Conformational Dynamics and the Binding of Specific and Nonspecific DNA by the Autoinhibited Transcription Factor Ets-1 (original) (raw)
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A single-stranded DNA binding protein (SSB), labeled with a fluorophore, interacts with singlestranded DNA (ssDNA), giving a 6-fold increase in fluorescence. The labeled protein is the adduct of the G26C mutant of the homotetrameric SSB from Escherichia coli and a diethylaminocoumarin {N-[2-(iodoacetamido)ethyl]-7-diethylaminocoumarin-3-carboxamide}. This adduct can be used to assay production of ssDNA during separation of double-stranded DNA by helicases. To use this probe effectively, as well as to investigate the interaction between ssDNA and SSB, the fluorescent SSB has been used to develop the kinetic mechanism by which the protein and ssDNA associate and dissociate. Under conditions where ∼70 base lengths of ssDNA wrap around the tetramer, initial association is relatively simple and rapid, possibly diffusion-controlled. The kinetics are similar for a 70-base length of ssDNA, which binds one tetramer, and poly(dT), which could bind several. Under some conditions (high SSB and/or low ionic strength), a second tetramer binds to each 70-base length, but at a rate 2 orders of magnitude slower than the rate of binding of the first tetramer. Dissociation kinetics are complex and greatly accelerated by the presence of free wild-type SSB. The main route of dissociation of the fluorescent SSB 3 ssDNA complex is via association first with an additional SSB and then dissociation. Comparison of binding data with different lengths of ssDNA gave no evidence of cooperativity between tetramers. Analytical ultracentrifugation was used to determine the dissociation constant for labeled SSB 2 3 dT 70 to be 1.1 μM at a high ionic strength (200 mM NaCl). Shorter lengths of ssDNA were tested for binding: only when the length is reduced to 20 bases is the affinity significantly reduced.
31P NMR analysis of the DNA conformation induced by protein binding : SRY/DNA complexes
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Complexes of the HMG box protein SRY with two duplexes of 8 and 14 base pairs have been studied by 31 P NMR and complete assignment of all phosphorus signals of the bound DNA duplexes are presented. While for the free DNA, all 31 P signals display limited spectral dispersion (, 0.8 p.p.m.) for the bound duplexes, 31 P resonances are spread over 2 p.p.m. Based on the previously published 3D structure of hSRY-HMG, with the 8 mer it is demonstrated that the upfield shifted resonances correspond to the site of partial intercalation of an isoleucine side chain into the DNA. Moreover, the observation of significant difference in linewidths between the two duplexes allows to estimate lifetime of the complexes from 31 P± 31 P 2D exchange experiments.
Correction of the NMR structure of the ETS1/DNA complex
Journal of biomolecular NMR, 1997
The ETS family of transcription factors consists of a group of proteins that share a highly conserved 85 amino acid DNA-binding domain (DBD). This family recognizes a consensus sequence rich in purine bases with a central GGAA motif. A comparison of the published three-dimensional structures of the DBD/DNA complexes of ETS1 by NMR [Werner et al. (1995) Cell, 83, 761-771] and the related Pu.1 by X-ray crystallography [Kodandapani et al. (1996) Nature, 380, 456-460] reveals an apparent discrepancy in which the protein domains bind with opposite polarity to their target sequences. This surprising and highly unlikely result prompted us to reexamine our NMR structure. Additional NMR experiments now reveal an error in the original interpretation of the spectra defining the orientation of the ETS1-DBD on DNA. It was originally reported that the ETS1-DBD bound to DNA with a bipartite motif involving major groove recognition via a helix-turn-helix element and minor groove recognition via pro...
Synthesis of isotope labeled oligonucleotides and their use in an NMR study of a protein-DNA complex
Nucleic Acids Research, 1992
The synthesis of an oligonucleotide labeled with 13C at the thymine methyls and 15N at the exocyclic amino groups of the cytosines is described. 13CH31 and 15NH40H were used as sources of the labels. The labeled oligonucleotide was characterized by several NMR techniques. The duplex possesses a labeled functional group in the major groove at every base pair which makes it a very suitable probe for the study of sequence-specific protein-DNA interaction. The labeled thymine methyl group facilitates the detection of hydrophobic contacts with aliphatic side-chains of proteins. This is demonstrated in an NMR study of a complex between the glucocorticoid receptor DNAbinding domain and the labeled oligomer, which revealed a hydrophobic contact between a thymine methyl group and the methyl groups of a valine residue. There are indications for small differences between the solution structure the X-ray structure of the complex.
31 P NMR Investigation of Backbone Dynamics in DNA Binding Sites †
The Journal of Physical Chemistry B, 2009
The backbone conformation of DNA plays an important role in the indirect readout mechanisms for protein-DNA recognition events. Thus, investigating the backbone dynamics of each step in DNA binding sequences provides useful information necessary for the characterization of these interactions. Here we use 31 P Dynamic NMR to characterize the backbone conformation and dynamics in the Dickerson Dodecamer, a sequence containing the EcoRI binding site, and confirm solid-state 2 H-NMR results showing that the C3pG4 and C9pG10 steps experience unique dynamics and that these dynamics are quenched upon cytosine methylation. In addition, we show that cytosine methylation affects the conformation and dynamics of neighboring nucleotide steps but this effect is localized to only near neighbors and base pairing partners. Lastly, we have been able to characterize the %BII in each backbone step and illustrate that the C3pG4 and C9pG10 favor the non-canonical BII conformation, even at low temperatures. Our results demonstrate that 31 P Dynamic NMR provides a robust and efficient method for characterizing the backbone dynamics in DNA. This allows simple, rapid determination of sequence-dependent dynamical information, providing a useful method for studying trends in protein-DNA recognition events. . Supporting Information Available Two tables containing the %BII as a function of temperature for the C3 and C9 methylated samples and a figure containing the barrier with respect to backbone step number with an extended chemical shift range. This material is available free of charge via the internet at
The Structural and Dynamic Basis of Ets1 DNA Binding Autoinhibition
Journal of Biological Chemistry, 2005
The transcription factor Ets-1 is regulated by the allosteric coupling of DNA binding with the unfolding of an ␣-helix (HI-1) within an autoinhibitory module. To understand the structural and dynamic basis for this autoinhibition, we have used NMR spectroscopy to characterize Ets-1⌬N301, a partially inhibited fragment of Ets-1. The NMR-derived Ets-1⌬N301 structure reveals that the autoinhibitory module is formed predominantly by the hydrophobic packing of helices from the N-terminal (HI-1, HI-2) and C-terminal (H4, H5) inhibitory sequences, along with H1 of the intervening DNA binding ETS domain. The intramolecular interactions made by HI-1 in Ets-1⌬N301 are similar to the intermolecular contacts observed in the crystal structure of an Ets-1⌬N300 dimer, confirming that the latter represents a domain-swapped species. 15 N relaxation studies demonstrate that the backbone of the N-terminal inhibitory sequence is mobile on the nanosecond-picosecond and millisecond-microsecond time scales. Furthermore, hydrogen exchange measurements reveal that amide protons in helices HI-1 and HI-2 exchange with water at rates only ϳ15and ϳ75-fold slower, respectively, than predicted for an unfolded polypeptide. These findings indicate that inhibitory helices are only marginally stable even in the absence of DNA. The energetic coupling of DNA binding with the facile unfolding of the labile HI-1 provides a mechanism for modulating Ets-1 DNA binding activity via protein partnerships, post-translational modifications, or mutations. Ets-1 autoinhibition illustrates how conformational equilibria within structural domains can regulate macromolecular interactions. The abbreviations used are: HTH, helix-turn-helix; CD, circular dichroism; HSQC, heteronuclear single quantum correlation; HX, hydrogen exchange; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser effect spectroscopy; pH*, the observed pH meter reading without correction for isotope effects; RDC, residual dipolar coupling; r.m.s.d., root mean-square deviation; PDB, Protein Data Bank.
Biochemistry, 2009
Fourier transform infrared (FTIR) spectroscopy and a library of FTIR marker bands have been used to examine the structure and relative flexibilities conferred by different flanking sequences on the EcoRI binding site. This approach allowed us to examine unique peaks and subtle changes in the spectra of d(AAAGAATTCTTT) 2 , d(TTCGAATTCGAA) 2 , and d(CGCGAATTCGCG) 2 , and thereby identify local changes in base-pairing, base-stacking, backbone conformation, glycosidic bond rotation and sugar puckering in the studied sequences. The changes in flanking sequences induce differences in the sugar puckers, glycosidic bond rotation and backbone conformations. Varying levels of local flexibility are observed within the sequences in agreement with previous biological activity assays. The results also provide supporting evidence for the presence of a splay in the G 4 -C 9 base pair of the EcoRI binding site and a potential pocket of flexibility at the G 4 cleavage site that have been proposed in the literature. In sum, we have demonstrated that FTIR is a powerful methodology for studying the effect of flanking sequences on DNA structure and flexibility, for it can provide information about the local structure of the nucleic acid and the overall relative flexibilities conferred by different flanking sequences.
Structural features of ϕ29 single-stranded DNA-binding protein
Journal of Biological …, 1997
The single-stranded DNA-binding protein (SSB) of Bacillus subtilis phage 29 is absolutely required for viral DNA replication in vivo. About ϳ95% of the intrinsic tyrosine fluorescence of 29 SSB is quenched upon binding to ssDNA, making tyrosine residues strong candidates to be directly involved in complex formation with ssDNA. Thus, we have studied the spectroscopic properties of the 29 SSB tyrosines (Tyr-50, Tyr-57, and Tyr-76) using steady-state and time-resolved fluorescence measurements. 29 SSB tyrosines do not seem to be highly restricted by strong interactions with neighbor residues, as suggested by (i) the high value of the average quantum yield of the 29 SSB fluorescence emission (⌽ F ؍ 0.067 ؎ 0.010), (ii) the fast motions of the tyrosine side chains (short ؍ 0.14 ؎ 0.06 ns), and (iii) the lack of tyrosinate emission at neutral pH. Stern-Volmer analysis of the quenching by acrylamide and I ؊ indicates that 29 SSB tyrosines are surrounded by a negatively charged environment and located in a relatively exposed protein domain, accessible to the solvent and, likely, to ssDNA. Changes in the intrinsic fluorescence upon ssDNA binding allowed us to determine that temperature has an opposite effect on the thermodynamic parameters K (intrinsic binding constant) and (cooperativity) defining 29 SSB-poly(dT) interaction, the effective DNA binding constant, K eff ؍ K, being largely independent of temperature. Altogether, the fluorescent properties of 29 SSB tyrosines are consistent with a direct participation in complex formation with ssDNA.