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T Härd

Publications and source records attributed to T Härd.

At least 19 recordsLinked to original sources
PubMed1998

Thermodynamic characterization of non-sequence-specific DNA-binding by the Sso7d protein from Sulfolobus solfataricus.

We used isothermal titration calorimetry and fluorescence spectroscopy to investigate the thermodynamics of non-sequence-specific DNA-binding by the Sso7d protein from the archaeon Sulfolobus solfataricus. We report the Sso7d-poly(dGdC) binding thermodynamics as a function of buffer composition (Tris-HCl or phosphate), temperature (15 to 45 degrees C), pH (7.1 to 8.0), osmotic stress and solvent (H2O/2H2O), and compare it to poly (dAdT) binding; and we have previously also reported the salt concentration dependence. Binding isotherms can be represented by the McGhee-von Hippel model for non-cooperative binding, with a binding site size of four to five DNA base-pairs and binding free energies in the range DeltaG degrees approximately -7 to DeltaG degrees approximately -10 kcal mol-1, depending on experimental conditions. The non-specific nature of the binding is reflected in similar thermodynamics for binding to poly(dAdT) and poly(dGdC). The native lysine methylation of Sso7d has only minor effects on the binding thermodynamics. Sso7d binding to poly(dGdC) is endothermic at 25 degrees C with a binding enthalpy DeltaH degrees approximately 10 kcal mol-1 in both phosphate and Tris-HCl buffers at pH 7.6, indicating that DeltaH degrees does not include large contributions from coupled buffer ionization equilibria at this pH. The binding enthalpy is temperature dependent with a measured heat capacity change DeltaCp degrees=-0.25(+/-0.01) kcal mol-1 K-1 and extrapolations of thermodynamic data indicate that the complex is heat stable with exothermic binding close to the growth temperature (75 to 80 degreesC) of S. solfataricus. Addition of neutral solutes (osmotic stress) has minor effects on DeltaG degrees and the exchange of H2O for 2H2O has only a small effect on DeltaH degrees, consistent with the inference that complex formation is not accompanied by net changes in surface hydration. Thus, other mechanisms for the heat capacity change must be found. The observed thermodynamics is discussed in relation to the nature of non-sequence-specific DNA-binding by proteins.

Archaeal Proteins↗
PubMed1998

Architecture of nonspecific protein-DNA interactions in the Sso7d-DNA complex.

Many biochemical processes, including DNA packing, maintenance and control, rely on non-sequence specific protein-DNA interactions. Nonspecific DNA-binding proteins have evolved to tolerate a wide range of DNA sequences, yet bind with a respectable affinity. The nonspecific binding requirement is in contrast to that imposed on, for example, transcription factors and implies a different structural basis for the biomolecular recognition process. To address this issue, and the mechanism for archaeal DNA packing, we determined the structure of the Sso7d protein from Sulfolobus solfataricus in complex with DNA. Sso7d binds DNA by placing a triple-stranded beta-sheet across the DNA minor groove. The protein is anchored in this position by the insertion of hydrogen bond-donating side chains into the groove and additionally stabilized by electrostatic and non-polar interactions with the DNA backbone. This structure explains how strong binding can be achieved independent of DNA sequence. Sso7d binding also distorts the DNA conformation and introduces significant unwinding of the helix. This effect suggests a mechanism for DNA packing in Sulfolobus based on negative DNA supercoiling.

Amino Acid Sequence↗
PubMed1997

Structure and dynamics of the glucocorticoid receptor DNA-binding domain: comparison of wild type and a mutant with altered specificity.

Nuclear magnetic resonance was used to compare parameters reflecting solution structure and dynamics of the glucocorticoid receptor DNA-binding domain (GRDBD), which binds specifically to a GRE binding site on DNA, and a triple mutant (GRDBDEGA), which binds to an ERE site. The studies were prompted by an earlier observation that the cooperativity for dimeric DNA-binding is 10 times higher for the GRDBDEGA-ERE association than for the GRDBD-GRE association (Lundbäck et al., 1994). The higher binding cooperativity of the mutant was unexpected since the triple mutation (G458E, S459G, and V462A) is made in the recognition helix and distant from the dimerization surface which is formed by residues in the fragment A477-N491. Sequential and long-range NOE connectivities and measured 3JHNHalpha coupling constants indicate that the overall structures of the two proteins are very similar, possibly with a less well-defined structure of the fragment K486-N491 in GRDBDEGA. However, chemical shift changes, line broadening, and increased amide proton exchange rates are observed for several residues at, or close to, the dimerization surface of the mutant. These observations are interpreted as a lower stability and/or several slowly interconverting folded conformations of this region of GRDBDEGA. The effects are likely to be due to the loss of a hydrogen bond which links S459 to the dimerization region in GRDBD. Different mechanisms for the increased binding cooperativity of the mutant are discussed, and it is noted that the properties of the GRDBDEGA dimerization region are reminiscent of those reported for the estrogen receptor DBD, which also binds to an ERE site.

Amino Acid Sequence↗
PubMed1996

Sequence-specific DNA-binding dominated by dehydration.

Fluorescence spectroscopy and isothermal titration calorimetry were used to study the thermodynamics of binding of the glucocorticoid receptor DNA-binding domain to four different, but similar, DNA-binding sites. The binding sites are two naturally occurring sites that differ in the composition of one base pair, i.e., an A-T to G-C mutation, and two sites containing chemical intermediates of these base pairs. The calorimetrically determined heat capacity change (Delta C(p)o(obs)) for glucocorticoid receptor DNA-binding domain binding agrees with that calculated for dehydration of solvent-accessible surface areas. A dominating effect of dehydration or solvent reorganization on the thermodynamics is also consistent with an observed linear relationship between observed enthalpy change (Delta Ho(obs)) and observed entropy change (Delta So(obs)) with a slope close to the experimental temperature. Comparisons with structural data allow us to rationalize individual differences between Delta Ho(obs) (and Delta So(obs)) for the four complexes. For instance, we find that the removal of a methyl group at the DNA-protein interface is enthalpically favorable but entropically unfavorable, which is consistent with a replacement by an ordered water molecule.

Animals↗
PubMed1995

Structural characterization of a short peptide fragment that mediates estrogen-receptor dimerization.

In a recent study it is suggested that a short peptide fragment within the estrogen receptor ligand-binding and dimerization domain might act as a constitutively active dimerization motif [Lees, J. A., Fawell, S. E., White, R. & Parker, M. G. (1990) Mol. Cell. Biol. 10, 5529-5531]. We used NMR and CD spectroscopies to characterize the structure and biophysical properties of a synthetic peptide comprising residues Thr500-His528 of the mouse estrogen receptor, including the putative dimerization motif. We found that residues Leu501-Asn523 form a nascent helix in water solution, whereas the C-terminal (Lys524-His528) has no propensity for alpha-helical conformation. We found no evidence for a strong homodimerization activity of the peptide. However, we observed concentration-dependent NMR chemical shifts of several residues that would be located on the same face of an alpha-helix. This observation suggests a weak, but specific dimerization/oligomerization of the peptide at millimolar concentrations.

Amino Acid Sequence↗
PubMed1995

DNA-binding surface of the Sso7d protein from Sulfolobus solfataricus.

We have used nuclear magnetic resonance (n.m.r.) spectroscopy to identify the DNA-binding surface of the abundant, small and basic protein Sso7d from the hyperthermophilic archaebacterium Sulfolobus solfataricus. The Sso7d protein was previously found to bind strongly to double-stranded DNA sequences and to protect DNA from thermal denaturation, indicating that it might assume a similar function in vivo. Several amide resonances in two-dimensional n.m.r. 1H, 15N correlation spectra of 15N-enriched Sso7d are shifted and broadened upon addition of small amounts of ten base-pair or 19 base-pair duplex DNA oligomers under conditions where Sso7d-DNA complexes exchange rapidly on the n.m.r. time scale. The locations of the corresponding amides in the Sso7d structure define the surface that interacts with DNA. This surface coincides with a continuous region of strong positive electrostatic potential, which was calculated by means of numerical solution of the Poisson-Boltzmann equation. A model of the non-specific Sso7d-DNA complex is suggested based on the present data and previously obtained evidence that Sso7d interacts with the DNA major groove. The protein-DNA interface consists of a triple-stranded beta-sheet, which interacts with the DNA major groove and a reverse turn connecting the two strands of a double-stranded beta-sheet, which interacts with the minor groove. We note that the five (of 14) lysine side-chains that are specifically subjected to N zeta-monomethylation in the cell are located on surfaces of Sso7d that are exposed to the solvent in the proposed Sso7d-DNA complex.

Archaeal Proteins↗
PubMed1995

Structural characterization of a minimal functional transactivation domain from the human glucocorticoid receptor.

A 58-amino acid polypeptide containing the functional core region, the tau 1 core, of the major transactivation domain of the human glucocorticoid receptor has been expressed in Escherichia coli and purified to homogeneity. The polypeptide retains 60-70% of the activity of the intact domain when assayed in vivo or in vitro. This report describes a structural characterization of the tau 1 core peptide fragment. Circular dichroism spectroscopy shows that the tau 1 core and a larger fragment encompassing the intact tau 1 domain are largely unstructured in water solution under a variety of pH conditions. The tau 1 core, however, acquires a significant alpha-helical structure when analyzed in the presence of trifluoroethanol, an agent that favors secondary structure formation in regions that have propensity for alpha-helical conformation. Two- and three-dimensional NMR spectroscopy of 15N-labeled tau 1 core, in the presence of trifluoroethanol, has allowed sequential assignment of 1H and 15N resonances and identification of three protein segments with alpha-helical character. Potentially helix-breaking proline substitutions, in proposed alpha-helical regions, lead to reduced activity, suggesting that alpha-helices are important for transactivation in vivo.

Amino Acid Sequence↗
PubMed1995

A method for production of 13C/15N double labelled RNA in E. coli, and subsequent in vitro synthesis of ribonucleotide 5' triphosphates.

In this paper we describe an enhanced method for the large scale production of high quality 13C/15N labelled NTPs. High amounts of labelled RNA was obtained from E. coli cells grown in 13C/15N enriched medium and treated with chloramphenicol. Total RNA was extracted from spheroplasted cells in the presence of SDS and proteinase K and subsequently degraded to NMPs by nuclease P1 and high concentrations of nuclease S1 in a low salt buffer. To avoid non-specific degradation of the RNA, nuclease digestion was performed in a short term reaction on native, not heat-denatured RNA. CMP, AMP, GMP and UMP were chromatographically separated and converted to the corresponding NTPs by a mixture of kinases in the presence of a coupled redox system based on thioredoxin and dithiothreitol. The quality of the 13C/15N labelled NTPs was tested by in vitro transcription.

Carbon Isotopes↗
PubMed1995

On the pH dependence of amide proton exchange rates in proteins.

We have analyzed the pH dependencies of published amide proton exchange rates (kex) in three proteins: bovine pancreatic trypsin inhibitor (BPTI), bull seminal plasma proteinase inhibitor IIA (BUSI IIA), and calbindin D9K. The base-catalyzed exchange rate constants (kOH) of solvent exposed amides in BPTI are lower for residues with low peptide carbonyl exposure, showing that the environment around the carbonyl oxygen influences kOH. We also examined the possible importance of an exchange mechanism that involves formations of imidic acid intermediates along chains of hydrogen-bonded peptides in the three proteins. By invoking this "relayed imidic acid exchange mechanism," which should be essentially acid-catalyzed, we can explain the surprisingly high pHmin (the pH value at which kex reaches a minimum) found for the non-hydrogen-bonded amide protons in the beta-sheet in BPTI. The successive increase of pHmin along a chain of hydrogen-bonded peptides from the free amide to the free carbonyl, observed in BPTI, can be explained as an increasing contribution of the proposed mechanism in this direction of the chain. For BUSI IIA (pH 4-5) and calbindin D9K (pH 6-7) the majority of amide protons with negative pH dependence of kex are located in chains of hydrogen-bonded peptides; this situation is shown to be consistent with the proposed mechanism.

Amides↗
PubMed1995

Molecular dynamics simulations of the glucocorticoid receptor DNA-binding domain in complex with DNA and free in solution.

Molecular dynamics simulations have been performed on the glucocorticoid receptor DNA binding domain (GR DBD) in aqueous solution as a dimer in complex with DNA and as a free monomer. In the simulated complex, we find a slightly increased bending of the DNA helix axis compared with the crystal structure in the spacer region of DNA between the two half-sites that are recognized by GR DBD. The bend is mainly caused by an increased number of interactions between DNA and the N-terminal extended region of the sequence specifically bound monomer. The recognition helices of GR DBD are pulled further into the DNA major groove leading to a weakening of the intrahelical hydrogen bonds in the middle of the helices. Many ordered water molecules with long residence times are found at the intermolecular interfaces of the complex. The hydrogen-bonding networks (including water bridges) on either side of the DNA major groove involve residues that are highly conserved within the family of nuclear receptors. Very similar hydrogen-bonding networks are found in the estrogen receptor (ER) DBD in complex with DNA, which suggests that this is a common feature for proper positioning of the recognition helix in ER DBD and GR DBD.

Amino Acid Sequence↗
PubMed1995

Solution structure of a mammalian PCB-binding protein in complex with a PCB.

Metabolites of polychlorinated biphenyls (PCBs) bind with high affinity to uteroglobin, a small homodimeric protein that also binds progesterone. We present the solution structure of the reduced form of rat uteroglobin in complex with a PCB methylsulphone, (MeSO2)2-TCB. The structure reveals the molecular basis for the accumulation of (MeSO2)2-TCB by uteroglobin. The structure also shows how ligand binding and release might be controlled by reduction/oxidation of two intermolecular disulphide bonds. Breakage of these bonds induces a local unfolding of the N- and C-termini and a separation of helices creating a channel into the binding site. These effects make the ligand binding cavity readily accessible to entry of the ligand.

Amino Acid Sequence↗
PubMed1994

Thermodynamics of sequence-specific glucocorticoid receptor-DNA interactions.

The thermodynamics of sequence-specific DNA-protein interactions provide a complement to structural studies when trying to understand the molecular basis for sequence specificity. We have used fluorescence spectroscopy to study the chemical equilibrium between the wild-type and a triple mutant glucocorticoid receptor DNA-binding domain (GR DBD wt and GR DBDEGA, respectively) and four related DNA-binding sites (response elements). NMR spectroscopy was used to confirm that the structure of the two proteins is very similar in the uncomplexed state. Binding to DNA oligomers containing single half-sites and palindromic binding sites was studied to obtain separate determinations of association constants and cooperativity parameters involved in the dimeric DNA binding. Equilibrium parameters were determined at 10-35 degrees C in 85 mM NaCl, 100 mM KCl, 2 mM MgCl2, and 20 mM Tris-HCl at pH 7.4 (20 degrees C) and at low concentrations of an antioxidant and a nonionic detergent. GR DBDwt binds preferentially to a palindromic consensus glucocorticoid response element (GRE) with an association constant of (7.6 +/- 0.9) x 10(5) M-1 and a cooperativity parameter of 10 +/- 1 at 20 degrees C. GR DBDEGA has the highest affinity for an estrogen response element (ERE) with an association constant of (2.2 +/- 0.3) x 10(5) M-1 and a cooperativity parameter of 121 +/- 17 at 20 degrees C. The difference in cooperativity in the two binding processes, which indicates significant differences in binding modes, was confirmed using gel mobility assays. van't Hoff analysis shows that DNA binding in all cases in entropy driven within the investigated temperature range. We find that delta H0obs and delta S0obs for the formation of a GR DBDwt-GRE versus GR DBDEGA-ERE complex are significantly different despite very similar delta G0obs values. A comparison of GR DBDwt binding to two similar GREs reveals that the discrimination between these two (specific) sites is due to a favorable delta(delta S0obs) which overcompensates an unfavorable delta(delta H0obs), i.e., the sequence specificity is in this case entropy driven. Thus, entropic effects are of decisive importance for the affinity as well as the specificity in GR-DNA interactions. The molecular basis for measured equilibrium and thermodynamic parameters is discussed on the basis of published structures of GR DBD-GRE and ER DBD-ERE complexes.

Amino Acid Sequence↗
PubMed1994

Solution structure and DNA-binding properties of a thermostable protein from the archaeon Sulfolobus solfataricus.

The archaeon Sulfolobus solfataricus expresses large amounts of a small basic protein, Sso7d, which was previously identified as a DNA-binding protein possibly involved in compaction of DNA. We have determined the solution structure of Sso7d. The protein consists of a triple-stranded anti-parallel beta-sheet onto which an orthogonal double-stranded beta-sheet is packed. This topology is very similar to that found in eukaryotic Src homology-3 (SH3) domains. Sso7d binds strongly (Kd < 10 microM) to double-stranded DNA and protects it from thermal denaturation. In addition, we note that epsilon-mono-methylation of lysine side chains of Sso7d is governed by cell growth temperatures, suggesting that methylation is related to the heat-shock response.

Amino Acid Sequence↗
PubMed1993

Refined solution structure of the glucocorticoid receptor DNA-binding domain.

A refined solution structure of the glucocorticoid receptor DNA-binding domain (GR DBD) has been determined using two- and three-dimensional nuclear magnetic resonance (NMR) spectroscopy on an 15N-labeled GR DBD fragment in conjunction with distance geometry and simulated annealing calculations. Thirty structures of the fragment C440-R510 of the rat GR were calculated based on 906 distance constraints obtained from NOE intensities (168 intraresidue and 738 interresidue NOEs) and 43 dihedral constraints. Average atomic root mean square (rms) differences between the 24 best structures and their geometric average are 0.70 A for backbone atoms and 1.44 A for all heavy atoms. Several regions that were not well defined in a previous NMR structure determination of a similar protein fragment [Härd, T., Kellenbach, E., Boelens, R., Maler, B.A., Dahlman, K., Freedman, L.P., Carlstedt-Duke, J., Yamamoto, K.R., Gustafsson, J.-A., & Kaptein, R. (1990b) Science 249, 157-160] are now well-defined. The refined structure of the uncomplexed GR DBD is very similar to the crystal structure of GR DBD in a sequence specific DNA complex [Luisi, B. F., Xu, W. X., Otwinowski, Z., Freeman, L. P., Yamamoto, K. R., & Sigler, P. B. (1991) Nature 352, 497-505], in particular with regard to the presence and relative positions of secondary structure elements. The backbone atom rms difference between the average NMR solution structure and the crystal structure of the DNA-complexed GR DBD is 1.8 A. The most pronounced differences between the free and DNA-complexed states are found within the fragment C476-C482 in the second zinc-coordinating domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Thermodynamics of the glucocorticoid receptor-DNA interaction: binding of wild-type GR DBD to different response elements.

We used fluorescence spectroscopy to study the chemical equilibria between an 82-residue protein fragment containing the core conserved region of the glucocorticoid receptor DNA-binding domain (GR DBD) and a palindromic glucocorticoid response element (GRE), a consensus GRE half-site, a consensus estrogen response element (ERE) half-site, and two intermediate half-sites (GRE2 and ERE2). Equilibrium parameters were determined at 20 degrees C and buffer conditions that approximate intracellular conditions. The association constants for GR DBD binding to the GRE (5'TGTTCT3') and GRE2 (5'TGTCCT3') half-sites at 85 mM NaCl, 100 mM KCl, 2 mM MgCl2, and 20 mM Tris-HCl at pH 7.4 and low concentrations of an antioxidant and a nonionic detergent are (1.0 +/- 0.1) x 10(6) M-1 and (5.1 +/- 0.2) x 10(5) M-1, respectively. The association constants for binding to the ERE (5'TGACCT3') and ERE2 (5'TGATCT3') half-sites are < 10(5) M-1. The implications of these numbers for the specificity and affinity for the binding of the intact GR to DNA are discussed. Comparison of GR DBD binding to a GRE half-site and a palindromic GRE sequence allowed us to estimate the cooperativity parameter, omega obs = 25-50, for GR DBD binding to GRE. The thermodynamics of the GR DBD interaction with a GRE half-site were also investigated by determining the temperature dependence of the observed association constant. The nonlinear dependence in ln Kobs as a function of 1/T is consistent with a change in standard heat capacity, delta Cp degree obs = 1.0 +/- 0.2 kcal mol-1 K-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1993

A comparison of 15N NMR relaxation measurements with a molecular dynamics simulation: backbone dynamics of the glucocorticoid receptor DNA-binding domain.

The rapid motions of the backbone of the DNA-binding domain of the glucocorticoid receptor (GR DBD) have been investigated using proton-detected heteronuclear NMR experiments on 15N-labeled protein at pH 6.0 and with a 200 psec molecular dynamics simulation of hydrated GR DBD. The experimental data were interpreted in terms of a generalized order parameter (S2) and an effective correlation time (tau e) for the internal motion of each amide bond. A back calculation, using the same model, yielded the [1H]-14N nuclear Overhauser effects (NOEs) and the 15N spin-lattice relaxation times (T1) from the simulated data. The rapid motions of the backbone turned out to be rather limited and uniform throughout the protein, with a somewhat reduced mobility in the two major alpha-helical regions and a slightly enhanced flexibility for some residues in the first zinc coordinating region. The agreement between the experimental and simulated S2-values was as good as quantitative for most of the residues, except for some residues that were subject to a more large-scale, and in the simulation thus poorly sampled, motion. Examples of such motions that were found in the simulation include jumps of the amide bond of Ile-487 between the charged oxygens of the side chain of Asp-485 and less distinct large scale motions for some of the residues in the extended regions, that were shown to give rise to noisy and/or fast decaying internal reorientational correlation functions. For these residues large differences in the simulated and experimental tau e-values were found, indicating that motions on different time scales were dominating in the experimental and simulated values. The lower (< 0.7) experimental NOEs for these residues could not be reproduced in the simulation and were shown to be a consequence of the lower tau e-values estimated in the simulation. By combining information from the simulation and the experiment a more complete picture of the motions for these residues can be obtained as is illustrated with an estimation of the jump angle and jump frequency for the amide bond of Ile-487.

Amino Acid Sequence↗