kDa type II DNA-binding protein (DBPII) that consists of two 99-residue monomers. An assignment strategy is employed that uses six complementary selectively deuterium-labeled TF1 variants and an uniformly 15N-labeled TF1 variant. Two-dimensional and three-dimensional homonuclear and heteronuclear NMR correlated spectra are analyzed and yield nearly complete assignments for the 1H and 15N resonances. Discrete protein secondary structure domains are also defined; in each monomer, three alpha-helices, an antiparallel beta-sheet, and an antiparallel beta-ribbon are identified. Analyses of two dimers formed from two distinct selectively deuteriated monomers serve to identify a number of interproton contacts as either intermonomeric or intramonomeric. An analysis of amide proton exchange reveals that the carboxy-terminal alpha-helix is less stable than the other two alpha-helices in each monomer. A previously proposed working structural model of the TF1 dimer [Geiduschek et al. (1990) J. Struct. Biol. 104, 84-90], based on the crystal structure of a highly homologous DBPII, the Bacillus stearothermophilus-encoded HU protein, is generally supported by our results. Several departures from this model, however, are noted. Most notably, the carboxy-terminal tail of TF1 adopts an alpha-helical conformation with a backbone distortion at Lys93.

Formation of multiple complexes between actinomycin D and a DNA hairpin: structural characterization by multinuclear NMR.

The solution conformations of a DNA oligomer and its complexes with the anticancer drug actinomycin D (ActD) were characterized using homo- and heteronuclear NMR techniques. Previous high-resolution NMR investigations of ActD-DNA complexes employed symmetric double-stranded DNA oligomers, yielding two identical symmetry-related complexes. In order to understand the important effects that neighboring base pairs and/or unusual nucleic acid structures may have on ActD binding specificity and orientation, we chose to study the oligonucleotide d(TCGCGTTTTCGCGA), which adopts a hairpin structure in solution. NOE cross-peak intensities were used to generate distance constraints for molecular dynamics simulations and structure determinations of the free oligonucleotide and for both complexes. A total of 86 intermolecular NOEs were identified for each complex, 27 of which involve exchangeable protons. These intermolecular NOEs along with changes in the phosphorus chemical shifts were used to determine the drug binding site on the DNA. As expected, ActD intercalated exclusively at the single d(GC) step in the DNA hairpin. Interestingly, although the two complexes, which differ by the orientation with which the asymmetric drug chromophore intercalates the DNA, were not formed in equal concentrations, their conformations are very similar. The RMS difference of the DNA hairpin in the two complexes is only 1.10 A. The structures of the minor groove binding pentapeptide rings are not affected by any of the changes in the normal double-helical structure imposed by the hairpin loop. The total pairwise RMS difference over all atoms for the four peptides (two per complex) in the calculated structures is 0.72 A. Conversely, the structure of the hairpin loop is not appreciably changed upon binding--the RMS difference between the free DNA loop region and the loop region in the two complexes is 1.68 A and only 0.43 A between the two complexes. Our data also support a possible conformation of the d(T)4 loop that does not possess a thymine-thymine "wobble" base pair.

A 1H-NMR study of the transcription factor 1 from Bacillus subtilis phage SPO1 by selective 2H-labeling. Complete assignment and structural analysis of the aromatic resonances for a 22-kDa homodimer.

1H-NMR experiments have been performed on transcription factor 1 (TF1) encoded by Bacillus subtilis phage SPO1. To study this 22-kDa homodimeric DNA-binding protein, a selective 2H-labeling strategy has been employed. Complete sequence-specific assignments of all the resonances from the five aromatic residues were determined by a modified standard sequential-assignment procedure. The reduced contribution of spin diffusion upon the long-mixing-time nuclear-Overhauser-enhancement spectroscopy for the selectively 2H-labeled variants, as opposed to the fully 1H-containing protein, has allowed for the identification of the spin systems and of the long-range dipolar contacts between Phe28 and Phe47 protons in the protein core and between Phe61 and Phe97 protons. The latter suggests an interaction between the proposed beta-ribbon DNA-binding arm and the carboxy terminus of the paired monomer. A previously proposed TF1 structural model [Geiduschek, E. P., Schneider, G. J. & Sayre, M. H. (1990) J. Struct. Biol. 104, 84-90)] has been modified using constrained-energy-minimization calculations incorporating the experimentally determined set of aromatic-to-aromatic contacts. This new model has been analyzed with regard to the relative mobility and the relative solvent accessibility of the aromatic residues which have been measured by the nonselective T1 relaxation times of the aromatic resonances for the fully 1H-containing protein and the relaxation time enhancements upon selective 2H-labeling, respectively.

The formation of A-DNA in NaDNA films is suppressed by netropsin.

Oriented films of NaDNA complexed with netropsin were studied with deuterium nuclear magnetic resonance (2H NMR), X-ray diffraction and ultraviolet (UV) linear dichroism to obtain information about the influence of netropsin on the structural arrangement of the DNA bases and on the B-A transition. The results of these studies clearly demonstrate a strong suppression of the formation of A-DNA at relative humidities (RHs) down to about 50%. The suppression was complete in the NaDNA-netropsin complex studied with 2H NMR which had a netropsin input ratio, r, of 0.22 drug/base pair. The sample used for UV linear dichroism had a similar input ratio while the X-ray diffraction samples had input ratios between 0.033 and 0.39 drug/base pair. Together, the results of these studies are in agreement with previous infrared (IR) linear dichroism studies of the conformation of the sugar-phosphate backbone in NaDNA-netropsin complexes, which showed that the B-A transition is suppressed for r-values down to approximately 0.1 drug/base pair (Fritzsche, H., Rupprecht, A. and Richter, M., Nucleic Acids Res. 12 (1984) 9165-9177).

Deuterium relaxation and internal motion in solid Li-DNA.

As part of an effort to explore the nature of the internal motion in solid polynucleotides, the spectral densities of motion J1(omega 0) and J2(2 omega 0) have been measured for oriented, partially hydrated samples of calf thymus Li-DNA deuterated in the guanine and adenine 8-positions. Both spectral densities increase with increasing hydration level, J1 is found to be 2-5 times larger than J2, and their frequency dependence appears to be omega-1 and omega-3/2, respectively. The large values of the ratio J1/J2 rule out any in-plane torsional motion as the dominant relaxation mechanism in these samples, but a drop in this ratio at high hydration levels (G13 H2O/nucleotide) may indicate increasing contributions from such torsional motion. Although a satisfactory fit to a particular motional model has yet to be achieved, our findings show that the librational motion of the C8-D bond at or below a hydration level of 10 H2O/nucleotide is approximately uniaxial, with correlation times for the motion in the range 0.2-3.0 microseconds.

Reduced DNA flexibility in complexes with a type II DNA binding protein.

We studied internal molecular motions in Bacillus subtilis phage SPO1 DNA using the time-resolved fluorescence polarization anisotropy (FPA) of intercalated ethidium. The torsional flexibility of this (hydroxymethyl)uracil-containing DNA is very similar to that of naturally occurring thymine-containing DNAs, as judged from fits of the time-resolved FPA decay to an elastic DNA model. Binding of transcription factor 1 (TF1), a type II procaryotic DNA binding protein encoded by the phage SPO1, enhances the FPA, indicating a substantial decrease in the average DNA torsional flexibility in the DNA-TF1 complex. The FPA increase is correlated with a reduced ethidium binding affinity. The effects can be noticed at TF1 binding ratios less than 1 TF1 dimer/500 DNA base pairs, and the measured torsional rigidity at high TF1 binding ratios (1 TF1 dimer/15-20 DNA base pairs) is about 7 times greater than in the absence of TF1. On the basis of a discussion of various mechanisms for the observed effect we argue that it is due to protein-induced DNA bending at low binding densities although other explanations are also possible. This interpretation might have implications for understanding the biological function of TF1.

A fluorescence study of the binding of Hoechst 33258 and DAPI to halogenated DNAs.

We have studied the time-resolved and the steady-state fluorescence of the DNA groove binders 4',6-diamidino-2-phenylindole (DAPI) and Hoechst 33258 with the double stranded DNAs poly(dA-dU) and poly(dI-dC) and their halogenated analogs, poly(dA-I5dU) and poly(dI-Br5dC). These studies were prompted by earlier observations that steady-state fluorescence of Hoechst 33258 is quenched on binding to halogenated DNAs (presumably due to an intermolecular heavy atom effect involving the halogen atom in the major groove), and recent studies which clearly point to a binding-site in the minor groove of DNA. Measurements of the time resolved fluorescence decay demonstrate that the fluorescence of Hoechst 33258 is quenched on binding to the halogenated DNAs, in agreement with previous observations. However, quenching studies carried out using the free halogenated bases IdUrd and BrdCyd in solution yielded bimolecular rate constants more than one order of magnitude larger than those expected for an intermolecular heavy atom effect. Moreover, the quenching of the Hoechst 33258 fluorescence was accompanied by an accelerated photochemical destruction of Hoechst 33258. We therefore conclude that the fluorescence quenching observed with halogenated DNAs is probably due to a photochemical reaction involving Hoechst 33258, rather than direct contact of Hoechst 33258 with the halogen substituents in the major groove of the DNA. The fluorescence decay measurements however, do provide clear evidence for at least two different modes of binding. Taking into account the alternating sequences used in this study and the possibility of two different conformations for bound dye, at least four different modes of binding are plausible. Our present data do not allow us to distinguish between these alternatives. The time-resolved fluorescence decays and fluorescence quantum yields of DAPI are not affected by the presence of the heavy atom substituents in the DNA major groove. Based on this observation and earlier reports that DAPI binds in one of the DNA grooves, we conclude that the high affinity sites for DAPI on DNA are located in the minor groove.

Interaction of water with oriented DNA in the A- and B-form conformations.

High resolution 2H nuclear magnetic resonance (NMR) was used to investigate the interaction of D2O with solid samples of uniaxially oriented Li-DNA (B-form DNA) and Na-DNA (A- and B-form DNA). At low levels of hydration, 0 approximately 4 D2O/nucleotide, the 2H spectra shows a very weak (due to short T2) broad single resonance, suggestive of unrestricted rotational diffusion of the water. At approximately 5 or more D2O/nucleotide, the Li-DNA (B-form) spectra suddenly exhibit a large doublet splitting, characteristic of partially ordered water. With increasing hydration, the general trend is a decrease of this splitting. From our analysis we show that the DNA water structure reorganizes as the DNA is progressively hydrated. The D2O interaction with Na-DNA is rather different than with Li-DNA. Below 10 D2O/nucleotide Na-DNA is normally expected to be in the A-form, and a small, or negligible splitting is observed. In the range 9-19 D2O/nucleotide, the splitting increases with increasing hydration. Above approximately 20 D2O/nucleotide Na-DNA converts entirely to the B-form and the D2O splittings are then similar to those found in Li-DNA. We show that the complex Na-DNA results obtained in the range 0-20 D2O/nucleotide are caused by a mixture of A- and B-DNA in those samples.

A type II DNA-binding protein genetically engineered for fluorescence spectroscopy: the "arm" of transcription factor 1 binds in the DNA grooves.

We examined the fluorescence properties of a mutant TF1 protein (transcription factor 1; a member of the type II class of DNA-binding proteins, DBPII) containing tryptophan in place of phenylalanine (TF1-W61) at position 61 in the "arms" of the protein dimer. The time-resolved fluorescence (excited at 295 nm) of Trp61 decays as a double exponential with lifetimes and amplitudes that are comparable to those found in other tryptophan-containing proteins and peptides, and the time-resolved fluorescence polarization decay indicates that the tryptophan residue possesses considerable internal flexibility, in agreement with crystal studies of the homologous HU protein. The tryptophan emission is quenched when TF1-W61 binds to DNA, and equilibrium studies based on fluorescence show that the nonspecific binding affinity of the TF1-W61 mutant to DNA is similar to that of wild-type TF1. Comparison of the time-resolved fluorescence decay and steady-state fluorescence intensity reveals at least two general classes of Trp61 in the DNA complexes. One class of tryptophans is partially quenched, and the extent of quenching in the complexes with various natural DNAs and synthetic double-stranded polynucleotides correlates with the spectral overlap between tryptophan emission and DNA absorption, indicating that through-space excitation energy transfer contributes to the observed quenching. Comparisons between experimentally determined energy transfer rates and model calculations suggest that the Trp61 is located in one of the DNA grooves at a distance of less than 7.5 A from the DNA helix axis. The second class of Trp61 is "totally" quenched, and we attribute this to tryptophan residues that are in direct contact with the DNA bases.(ABSTRACT TRUNCATED AT 250 WORDS)