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G D Fasman

Publications and source records attributed to G D Fasman.

At least 73 records · Page 4Linked to original sources
PubMed1982

Sodium butyrate induced structural changes in HeLa cell chromatin.

Postsynthetic modifications of core histones by treatment of HeLa S3 cells with 5mM sodium butyrate lead to alterations in the structure of high molecular weight chromatin. Whole chromatin from butyrate-treated cells, which results in highly acetylated core histones, has an ellipticity [theta]282.5 of 3700 deg cm2 dmol-1 (0.2 mM EDTA, pH 7.4) that is 1200 deg c m2 dmol-1 less than chromatin from untreated HeLa cells, suggesting a more condensed structure. No difference in the circular dichroism spectra was observed in H1-stripped, high molecular weight chromatin obtained from control and butyrate-treated cells at low (0.2 mM EDTA, pH 7.4) ionic strength. Thermal denaturation profiles of high molecular weight chromatin were resolved into three transitions and exhibited a shifting of hyperchromicity from transition I to transitions III, at a higher Tm, with butyrate treatment of HeLa cells, further indicating a more compact structure. Thermal denaturation profiles of H1-stripped chromatin were not affected by butyrate treatment. Ionic strength studies in the range of 0-5 mM NaH2PO4, 0.2 mM EDTA, pH 7.4, of high molecular weight chromatin exhibited a decrease in [theta]282.5 and a shifting of hyperchromicity from transition I to transition III with increasing ionic strength. Control high molecular weight chromatin was more sensitive to changes in ionic strength than its highly acetylated counterpart. These results suggest that acetylation of histones alone does not result in a change in histone-DNA interactions but other changes associated with butyrate treatment most probably cause a more condensed structure, of the fraction studied herein, which is mediated by H1 or other materials removed during stripping in 0.35 M NaCl.

Acetylation↗
PubMed1981

Study of conformational states and reversibility of histone complexes.

The core histone complex (H3:H4:H2A:H2B)2 and products of dissociation, the H2A:H2B: dimer and the H3:H4 tetramer, were isolated from chicken erythrocyte chromatin by several literature methods as well as gel filtration on Bio-Gel A15m at various salt concentrations. The conformational and oligomeric characteristic of these histone complexes were compared to analogous histone complexes prepared by renaturation of individually acid-extracted histones by circular dichroism (CD) and analytical gel filtration chromatography. The salt-extracted core histone complex (independent of method of preparation), the purified dissociation products, the H2A:H2B dimer, and the H3:H4 tetramer in 2 M NaCl, 10 mM sodium phosphate, 0.25 mM EDTA and 0.1 mM DTT, pH 7.0, have conformation which are identical, by the criteria of similar CD spectra, with complexes prepared from acid-extracted histones, Likewise, the salt-extracted complexes may be cycled through solvents of low ionic strength (10 mM sodium phosphate, pH 7.0 or 50 mM NaOAc, pH 5.0) or 1 mM HCl and returned to 2.0 M NaCl, 10 mM sodium phosphate, 0.25 mM EDTA, and 0.1 mM DTT, pH 7.0, in a completely reversible manner. Thus it would appear that acid-denatured histones are capable of being fully renatured to yield native-like complexes.

Animals↗
PubMed1980

Conformational changes in the H3 . H4 histone complex. Serological and circular dichroism studies.

The H3.H4 complex has been extracted from calf thymus chromatin in either 0.05 M NaOAc, pH 5.0, or in 2.0 M KCl, 0.1 M KPO4, pH 6.7, and both forms have been shown to consist solely of histones H3 and H4 when examined on sodium dodecyl sulfate-polyacrylamide gels. Serological and circular dichroism analyses indicate the existence of structural differences between the two forms. Dilution of the high salt form of the complex into low salt produces a time-dependent conformational change which is related to reduction in the alpha helix content of the complex and which exposes antigenic sites on the complex. The existence of multiple forms of the H3.H4 complex may be related to the dynamic equilibrium of nucleosome structure in vivo.

Animals↗
PubMed1980

Dependence of mononucleosome deoxyribonucleic acid conformation on the deoxyribonucleic acid length and H1/H5 content. Circular dichroism and thermal denaturation studies.

Four mononucleosome preparations were isolated from micrococcal nuclease digests of chicken erythrocyte nuclei which differed in average deoxyribonucleic acid (DNA) length and in H1 and H5 content. The circular dichroism properties of the unperturbed mononucleosome preparations and the corresponding H1- and H5-depleted species demonstrate that the nucleoprotein spectra above 250 nm are all altered relative to protein-free DNA by the addition of a single negative band at 275 nm, similar to the band observed for psi-DNA. The quantitative analysis of the psi-type band intensity for any of the higher molecular weight unperturbed samples relative to core particle mononucleosomes yielded a constant number of DNA base pairs (approximately 140) contributing to this new band. Upon removal of H1 and H5 from the mononucleosome preparations which have sufficiently long linker DNA, the psi-type band intensity indicates an approximately 30 base pair reduction in the number of core DNA base pairs contributing to the altered circular dichroism properties. The psi-type band is proposed to be due to the compact DNA tertiary structure, i.e., the manner in which the DNA is wound around the histone core allowing interactions between adjacent turns of the superhelix. This interpretation implies that approximately 30 base pairs of core DNA are removed from the unique core tertiary structure when the linker DNA is not bound by H1 or H5. The circular dichroism analysis correlates well with the thermal denaturation properties of mononucleosomes. Removal of H1 and H5 causes an overall reduction in the thermal stability of both core and linker DNA. The degree of destabilization is greatest when the average DNA length is maximum. Some core DNA is lost from the highest temperature melting bands when histone-free DNA is present. These results indicate two regions of different conformational and thermodynamic stability in core DNA. The length of attached linker DNA and its histone content influence the two regions of the core to differing extents.

Animals↗
PubMed1980

Conformational studies of the synthetic precursor-specific region of preproparathyroid hormone.

the secondary structure of a synthetic peptide representing the NH2-terminal, precursor-specific extension sequence of preproparathyroid hormone was studied. NH2-terminal extensions, or leader sequences, may serve a critical role in determining and facilitating the cellular secretion of proteins. These precursor regions, including the synthetic hormonal fragment studied, share common features of amino acid sequence and also may be similar in secondary structure. The secondary structure of the synthetic precursor peptide was predicted as described [Chou, P. Y. & Fasman, G. D. (1978) Adv. Enzymol. 47, 45-148]. The secondary structure was derived from circular dichroism spectra in both an aqueous buffer at physiological pH and in a nonpolar solvent selected to approximate the intramembranous environment. Two highly structured conformations were observed. In the aqueous buffer the secondary structure was 27% alpha-helix, 43% beta-sheet, and 30% random coil. In the nonpolar solvent the secondary structure was 46% alpha-helix, 0% beta-sheet, and 54% random coil. These findings correlated well with the two highest-probability structures predicted from the amino acid sequence. Both the relatively high content of secondary structure in a peptide of this size (30 amino acids) and the conformational transition observed in changing from aqueous to nonpolar environments may reflect structural properties critical to the physiological function of NH2-terminal extension sequences, and both are consistent with current theories regarding the role of precursor regions in the intracellular transport and secretion of proteins.

Circular Dichroism↗
PubMed1979

Quenching of intrinsic tryptophan fluorescence in membranes of rat pituitary cells.

The GH4C1 strain of hormone-producing rat pituitary cells has specific receptors for the tripeptide thyrotropin-releasing hormone (TRH). Membranes prepared from GH4C1 cells show intrinsic tryptophan fluorescence which was quenched by low concentrations (10--100 nM) of TRH and Ntau-methyl TRH but not by biologically inactive analogs of TRH. Membranes from GH4C1 cells were subjected to thermal denaturation. A conformational transition was noted above 40 degrees C and an irreversible denaturation was observed at 52 degrees C. TRH-induced quenching of intrinsic fluorescence was lost completely in membranes previously incubated for 10 min at 30 degrees C while loss of [3H]-TRH binding was only about 20% at this temperature. Collisional quenching by iodide revealed that about 38% of the tryptophanyl residues in GH4C1 membranes were exposed to solvent. Quenching by TRH occurred with a shift in wavelength maximum from 336 to 342 nm suggesting that few of the tryptophanyl residues quenched by the tripeptide are totally exposed. Membranes prepared from cells preincubated with 20 nM TRH for 48 h, in which TRH receptors were decreased to 30% of control values, showed no quenching of tryptophan fluorescence in response to freshly added TRH. We conclude that the TRH-receptor interaction in GH4C1 cells is associated with a change in membrane conformation that can be measured by differential spectrofluorometry of intrinsic tryptophan fluorescence.

Animals↗
PubMed1979

Analysis of chromatin reconstitutiion.

The ability of high molecular weight chicken erythrocyte chromatin to spontaneously self-assemble into native-like material, after dissociation by high ionic strength and reassociation by salt gradient dialysis, was critically examined. The native conformational state of the reassembled nucleoprotein complex was regenerated to the extent reflected by circular dichroism spectra and thermally induced helix--coil transition of the nucleoprotein DNA. However, internucleosomal packing of approximately 205 base pairs of DNA per repeating unit, as probed by digestion with micrococcal nuclease, was not regenerated upon reassembly and was replaced by a packing of approximately 160 base pairs per repeating unit. Thus, high molecular weight chromatin containing only lysine-rich histones (H1 and H5) and core histones (H2A, H2B, H3, and H4) is not a true self-assembling system in vitro using the salt gradient dialysis system used herein. Circular dichroism and thermal denaturation studies on core chromatin (lysine-rich histones removed) showed that core histones alone are not capable of reassembling high molecular weight DNA into native-like core particles at low temperature (4 degree C). Reassembly at 21 degree C restored the circular dichroism but not the thermal denaturation properties to those characteristic of undissociated core chromatin. Nonetheless, micrococcal nuclease digestions of both reassembled core chromatin products were identical with undissociated native core chromatin. Ressembly in the presence of the complete complement of histones, followed by removal of the lysine-rich histones, did regenerate the thermal denaturation properties of undissociated native core particles. These results indicated multiple functions of the lysine-rich histones in the in vitro assembly of high molecular weight chromatin.

Animals↗
PubMed1979

Prediction of beta-turns.

An automated computer prediction of the chain reversal regions of globular proteins is described herein using bend frequencies and beta-turn conformational parameters (Pt) determined from 408 beta-turns in 29 proteins calculated from x-ray atomic coordinates. The probability of bend occurrence at residue i is pt = fi X fi+1 X fi+2 X fi+3 with the average bend probability less than Pt greater than = 0.55 X 10(-4). Tetrapeptides with pt greater than 0.75 X 10(-4) ( approximately to 1.5 X less than pt greater than) as well as less than Pt greater than 1.00 and less than Pa greater than less than less than Pt greater than greater than less than P beta greater than are selected by the computer as probable bends. Adjacent probable bends (i.e., 11-14, 12-15, 13-16) are compared pairwise by the computer, and the tetrapeptide with the higher pt value is predicted as a beta-turn. The percentage of bend and nonbend residues predicted correctly for 29 proteins by this computer algorithm is %t+nt = 70%, whereas 78% of the beta-turns were localized correctly within +/- 2 residues. The average beta-turn content in the 29 proteins is 32%, with helical proteins having fewer bends (17%) than beta-sheet proteins (41%). Three proteins having iron-sulfur clusters were found with the highest percentages of beta-turns: Chromatium high potential iron protein (65%), ferredoxin (57%), and rubredoxin (65%). Finally, the bend frequencies at all 12 positions from 457 beta-turns in 29 proteins (Chou and Fasman, 1977) were used to test the effectiveness of predicting bends using 2, 4, 8, and 12 residues as well as different cut-off pt values. The computer analysis showed that 1.25 less than pt greater than to be the best cut-off yielding 70% accuracy in %t+nt for 4 residues and %t+nt = 73% for 12 residues in predicting the bend and nonbend regions of proteins.

Amino Acid Sequence↗
PubMed1979

Conservation of chain reversal regions in proteins.

Using the bend frequencies based on 29 proteins in the previous paper (Chou and Fasman, 1979), beta-turn probability profiles were calculated for the C-peptides of 10 mammalian proinsulins, for 7 proteinase inhibitors, and for 12 species of pancreatic ribonucleases. Beta-turn correlation coefficient matrix tables were also computed to obtain the statistical mean between 45 pairs of proinsulin C-peptides, less than Ct greater than = 0.57 +/- 0.31; 21 pairs of proteinase inhibitors, less than Ct greater than = 0.73 +/- 0.13; and 66 pairs of ribonucleases, less than Ct greater than = 0.83 +/- 0.08. Despite relatively low sequence conservation in these three sets of proteins, beta-turns were predicted to be highly conserved: 33% sequence vs. 78% bend for the proinsulins, 20% sequence vs. 85% bend for the proteinase inhibitors, and 65% sequence vs. 92% bend for the ribonucleases. These results suggest that chain reversal regions play an essential role in keeping the active structural domains in hormones and enzymes intact for their specific biological function.

Amino Acid Sequence↗
PubMed1978

High resolution proton magnetic resonance spectroscopy of histones and histone--histone complexes in aqueous solution.

Low molecular weight histone complexes of H2A (congruent to dimer), H2B (congruent to tetramer), H3--H4 (congruent to tetramer), H2A--H2B (congruent to dimer), and H2B--H4 (congruent to dimer) have been prepared in 2 M NaCl and neutral pH at 4 degrees C. These materials are free of nonspecific aggregate and are suitable for study by high resolution proton magnetic resonance spectroscopy. Such spectra have been recorded in aqueous solutions under conditions allowing a study of the exchangeable proton resonances of histone complexes for the first time and indicate that the structured regions are rich in hydrophobic amino acids, as well as arginine and some acidic amino acids. Most of the lysine and probably alanine residues remain in a motile, random coil-like state after formation of the complexes. It is suggested that arginine residues may be important in inter- and/or intra-subunit interactions in histone complexes.

Animals↗
PubMed1978

Circular dichroism analysis of mononucleosome DNA conformation.

Mononucleosomes were isolated from micrococcal nuclease digests of chicken erythrocyte nuclei. The circular dichroism properties of mononucleosome preparations, differing in average DNA length and in H1 and H5 content, demonstrate that the spectrum of chromatin is due only to the complete structure of its repeating subunits. The nucleoprotein spectra are all altered relative to protein-free DNA by the emergence of a single negative band at 275 nm, similar to the band observed for psi DNA. The intensity of the psi-type band depends on the proportion of DNA condensed in a specific manner. The psi-type band is proposed to be due to the compact DNA tertiary structure; i.e., the manner in which the DNA is wound around the histone core allowing interactions between adjacent turns of the superhelix. This interpretation attributes changes and variability in nucleoprotein circular dichroism spectra under different experimental conditions to alterations in DNA tertiary structure rather than secondary structure.

Chromatin↗