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G D Fasman

Publications and source records attributed to G D Fasman.

At least 91 records · Page 5Linked to original sources
PubMed1977

Interaction of DNA with poly(L-Lys-L-Ala-Gly) and poly(L-Lys-L-Ala-L-Pro). Circular dichroism and thermal denaturation studies.

Complexes of DNA with polypeptides composed of Lys, Ala, and Gly in both a sequential order, poly(L-lysine-L-alanine-glycine), and a statistical distribution, poly(L-lysine36-L-alanine28-glycine), were prepared using gradient dialysis. These polypeptide-DNA complexes were studied using ultraviolet absorption (UV) and circular dichroism (CD) to probe the conformation, binding, and melting behavior of DNA in the complex. Complexes with the sequential polypeptide showed no structural change in the DNA; however, the complexes with the random polypeptide yield CD spectra similar to phi DNA [Maniatis, T., Venable, Jr., J.S., and Lerman, L.S. (1974), J. Mol. Biol. 84, 37]. A second sequential polypeptide, poly(L-Lys-L-Ala-L-Pro)n, -DNA complex was also studied. It was found to exhibit pronounced structural changes as a function of ionic strength and poly-peptide-DNA ratio, more similar to the random sequence that the ordered sequence of the Lys, Ala, Gly polymer. Thus the importance of the composition and amino acid sequence in polypeptides which bind to DNA, even in such simple systems, is demonstrated. Evidence from thermal denaturation, employing simultaneous monitoring of CD and UV changes, supports a model in which specific polypeptides cause condensation of the DNA in the complex into an asymmetric tertiary structure. The relevance of these model systems to chromatin is discussed.

Alanine↗
PubMed1976

Chromatic models. Interactions between DNA and polypeptides containing L-lysine L-valine: circular dichroism and thermal denaturation studies.

The interaction of calf thymus DNA with statistical copolymers of L-lysine and L-valine [poly(L-Lys100f-Lvalf)] and block copolymers [poly(L-Lys)100f-poly(L-Val)f] were investigated as a function of ionic strength using circular dichroism (CD) spectroscopy. It was found that valine suppresses the ability of the copolymer-DNA complexes to yield a psi-type CD spectra as found for poly(L-Lys)-DNA [Jordan, C.F., Lerman, L.S., and Venable, J.N. (1972), Nature (london), New Biol. 236, 67] and lowers the ionic strength at which CD distortion occurs. Thermal denaturation, simultaneously monitoring 280-nm ellipticity, [theta]280, and hyperchromicity, h280, was carried out on annealed complexes of poly(L-Lys)-DNA, poly(L-Lys84.5-L-Val15.5)-DNA, poly(L-Lys)87.2-poly(L-Val)12.8-DNA, and directly mixed complexes of poly(L-Lys)-DNA, IN 2.5 X 10(-4) MEDTA, pH 7.0 solution. The CD denaturation of uncomplexed DNA at several ionic strengths was also determined to examine pre-melting. Despite the inability of both statistical and block copolymers of L-Lys and L-Val to form psi-type complexes with DNA, they bind as well to DNA as does poly(L-Lys) and give rise to a thermal denaturation pattern showing bound peaks between 90 and 100 degrees C, seen clearly with CD denaturation. The thermal denaturation of mixed and annealed complexes of poly(L-Lys)-DNA shows similar patterns in hyperchromicity changes as a function of temperature but very different CD melts. From the CD melt of annealed poly(L-Lys)-DNA, it appears that aggregation and long-range order of the complex are significant in low salt (2.5 X 10(-4) MEDTA) as well as in 1.0 M NaCl. These studies further illustrate the importance of the nature of nonionic interactions (hydrophobic) between polypeptides and DNA in determining the behavior of their complexes, such as causing condensation into higher order asymmetric structures. In light of these observations, the possible significance to the CD melting of chromatin and the validity of identification of C-form DNA by CD spectroscopy are discussed.

Chromatin↗
PubMed1976

Chromatin models. The ionic strength dependence of model histone-DNA interactions: circular dichroism studies of lysine-leucine polypeptide-DNA complexes.

The ionic strength dependence of the complexes between DNA and both random, (Lysx, Leuy)n, and block copolymers, (Lysx)n(Leuy)m, of lysine and leucine, with different amino acid compositions, was studied using circular dichroism (CD) as the probe to detect conformational differences in these complexes relative to native DNA. It was found that the CD spectra of complexes of both the random (Lys84, Leu16)n and block (Lys85)n(Leu15)m copolymers with DNA show a very sharp ionic strength dependence. The maximum altered CD spectrum for the complexes with the block copolymer was found to occur at the same ionic strength as that for poly(L-lysine)-DNA complexes, while the maximum CD change for the random copolymer complex occurred at a slightly lower ionic strength. This sharp dependence of the CD change on the ionic strength was found to be independent of the polymer/DNA ratio, r, for each individual copolymer. The CD spectra for these complexes at optimum NaCl concentration resemble those of the psi spectra of DNA [Jordan, C. F., Lerman, L.S., and Venable, J.H. (1972), Nature (London), New Biol. 236, 67]. The complexes of the random copolymer, (Lys68, Leu32)n, with DNA (r=0.25) at 0.15 M NaCl and below have CD spectra that resemble the A-form DNA spectra. The ionic strength dependence of the CD spectra of this complex is not as sharp as observed with the above polymers and has a broad positive plateau. It is suggested that both the CD spectra of these complexes reflect the phenomena of DNA condensation into a higher order asymmetric structure (folded and compact). The block copolymer, (Lys77)n(Leu23)m, complexes with DNA show very slight alterations in the CD spectra, with respect to native DNA. It appears that the long Leu sequence at one end of such copolymers may be unpropitious for causing the polypeptide-DNA complex to condense into a higher order asymmetric structure. Thus the importance of the distribution of hydrophobic residues, in the copolypeptides of Lys, is shown for causing condensation of complexes with DNA. The relevance of these findings to histone-DNA complexes in chromatin is discussed.

Binding Sites↗
PubMed1976

Chromatin models. Thermal denaturation studies of (Lysx, Leuy)n-and (Lys)n(Leu)m-DNA complexes.

The structural transitions of (Lysx, Leuy)n-DNA and (Lysx)n(Leuy)m-DNA complexes have been studied by thermal denaturation utilizing simultaneous absorption and circular dichroism (CD) measurements [R. Mandel and G.D. Fasman (1974), Biochem. Biophys. Res. Commun. 59, 672]. These complexes are used as models for nucleohistones. At amino acid/nucleotide ratios r less than 1, the copolymers bind to DNA in a ratio of one amino acid residue per nucleotide, and such binding stabilizes the DNA double helix against thermal denaturation relative to the unbound regions. The leucine residues in the copolymers stabilize the bound portion of the complex against thermal denaturation but to a lesser degree than does poly(L-lysine). This study confirms the hypothesis that absorption melting profiles reflect only the change in secondary structure (helix-coil transition) of DNA. It was found that, in the absence of a higher ordered structure (condensed), the CD melting profile also reflects this same conformational transition, and the melting temperatures, Tm, in CD are equal to those in absorption. However, when a higher ordered structure (tertiary) exists in the complex, then the CD melting profile will be dominated by the structural transitions related to the melting of the higher ordered asymmetric structure in the condensed state, followed by the melting of the secondary structure. Under such circumstances, the Tm obtained from absorption may be slightly different from that of the CD, since only the secondary structural changes are being reflected in absorption. The relevance of these studies to the structure of chromatin is discussed.

Binding Sites↗
PubMed1976

Prediction of the conformation of the histones.

The secondary structures of the histones, H1, H2A, H2B, H3, and H4 have been predicted utilizing the predictive scheme of Chou and Fasman (Biochemistry 13:211, 222[1974]) and a new set of conformational parameters based on the X-ray data of 29 protein structures. The alpha-helical, beta-sheet, reverse beta-turns, and random coil regions of these proteins are carefully delineated. Structures are specified which are most probably under various environmental conditions, i.e., for changes in ionic strength, association between histones and in association with DNA. Potential conformational changes within these histones are also predicted.

Amino Acid Sequence↗
PubMed1976

Chromatin and nucleosome structure.

Chromatin nucleosomes (mononucleosomes through pentanucleosomes) have been isolated by staphylococcal nuclease digestion of calf thymus nuclei. The peak value ellipticity is the same for all oligomers, 1900 deg cm2, mol-1 at 280-nm, 23 degrees C. The dh280/dT vs T show a progressive increase in Tm of the main thermal band (73.5 degrees C, monomer; 79 degrees C, pentamer). Very small amounts of free DNA can be observed in the melting profiles, and shoulders at 60 degrees C and 93 degrees C appear and increase in magnitude as the particle size increases. The magnitude of the change, delta[theta]280, increases with oligomer size. This pattern could result from an initial unfolding of an asymmetric assembly of nucleosomes (polynucleosome superhelix) in addition to the denaturation of the internal nucleosome structure, and a subsequent or simultaneous denaturation of the double strand DNA. The extent of this unfolding appears to depend upon the size of the oligomer and therefore implies interactions between asymmetrically assembled neighboring nucleosomes.

Animals↗
PubMed1975

Complexes of DNA with histones f2a2 and f3. Circular dichroism studies.

Two histones from calf thymus, the slightly lysine-rich histone f2a2 and the arginine-rich f3, were combined separately, with homologous DNA. The complexes were reconstituted by means of guanidine hydrochloride gradient dialysis, and their circular dichroic (CD) spectra were examined in 0.14 M NaCl. The CD spectra of f2a2-DNA complexes are characterized by a positive band at 272 nm which is blue-shifted and greatly enhanced relative to the corresponding band for native DNA. This type of CD change was noted previously with f2a1-DNA and f2b-DNA complexes. In contrast, f3 histone causes only minor distortions in the DNA CD spectrum, and their character depends upon the state of the two sulfhydryl groups in f3. When the cysteines are reduced, f3-DNA complexes have a slightly increased positive band with a small blue shift; when oxidized disulfide is the predominant form, this CD band becomes slightly smaller than native DNA value. This laboratory has now examined complexes reconstituted from DNA and all five histones of calf thymus. The sum of the CD spectra of these complexes, although very similar to the CD curve for reconstituted complexes containing whole histone, does not approximate that of chromatin; the consequence of this observation is discussed.

Animals↗
PubMed1975

The conformation of glucagon: predictions and consequences.

It is proposed that glucagon, a polypeptide hormone, is delicately balanced between two major conformational states. Utilizing a new predictive model [Chou, P.Y., and Fasman, G.D. (1974), Biochemistry 13, 222] which considers all the conformational states in proteins (helix, beta sheet, random coil, and beta turns), the secondary structural regions of glucagon are computed herein. The conformational sensitivity of glucagon may be due to residues 19-27 which have both alpha-helical potential (mean value of Palpha = 1.19) as well as beta-sheet potential (mean value of Pbeta = 1.25). Two conformational states are predicted for glucagon. In predicted form (a), residues 5-10 form a beta-sheet region while residues 19-27 form an alpha-helical region (31% alpha, 21% beta) agreeing well with the circular dichroism (CD) spectra of glucagon. The similarity in the CD spectra of glucagon and insulin further suggests the presence of beta structure in glucagon, since X-ray analysis of insulin showed 24% beta sheet. In predicted form (b), both regions, residues 5-10 and residues 19-27, are beta sheets sheets (0% alpha, 52% beta) in agreement with the infrared spectral evidence that glucagon gels and fibrils have a predominant beta-sheet conformation. Since three reverse beta turns are predicted at residues 2-5, 10-13, and 15-18, glucagon may possess tertiary structure in agreement with viscosity and tritium-hydrogen exchange experiments. A proposal is offered concerning an induced alpha yields beta transition at residues 22-27 in glucagon during receptor site binding. Amino acid substitutions are proposed which should disrupt the beta sheets of glucagon with concomitant loss of biological activity. The experimental findings that glucagon aggregates to form dimers, trimers, and hexamers can be explained in terms of beta-sheet interactions as outlined in the present predictive model. Thus the conflicting conclusions of previous workers, concerning the conformation of glucagon in different environments, can be rationalized by the suggested conformational transition occurring within the molecule.

Amino Acid Sequence↗
PubMed1975

Interaction of histone f2al fragments with deoxyribonucleic acid. Circular dichroism and thermal denaturation studies.

The glycine-arginine-rich histone, f2al (IV) (102 amino acids), from calf thymus was cleaved at residue 84 with cyanogen bromide. Complexes containing homologous DNA and each f2al fragment were reconstituted by means of Gdn-HC1 gradient dialysis. The circular dichroic (CD) spectra of these complexes were all examined in 0.14 M NaC1. The CD spectra of the DNA-f2al fragment complexes did not differ appreciably from that of DNA alone in the wavelength region above 240 nm. However, intact f2al-DNA complexes yield CD spectra which differ significantly (enhanced, blue-shifted, 273-nm band) from that of native DNA (Shih and Fasman, 1971). The small C-terminal fragment (85-102) was bound weakly to DNA under the conditions used. However, the large basic N-terminal fragment (1-83) was bound as well to DNA as was whole f2al, but produced no CD distortion. The conformation of the N-terminal fragment, unlike intact f2al, was not changed upon increasing the ionic strength to 0.14 M NaF. These results complement previous studies on f2al and its N-terminal CNBr fragment (Ziccardi and Schumaker, 1973). Thermal denaturation of the complexes in 2.5 X 10(-4) M EDTA was monitored simultaneously by changes in the absorption and CD spectra. All complexes showed a thermal transition at 45 degrees (Tml), attributable to the melting of free, double-stranded DNA. In addition, f2al-DNA and N fragment-DNA complexes displayed melting phenomena at 88 and 78 degrees (Tm2), respectively, caused by the denaturation of the histone-bound DNA. This difference in Tm2 constitutes further evidence that loss of the 18-amino-acid carboxyl end segment of f2al prohibits the unique type of interaction which occurs between DNA and the intact histone.

Animals↗