Biomedical subjects
Publications and source records attributed to G D Fasman.
The prediction of the secondary and tertiary structure of globular and membrane proteins is reviewed. Prospects are encouraging for future developments, but present algorithms require cautious interpretation.
Peptides containing 13 and 39 amino acid residues and serine-side-chain-phosphorylated (P) analogues thereof, corresponding to human neurofilament protein middle-sized subunit (NF-M), have been synthesized in order to localize the phosphorylation site of this protein. The secondary structure of the nonphosphorylated peptides, determined by circular dichroism (CD) measurements, predicted secondary structural calculations and energy conformational calculations, was suggested to be a series of alternating type I (III). beta-turns and 3(10) or alpha-helices. By contrast, the phosphorylated peptides exhibit a unique conformation, probably due to salt bridges between the phosphoserine and the lysine residues. This has provided the first clear evidence that phosphorylation induces conformational changes among these synthetic peptides and presumably, in NF proteins as well. These phosphorylation loops might be the major recognition sites of the neurofilament protein-directed kinases.
The conformation of the histone octamer complex in solution has been shown, by circular dichroism studies, to be highly dependent on the nature of the salt milieu and its concentration. In 2 M NaCl, the complex has 43.5% alpha-helix, 16% beta-sheet, and 40.5% random structure. In 2.3 M (NH4)2SO4, the octamer has 49.0% alpha-helix and 51% random structure. These results partially explain the discrepant results obtained by the X-ray analysis of crystals obtained under varying conditions.
The role of acetylation on the conformation and association state of the inner core histone octamer isolated from HeLa cells was examined. Preparation of suitable quantities of pure acetylated and control inner core histones from HeLa cells required the development of a new preparative procedure. The results from size-exclusion high-performance liquid chromatography and sedimentation equilibrium studies indicated that acetylated inner core histones associate to species larger than the octamer and form a more stable complex. Circular dichroism studies demonstrated that the amount of alpha-helix increases with increasing association of the histones. Furthermore, acetylation results in an increase in the amount of alpha-helix, perhaps coupled through its effect on the association state. At high protein concentration and elevated temperature, the acetylated sample displays a greater increase in beta-sheet content, relative to the control sample. This increase in beta-sheet content may be induced during the association of the acetylated sample to species larger than the octamer. There is a marked effect on the conformation of both acetylated and control inner core histones as a function of protein concentration, ionic strength, and temperature. The difference in conformational flexibility and association state of the acetylated vs. the control inner histone core may play a significant role in the control of transcription in the nucleus.
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Chicken erythrocyte chromatin was prepared according to two different methods [Fulmer, A. W., & Bloomfield, V. A. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 5968-5972; Ausio, J., Borochov, N., Seger, D., & Eisenberg, H. (1984) J. Mol. Biol. 177, 373-398] to give three main common fractions, according to its solubility (S) or insolubility (I) in 0.15 M NaCl buffers or to its further solubility in 0.25 mM ethylenediaminetetraacetic acid (E). From the biochemical point of view, all of them have been found to be undistinguishable. Analytical ultracentrifugation shows that all of these fractions can reversibly undergo the transition from the low to the higher order structure, through a nearly identical way of folding. Thermal denaturation profiles yielded three transitions having the same Tm's for the three fractions. The percentage of DNA melting in the first transition decreased in the order S greater than I greater than E, and the amount in the second transition increased in the same order. Together with the different solubility of these fractions in the presence of divalent ions, these results indicate that in the three fractions of chromatin studied, the amount of linker DNA bound to the nucleosome varied.
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Synthetic peptides corresponding to the first 23 amino acids of the glycoprotein D molecule of herpes simplex virus have been used to immunize mice and examine the role of antigen conformation in T cell responses. The structure of the peptides was determined by circular dichroism studies and was shown to be consistent with theoretical structure predictions. T cell clones were found that could respond to peptides that were nonhelical, and this response, which was directed to the C-terminal determinant (residues 8-23), could be modified by a constrained N-terminal sequence (residues 1-7) of the peptide. Namely, substitution of Ala for Pro at position 7 induced an alpha-helix and inhibited the response. Furthermore, a response to the N-terminal part of the molecule seemed to be directed to the alpha-helix and correlated positively with calculated degree of helicity. This response was also modified, in this case, by the C-terminal part of the molecule. These results suggest that local secondary structure of an antigen may regulate T cell responses and that structural changes in the peptide antigen downstream from the determinant modify recognition of that determinant. Furthermore, antigen processing by the macrophage and antigen presentation of T cells appears to conserve antigen structural integrity.
The effect of binding enzymatically phosphorylated H1 histone on the structure of reconstituted chromatin was determined by circular dichroism and thermal denaturation studies at low ionic strength. Procedures were developed for the selective removal of the lysine-rich histones H1 and H5 from chicken erythrocyte chromatin (stripped chromatin) and for the specific reconstitution of chromatin, without nucleosome sliding. Reconstitution was carried out with either rat thymus H1, phosphorylated rat thymus H1 (containing an average of 5.3 phosphates/molecule, or chicken erythrocyte H5. The maximum ellipticity in 1.0 mM phosphate buffer, pH 7.4, of stripped chromatin [( theta]282.5 = 4800 degree cm2/dmol) was effectively reduced to the value for native chromatin [( theta]282.5 = 3900 degree cm2/dmol) through reconstitution with approximately one molecule of either H1 [( theta]282.5 = 4050 degree cm2/dmol) or phosphorylated H1 [( theta]282.5 = 4100 degree cm2/dmol) per 200 base pairs of DNA. As the circular dichroic spectra for chromatin reconstituted with H1 and phosphorylated H1 are similar, it appears that phosphorylation per se does not induce a major structural alteration of chromatin structure. Thermal denaturation studies at low ionic strength of chromatin reconstituted with either H1 or phosphorylated H1 revealed a transition that occurs at a slightly lower temperature than the high temperature transition of native chromatin. This transition was shifted approximately 1 degree C to lower temperature when the chromatin was reconstituted with phosphorylated H1 as compared to H1. Thus, phosphorylation of H1 caused a significant destabilization of the chromatin structure at low ionic strength. When thermal denaturation was carried out on chromatin reconstituted with H5, native-like profiles were obtained.
The effect of binding a high mobility group protein (HMG 17) on the stability and conformation of acetylated and control HeLa high molecular weight core chromatin (stripped of H1 and non-histone chromosomal proteins) was studied by circular dichroism and thermal-denaturation measurements. Previously it had been shown that conformational differences exist between native whole chromatin derived from butyrate-treated (acetylated) and control HeLa cells and that these conformational differences disappear by removing H1 and non-histone chromosomal proteins ( Reczek , P.R., Weissman , D., Huvos , P.E. and Fasman, G.D. (1982) Biochemistry 21, 993-1002). The circular dichroism spectra and the thermal denaturation profiles of control and acetylated core chromatin were found to be similar. The circular dichroism properties of HMG 17 reconstituted highly acetylated and control core chromatin indicated the same alteration of chromatin structure at low ionic strength (1 mM sodium phosphate/0.25 mM EDTA, pH 7.0). The magnitudes of the decrease in ellipticity were proportional to the amount of HMG 17 bound and were found to be the same for both the acetylated and control core chromatin. Thermal denaturation profiles confirmed this change in structure induced by HMG 17 on control and highly acetylated core chromatin. The thermal denaturation profiles, which were resolved into three component transitions, exhibited a shifting of hyperchromicity from the lower melting transitions to the higher melting transitions, with a concomitant rise in Tm, on HMG 17 binding to both control and acetylated chromatin. The natures of the interactions of HMG 17 at higher ionic strength (50 mM NaCl/0.25 mM EDTA/1 mM sodium phosphate, pH 7.0) with acetylated and control core chromatin were slightly different, as measured by circular dichroism; however, a decrease in ellipticity was observed for both samples upon binding of HMG 17. These observations suggest that acetylation coupled with HMG 17 binding to core chromatin does not loosen chromatin structure. HMG 17 binding to control and acetylated core chromatin produces an overall stabilization and compaction of chromatin structure.
The technique of nick translation of nuclei (Levitt, A., Axel, R., and Cedar, H. (1979) Dev. Biol. 69, 496-505) has been used in HeLa cells to label DNase I-sensitive regions. Micrococcal nuclease digestion of the nick translated nuclei was followed by a low ionic strength gel electrophoresis system which separates different types of mononucleosomes. The major label was observed in the vicinity of high mobility group protein containing mononucleosomes. However, further analysis revealed that the particle does not sediment in the position of mononucleosomes on a sucrose gradient. Two alternative explanations are discussed as the possible source of this particle. It is either a high mobility group protein containing nucleosome in some unfolded conformation or the labeled particle originates from discrete DNA fragments, wrapped around some nonhistone proteins, located in a highly DNase I-sensitive region, which is resistant to micrococcal nuclease digestion.
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The circular dichroism spectra of four beta-turn model peptides, Z-Aib-Pro-Aib-Pro-OMe (1), Piv-Pro-Aib- NHMe (2), Piv-Pro-D-Ala- NHMe (3) and Piv-Pro-Val- NHMe (4) have been examined under a wide range of solvent conditions, using methanol, hexafluoroisopropanol and cyclohexane. Type I and Type II beta-turns have been observed for peptides 1 and 2 respectively, in the solid state, while the Pro-D-Ala sequence adopts a Type II beta-turn in a related peptide crystal structure. A class C spectrum is observed for 1 in various solvents suggesting a variant of a Type I (III) structure. The Type II beta-turn is characterized by a CD spectrum having two positive CD bands at approximately 230 nm and approximately 202 nm, a feature observed in Piv-Pro-D-Ala- NHMe in cyclohexane and methanol and for Piv-Pro-Aib- NHMe in methanol. Peptide 2 exhibits solvent dependent CD spectra, which may be rationalized by considering Type II, III and V reverse turn structures. Piv-Pro-Val- NHMe adopts non-beta-turn structures in polar solvents, but exhibits a class B spectrum in cyclohexane suggesting a population of Type I beta-turns.
Core histones (H2A,H2B,H3,H4)2, were reconstituted with the synthetic polynucleotides poly(dA-dT) X poly(dA-dT) and poly(dG-dC) X poly(dG-dC) to yield synthetic chromatins containing 200 basepairs per octamer. These synthetic chromatins displayed a 36% decrease in the circular dichroism (CD) peak ellipticity from the value of the polynucleotide free in solution; the poly(dA-dT) X poly(dA-dT)/chromatin showed an increase in the complexity of the thermal denaturation profile compared to that of the polynucleotide. Both the temperature of maximum dh/dT for each transition (Tm) and the relative amount of poly(dA-dT) X poly(dA-dT) in the synthetic chromatin melting in each of the four thermal transitions is a function of the ionic strength over the 0-5 mM sodium phosphate range (0.25 mM EDTA, pH 7.0); a shift of material toward higher melting transitions was observed with increasing ionic strength. The CD peak ellipticity value for both synthetic chromatins was ionic strength-independent over the 0-5 mM sodium phosphate range. These results are in contrast to those observed with H1/H5 stripped chicken erythrocyte chromatin (Fulmer, A. and Fasman, G.D. (1979) Biopolymers 18, 2875-2891), where an ionic strength dependence was found. Differences in the CD spectra between poly(dA-dT) X poly(dA-dT)/chromatin, poly(dG-dC) X poly(dG-dC)/chromatin and H1/H5 stripped chicken erythrocyte chromatin suggest subtle differences in assembly. Finally, the temperature dependence of the CD spectra of poly(dA-dT) X poly(dA-dT)-containing synthetic chromatin, which is similar to that for the polynucleotide, suggests the core histone bound polynucleotide has a large degree of conformational flexibility allowing it to undergo the premelt transition.
The nature of the binding of a high mobility group protein (HMG 17) to native and H1-H5-depleted chicken erythrocyte chromatin was studied, as a function of ionic strength, using circular dichroism and thermal denaturation techniques. The circular dichroism properties of the HMG 17-reconstituted whole chromatin and H1-H5-depleted chromatin demonstrated that a condensation of chromatin structure occurred upon HMG 17 binding at low ionic strength (1 mM Na phosphate, 0.25 mM EDTA, pH 7.0). Thermal denaturation profiles confirmed this change in the structure of chromatin induced by HMG 17. Thermal denaturation profiles were resolved into three-component transitions. In general, a shift in the temperature of maximum dh/dT for each transition (Tm) was observed for all transitions upon HMG 17 binding. DNA melting in the first transition, originating from linker regions of whole chromatin, was nearly totally depleted and was distributed mainly into the highest melting transition. The same trends were also observed in H1-H5-depleted chromatin. These results indicate that the binding sites of HMG 17 are situated in the linker regions immediately adjacent to the core. The nature of the interaction of HMG 17 at higher ionic strength (50 mM NaCl, 1 mM Na phosphate, 0.25 mM EDTA, pH 7.0) with whole chromatin and H1-H5-depleted chromatin was found to be different but a decrease in [theta] values was found in both chromatins. These observations suggest that HMG 17 does not loosen chromatin structure but produces an overall stabilization and condensation of structure. The implications of these results to the currently accepted models of transcriptionally active chromatin are discussed.
Changes in the conformations of H3-H4, H2A-H2B, and the core histone complex brought about by the addition of organic solvents have been examined by circular dichroism spectroscopy. All three complexes assume increased alpha helicity with increasing amounts of the organic solvent. An amount of secondary structure equal to that obtained in phosphate-buffered 2 M NaCl solution can be induced in low-salt solutions of the complexes by the addition of 40-50% ethylene glycol, 50% glycerol, or approximately 2% hexafluoro-2-propanol. H3-H4 was found to be somewhat more flexible than H2A-H2B in its response to changes in solvent polarity. Upon being heated, H3-H4 and the core histone complex both undergo irreversible alpha leads to beta transitions in 50% ethylene glycol under low-salt conditions, while H2A-H2B undergoes an essentially reversible alpha leads to beta random-coil transition under the same conditions. These results are discussed in terms of the dynamics of the nucleosome particle.