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Biomedical subjects

M G Fried

Publications and source records attributed to M G Fried.

At least 19 recordsLinked to original sources
PubMed2001

Host-guest study of left-handed polyproline II helix formation.

The importance of the left-handed polyproline II (PPII) helical conformation has recently become apparent. This conformation generally is involved in two important functions: protein-protein interactions and structural integrity. PPII helices play vital roles in a variety of processes including signal transduction, transcription, and cell motility. Proline-rich regions of sequence are often assumed to adopt this structure. Remarkably, little is known about the physical determinants of this secondary structure type. In this study, we have explored the formation of PPII helices by a short poly(proline) peptide. In addition, the results from experiments used to determine the propensities for apolar residues, plus glycine, asparagine, and glutamine, to adopt this structure in a poly(proline)-based host peptide are reported here. Proline possesses the highest intrinsic propensity, with glutamine, alanine, and glycine having surprisingly high propensities. beta-Branched residues possess the lowest propensities of the residues examined. It is postulated that propensities possessed by apolar residues are due in part to peptide-solvent interactions, and that the remarkably high propensity possessed by glutamine may be due to a side chain to backbone hydrogen bond. These data are the first step toward a molecular understanding of the formation of this important, and yet little studied, secondary structure.

Circular Dichroism↗
PubMed2001

In vitro interaction of the Escherichia coli cyclic AMP receptor protein with the lactose repressor.

Sedimentation equilibrium studies show that the Escherichia coli cyclic AMP receptor protein (CAP) and lactose repressor associate to form a 2:1 complex in vitro. This is, to our knowledge, the first demonstration of a direct interaction of these proteins in the absence of DNA. No 1:1 complex was detected over a wide range of CAP concentrations, suggesting that binding is highly cooperative. Complex formation is stimulated by cAMP, with a net uptake of 1 equivalent of cAMP per molecule of CAP bound. Substitution of the dimeric lacI-18 mutant repressor for tetrameric wild-type repressor completely eliminates detectable binding. We therefore propose that CAP binds the cleft between dimeric units in the repressor tetramer. CAP-lac repressor interactions may play important roles in regulatory events that take place at overlapping CAP and repressor binding sites in the lactose promoter.

Bacterial Proteins↗
PubMed2001

A histone fold TAF octamer within the yeast TFIID transcriptional coactivator.

Gene activity in a eukaryotic cell is regulated by accessory factors to RNA polymerase II, which include the general transcription factor complex TFIID, composed of TBP and TBP-associated factors (TAFs). Three TAFs that contain histone fold motifs (yTAF17, yTAF60 and yTAF61) are critical for transcriptional regulation in the yeast Saccharomyces cerevisiae and are found in both TFIID and SAGA, a multicomponent histone acetyltransferase transcriptional coactivator. Although these three TAFs were proposed to assemble into a pseudooctamer complex, we find instead that yTAF17, yTAF60 and yTAF61 form a specific TAF octamer complex with a fourth TAF found in TFIID, yTAF48. We have reconstituted this complex in vitro and established that it is an octamer containing two copies each of the four components. Point mutations within the histone folds disrupt the octamer in vitro, and temperature-sensitive mutations in the histone folds can be specifically suppressed by overexpressing the other TAF octamer components in vivo. Our results indicate that the TAF octamer is similar both in stoichiometry and histone fold interactions to the histone octamer component of chromatin.

Alleles↗
PubMed2000

Interactions of human fibrinogens with factor XIII: roles of calcium and the gamma' peptide.

Plasma factor XIII is the zymogen of the transglutaminase factor XIIIa. This enzyme catalyzes the formation of isopeptide cross-links between fibrin molecules in nascent blood clots that greatly increase the mechanical stability of clots and their resistance to thrombolytic enzymes. We have characterized the solution interactions of factor XIII with two variants of fibrinogen, the soluble precursor of fibrin. Both the predominant fibrinogen gamma(A)/gamma(A) and the major variant gamma(A)/gamma' form complexes with a 2 fibrinogen:1 factor XIII ratio. The absence of detectable concentrations of 1:1 complexes in equilibrium mixtures containing free factor XIII and 2:1 complexes suggests that this interaction is cooperative. Factor XIII binds fibrinogen gamma(A)/gamma' approximately 20-fold more tightly than fibrinogen gamma(A)/gamma(A), and the interaction with fibrinogen gamma(A)/gamma' (but not fibrinogen gamma(A)/gamma(A)) is accompanied by a significant release of Ca(2+). Taken together, these results suggest that the strikingly anionic gamma' C-terminal sequence contains features that are important for factor XIII binding. Consistent with this notion, a synthetic 20-residue polypeptide containing the gamma' sequence was found to associate with factor XIII in a 2:1 molar ratio and act as an efficient competitor for fibrinogen gamma(A)/gamma' binding.

Binding, Competitive↗
PubMed2000

Participation of the amino-terminal domain in the self-association of the full-length yeast TATA binding protein.

The association of monomeric TATA binding protein with promoter DNA is an essential first step in many current models of eukaryotic transcription initiation. This step is followed by others in which additional transcription factors, and finally RNA polymerase, assemble at the promoter. Here we characterize the quaternary interactions of the Saccharomyces cerevisiae TATA-binding protein (yTBP), in the absence of other proteins or DNA. The data reveal a robust pattern in which yTBP monomers equilibrate with tetramers and octamers over a broad span of temperatures (4 degrees C </= T </= 37 degrees C) and salt concentrations (60 mM </= [KCl] </= 1 M), that includes the physiological range. Association is highly cooperative, with octamer formation favored by approximately 9 kcal/mol over tetramer formation. Changes in association constant with [KCl] are consistent with an assembly-linked release of ions at low salt and an assembly-linked uptake of ions at high salt, for both monomer right arrow over left arrow tetramer and tetramer right arrow over left arrow octamer reaction steps. Fluorescence emission spectra and steady-state anisotropies reveal that the amino-terminal domain changes conformation and dynamics at both association steps and that the polarity of the environment near tryptophan 26 is sensitive to changes in [KCl] in the monomeric and tetrameric states but not the octameric state. These results are consistent with a [salt]-dependent change in the assembly mechanism near 300 mM KCl and suggest that the amino-terminal domain may modulate the self-association of the full-length protein. TBP self-association may regulate many of its cellular functions, including transit of the nuclear membrane and participation in transcription initiation.

Allosteric Regulation↗
PubMed2000

Abortion in the United States: barriers to access.

The legalization of abortion in the United States has brought a dramatic improvement in women's health and reductions in maternal and infant mortality. For young women, low-income women, and women of color, however, access to abortion has been increasingly restricted. This article describes the obstacles to abortion access, including lack of federal funding; restrictive laws, encompassing those requiring parental consent or notification for a minor seeking an abortion, as well as those attempting to ban a certain procedure; stigmatization and marginalization of abortion; decreasing abortion services; and a shortage of providers. The article connects the erosions in rights relating to abortion to policies undermining poor women's rights in relation to having children.

Abortion, Legal↗
PubMed1999

The TATA-binding protein from Saccharomyces cerevisiae oligomerizes in solution at micromolar concentrations to form tetramers and octamers.

Equilibrium analytical ultracentrifugation has been used to determine the stoichiometry and energetics of the self-assembly of the TATA-binding protein of Saccharomyces cerevisiae at 30 degreesC, in buffers ranging in salt concentration from 60 mM KCl to 1 M KCl. The data are consistent with a sequential association model in which monomers are in equilibrium with tetramers and octamers at protein concentrations above 2.6 microM. Association is highly cooperative, with octamer formation favored by approximately 7 kcal/mol over tetramers. At high [KCl], the concentration of tetramers becomes negligible and the data are best described by a monomer-octamer reaction mechanism. The equilibrium association constants for both monomer <--> tetramer and tetramer <--> octamer reactions change with [KCl] in a biphasic manner, decreasing with increasing [KCl] from 60 mM to 300 mM, and increasing with increasing [KCl] from 300 mM to 1 M. At low [KCl], approximately 3 mole equivalents of ions are released at each association step, while at high [KCl], approximately 3 mole equivalents of ions are taken up at each association step. These results suggest that there is a salt concentration-dependent change in the assembly mechanism, and that the mechanistic switch takes place near 300 mM KCl. The possibility that this self-association reaction may play a role in the activity of the TATA-binding protein in vivo is discussed.

DNA-Binding Proteins↗
PubMed1998

Electrophoretic analysis of multiple protein-DNA interactions.

Under favorable conditions, native gel electrophoresis allows the resolution of protein-DNA complexes that differ in stoichiometry, identities of occupied DNA sequences (configuration), and macromolecular conformation. This technique provides a unique opportunity to analyze, in thermodynamic terms, the molecular interactions that govern the equilibrium distributions of species in protein-DNA mixtures. Here we describe a general theoretical approach to the analysis of electrophoretic band intensities, and provide examples of its application to the analysis of several interacting systems.

Bacterial Proteins↗
PubMed1997

Role of macromolecular hydration in the binding of the Escherichia coli cyclic AMP receptor to DNA.

The osmotic stress technique was used to measure the changes in macromolecular hydration that accompany binding of the Escherichia coli CAP protein to its transcription-regulatory site (C1) in the lactose promoter and that accompany the transfer of CAP from site C1 to nonspecific genomic DNA. Formation of the C1 complex is accompanied by the net release of 79 +/- 11 water molecules. If all water molecules were released from macromolecular surfaces, this result would be consistent with a net reduction of solvent-accessible surface area of 711 +/- 189 A2. This area is only slightly smaller than the solvent-inaccessible macromolecular interface in crystalline CAP-DNA complexes. The transfer of CAP from site C1 to nonspecific sites is accompanied by the net uptake of 56 +/- 10 water molecules. Taken with the water stoichiometry of sequence-specific binding, this value implies that formation of a nonspecific complex is accompanied by the net release of 2-44 water molecules. The enhanced stabilities of CAP-DNA complexes with increased osmolality (decreased water activity) may contribute to the ability of E.coli cells to tolerate dehydration and/or high external salt concentrations.

DNA, Bacterial↗
PubMed1997

Sequestration stabilizes lac repressor-DNA complexes during gel electrophoresis.

The gel electrophoresis mobility shift assay is widely used for both qualitative and quantitative characterization of protein-nucleic acid interactions. Often it is found that protein-nucleic acid complexes persist within gels for much longer than would be expected on the basis of their free solution lifetimes. Excluded volume and matrix-interaction mechanisms have been proposed to account for the enhanced stabilities of complexes within gels. To test these mechanisms, we have investigated the influences of gel composition and concentration on the pseudo first-order dissociation kinetics of complexes containing the Escherichia coli lactose (lac) repressor protein and lactose promoter DNA. In both polyacrylamide and agarose gels, dissociation rates were slower than those in free solution and decreased with increasing gel concentration. This result is inconsistent with mechanisms of stabilization that require specific interactions with the gel matrix. Under standard reaction conditions, free solution values of kdiss were proportional to [DNA]0.83 +/- 0.11, while in 10% polyacrylamide gels kdiss values were proportional to [DNA]0.48 +/- 0.09. These results suggest that the lifetimes of lac repressor-DNA complexes in free solution are limited by their encounter frequency with molecules of DNA or with protein-DNA complexes; some or all of the stabilization observed in gels may be due to a reduction in this frequency.

Bacterial Proteins↗
PubMed1997

Factors that affect the stability of protein-DNA complexes during gel electrophoresis.

The gel electrophoresis mobility shift assay is widely used for qualitative and quantitative characterization of protein complexes with nucleic acids. Often it is found that complexes persist within electrophoresis gels for much longer than expected on the basis of their free-solution lifetimes. Volume exclusion, direct interaction with gel matrices and the reduction of water activity by the gel have been proposed as mechanisms enhancing the stability of complexes during electrophoresis. We have used the well-characterized interaction of the E. coli cyclic AMP receptor protein (CAP) with lactose promoter DNA to test these proposals. We found that the activity of water within polyacrylamide gels differs little from that of the buffer in which they were cast and that the dependence of the dissociation rate constant on water activity is too small for osmotic stabilization to contribute significantly to the lifetimes of CAP-DNA complexes. In addition, we found that a cross-linked gel matrix is not required for the stabilization of CAP-DNA complexes, that comparable stabilization is produced by three dissimilar polymers (linear polyacrylamide, dextran and polyethylene glycol), and that these polymers stabilize complexes more effectively than equivalent weight concentrations of their cognate monomers. While these results challenge the notion that direct interaction with the gel matrix contributes to the stability of protein-DNA complexes, they are all features expected of excluded volume mechanisms.

Acrylic Resins↗
PubMed1996

DNA binding mechanism of O6-alkylguanine-DNA alkyltransferase: stoichiometry and effects of DNA base composition and secondary structure on complex stability.

O6-Alkylguanine-DNA alkyltransferase (AGT) is an important cellular defense against the mutagenic effects of DNA alkylating agents. In humans this defense can contribute to the ability of some tumors to resist the effects of chemotherapeutic agents that act through DNA alkylation. We report here studies that characterize the interaction of AGT with DNA. We show that although AGT sediments as a monomer in the absence of DNA, it binds cooperatively to single stranded deoxyribonucleotides. The stoichiometries of complexes formed with 16-, 30-, and 80-base oligodeoxyribonucleotides are 3.8 +/- 0.3, 5.3 +/- 0.2, and 8.9 +/- 0.2, respectively; the binding density decreasing from approximately 4 nt/monomer to approximately 9 nt/monomer as DNA length increases over this range. Binding competition assays show that DNA affinities depend only weakly on base composition or secondary structure, although in general G + C-rich sequences are bound with greater affinity than are A + T-rich ones and single-stranded DNA is bound with greater affinity than duplex forms. These results suggest mechanisms by which AGT may search for alkylated sites and interact with them to effect DNA repair.

Base Composition↗
PubMed1996

The mechanism of CAP-lac repressor binding cooperativity at the E. coli lactose promoter.

The cyclic AMP receptor protein (CAP) and lactose repressor bind their regulatory sites in the lactose promoter with moderate cooperativity (omega C101 = 11.8(+/- 3.7)). This cooperativity is significantly reduced by the removal of DNA located upstream of the CAP binding site or by substitution of the dimeric lacI-18 mutant repressor for the wild-type tetrameric protein. These results are consistent with a mechanism of interaction in which CAP bends the DNA and the lac repressor binds simultaneously to its operator site and to promoter-distal sequences. Similar values of omega C101 were obtained with a promoter truncation containing the O3 pseudooperator site and one in which the site is destroyed, suggesting that DNA contacts distal to the O3 site are necessary for cooperative binding.

Binding Sites↗
PubMed1994

Analysis of the thermodynamic linkage of DNA binding and ion binding for dimeric and tetrameric forms of the lac repressor.

The salt concentration dependences of the observed association constants (Kobs) for the binding of wild-type lac repressor tetramer and the dimeric lacI-18 mutant repressor to lactose operator DNA were compared. For both proteins, the data are consistent with a class of linkage models in which ion binding by the protein is driven by differences in the ionic concentrations in bulk solution and in the volume near the DNA surface. The models that best agree with the data are those in which ion-binding reactions are cooperative. In spite of close agreement between these models and the data, the determination of ion stoichiometries and apparent ion-binding affinities requires additional mechanistic or structural information. The simplest ion-binding mechanism consistent with the data is compatible with a current structural model of the repressor-operator complex. At salt concentrations in excess of 50 mM, at which cation displacement from the DNA and anion displacement from the protein are expected to dominate, similar ion stoichiometries are found for the DNA binding of dimeric and tetrameric repressors. This supports the notion that the DNA contacts of these proteins are homologous. At lower salt concentrations, in which cation binding by the proteins is expected to be significant, the net ion stoichiometry of wild-type repressor binding is slightly greater than that of the lacI-18 mutant. This difference may reflect the availability of ion-binding sites in the distal subunits of tetramer that are not present in the dimer, or may be a consequence of the involvement of ion binding in the dimer/monomer equilibrium.

Anions↗
PubMed1994

Molecular sequestration stabilizes CAP-DNA complexes during polyacrylamide gel electrophoresis.

The gel electrophoresis mobility shift assay is widely used for qualitative and quantitative characterization of protein complexes with nucleic acids. Often it is found that complexes that are short-lived in free solution (t1/2 of the order of minutes) persist for hours under the conditions of gel electrophoresis. We have investigated the influence of polyacrylamide gels on the pseudo first-order dissociation kinetics of complexes containing the E.coli cyclic AMP receptor protein (CAP) and lactose promoter DNA. Within the gel matrix, kdiss decreased with increasing [polyacrylamide] and the order of the reaction was changed. In free solution, kdiss was proportional to [DNA]2, while in 5% gels, kdiss was proportional to [DNA]0.3. In gels of [polyacrylamide] > or = 10%, kdiss was nearly independent of [DNA] until fragment concentrations exceeded 0.1 microM. Even in the absence of competing DNA, kdiss(gel) < kdiss(solution). These results suggest that the lifetime of CAP-DNA complexes in free solution is limited by their encounter frequency with molecules of DNA or with protein-DNA complexes; some or all of the stabilization observed in gels may be due to a reduction in this frequency.

Cyclic AMP Receptor Protein↗
PubMed1994

Free DNA concentration in E. coli estimated by an analysis of competition for DNA binding proteins.

Transcription in E. coli is often controlled by the binding of specific gene-regulatory proteins. Binding of these proteins to their specific DNA binding sites occurs in the presence of a large excess of "nonspecific" genomic DNA. Binding to a specific DNA site thus depends on the concentration of regulatory protein, on its affinities for specific and competing nonspecific binding sites, and on the free concentrations of those sites. Although it is probable that genomic DNA is largely occluded by protein binding or by condensation in vivo, the actual extent to which the DNA is available to act as a competitor for specific binding (i.e. the effective concentration of nonspecific DNA) is not known. Because many regulatory interactions occur simultaneously in a cell, it is reasonable to expect that they will have evolved to function at equilibrium with a shared concentration of competing nonspecific DNA. This premise was the basis for this study. In vitro binding data were compiled for six regulatory proteins that function in E. coli, and used to calculate theoretical equilibrium binding distributions. The calculated distributions were used to evaluate the regulatory states of promoters according to models based on the equilibrium occupancies of regulatory sites. For four proteins whose DNA-binding affinities are modulated by ligand binding (CAP, lac repressor, trp repressor and araC), regulation was assessed as the extent to which the presence of the modulator could affect the occupancy by protein of the specific sites (e.g. the difference in equilibrium occupancy by CAP of CAP binding sites between conditions of high and low concentrations of CAP's affinity modulator, cAMP). For two proteins whose site affinities are not modulated by ligand binding (lambda repressor and lambda-cro), regulation was assessed by specific site occupancy at equilibrium. These regulation profiles were compared to determine whether a single concentration of nonspecific competing DNA is compatible with effective regulation as defined for all of the systems. For five of the six modeled systems (CAP, trp repressor, araC, lambda repressor and lambda-cro), a free nonspecific DNA concentration on the order of 10(-4) M base pairs is compatible with regulation based on equilibria of the protein-DNA interactions. The lac repressor-operator system is an exception to these results: as has been shown previously, the regulation of operator binding by low molecular weight inducers increases with increasing concentrations of nonspecific DNA (von Hippel et al., 1974 Proc. natn. Acad. Sci. U.S.A. 71, 4808-4812).(ABSTRACT TRUNCATED AT 400 WORDS)

DNA, Bacterial↗