Proteases as structural probes for chromatin: the domain structure of histones.
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Amino Acid Sequence↗
Biomedical subjects
C Crane-Robinson
Publications and source records attributed to C Crane-Robinson.
Proteins from the sperm of the bivalve mollusc Ensis minor. Co-existence of histones and a protamine-like protein.
Analysis of the total protein of the mature sperm of the bivalve mollusc Ensis minor (razor shell) using gel electrophoresis, amino acid analysis, nuclear magnetic resonance, circular dichroism and trypsin digestion, show it to contain all five histones plus a protamine-like protein. The histones H3, H4 and probably H2A are similar to those from calf thymus or sea urchin sperm, but the putative H2B appears to have a very high molecular mass (about 20 kDa). The histone H1 molecule is unusual, having little or no proline and 8-10 residues of histidine. The protamine-like species is rich in both lysine as well as arginine and is of much higher molecular mass than fish sperm protamines. Nucleosomes containing the four core histones have been prepared and the nucleosomal repeat shown to be 200 +/- 5 base pairs. Checks for the absence of contaminating cells reinforce the conclusion that a histone-containing nucleosomal structure co-exists with a protamine-like protein in this sperm chromatin.
Amino Acids↗
Nuclease digestion patterns as a criterion for nucleosome orientation in the higher order structure of chromatin.
Nuclease digestion patterns have been used to discriminate between possible orientations of nucleosomes in the higher order structure of chromatin. Computer simulations were done assuming 3 basically different orientations of nucleosomes which include all proposed models for the '30 nm fibre'. It is found that only alternating exposure of consecutive nucleosomes can explain the DNase I and DNase II digestion patterns.
Animals↗
Isolation of a 167 basepair chromatosome containing a partially digested histone H5.
A test has been made of the proposal that protection of the 167 basepair DNA length in the 'chromatosome' is due only to the central globular domain of the lysine-rich histones. Chicken erythrocyte chromatin was treated with trypsin to leave only the limit peptide from histones H1 and H5. Nucleosome monomers were then isolated on sucrose gradients following micrococcal nuclease digestion and were found to contain the 167 basepair DNA band as in intact chromatin. The presence of the limit peptide from H5 on the monomers was confirmed using an antibody to H5. It is concluded that the trypsin-susceptible domains of the lysine-rich histones are not involved in the protection of the 2-turn 167 basepair length of DNA in the nucleosome.
Animals↗
Conformation and domain structure of the non-histone chromosomal proteins, HMG 1 and 2. Isolation of two folded fragments from HMG 1 and 2.
Proteins HMG 1 and 2 have been digested with trypsin and two major products, stable to further digestion between 8 min and 2 h, have been purified (peptides A and B). Peptide B from HMG 1 has been identified as residues 12-75 and peptide A as residues 94/96-169 by amino acid analyses and Edman degradations. Peptide B spontaneously folds with the formation of 51% helix and exhibits the majority of the perturbed NMR resonances characteristic of folded intact HMG 1. Peptide B is stably folded in the presence of 150 mM NaCl between pH 3 and 10, like intact HMG 1. Peptide A forms 30% alpha-helix and also exhibits tertiary folding but is denatured by pH 10. The 11 N-terminal residues removed by trypsin contain both sites of post-synthetic acetylation (residues 2 and 11), a situation very similar to that found with core histones. It is proposed that HMG 1 and 2 consist of four structural domains, viz: (a) residues 1-11, (b) residues 12 to approximately 75, (c) residues 94-169 and (d) the very acidic region beyond residue 169. The instability of peptide A may mean that it is not a truly independent domain. No structural similarities to histone H1 are therefore observed in HMG 1 and 2.
Animals↗
Conformation and folding in histones H1 and H5.
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Animals↗
Histone H1 and chromatin higher order structure. Does histone H1 exhibit specific self-association?
1. Histones H1 and H5 in chromatin and in free solution can be cross-linked to higher multimers. Is this due to a specific protein/protein interaction? If so, this interaction might be the structural basis of the condensation of the chromosomal nucleofilament, known to be mediated by histones H1 and H5. 2. Since only the central domain of H1 and H5 exhibits tertiary folding and globular structure, this is the most likely site of specific interaction. 3. Formaldehyde has been used to test whether the central domains of histone H1 from calf thymus or from sea urchin sperm or histone H5 from chicken erythrocytes self-interact. 4. The cross-linking shown by each globular peptide was compared with that of its parent histone. 5. In all three cases the peptide cross-linked to a much lower extent than its intact parent histone and the observed cross-linked rates were roughly in proportion to the relative number of lysine residues parent histone and peptide. 6. It is concluded that there is no specific self-interaction between the globular domains of either H1 or H5 molecules in free solution. 7. This result suggests that specific H1/H1 protein/protein interactions are not the basic cause of chromatin condensation.
Animals↗
Precise elimination of the N-terminal domain of histone H1.
The proteinase from mouse submaxillary gland was used to cleave total calf thymus histone H1 between residues 32 and 33. The C-terminal peptide, comprising residues 33 to the C-terminus, was purified and identified by amino acids analysis and Edman degradation. Spectroscopic characterization by n.m.r. for tertiary structure and by c.d. for secondary structure shows the globular domain of the parent histone H1 to be preserved intact in the peptide. It has therefore lost only the N-terminal domain and is a fragment of histone H1 comprising the globular plus C-terminal domains only. Precise elimination of only the N-terminal domain makes the fragment suitable for testing domain function in histone H1.
Amino Acids↗
Proteolytic digestion studies of chromatin core-histone structure. Identification of limit peptides from histone H2B.
Trypsin digestion of chicken erythrocyte chromatin results in a well-defined set of limit peptides derived from the core histones (P1-P5). Two peptides running in sodium dodecyl sulphate gel electrophoresis at the positions of P2 and P3 have been identified as histone H2B residues 21-125 and 24-125. No C-terminal residues are therefore lost from histone H2B. The N-terminal sequence removed correlates well with that having low sequence conservation and not with that showing the greatest basicity. The same correlation was reported previously by us for histone H2A.
Amino Acid Sequence↗
The central tryptic fragment of histones H1 and H5 is a fully compacted domain and is the only folded region in the polypeptide chain. A thermodynamic study.
Scanning microcalorimetry has been used to show that the globular structure in histones H1 and H5 melts reversibly over a relatively narrow temperature range as a single domain with enthalpy of melting very close to the van't Hoff enthalpy. These proteins therefore exhibit macroscopic cooperativity like that of many well-studied small proteins. For histone H5 the observed melting temperature and melting enthalpy coincide with those previously obtained [Eur. J. Biochem. 67, 379-388 (1976)] using intrinsic Cotton effects in the circular dichroism spectrum, but not with those from extrinsic Cotton effects or NMR chemical shifts. Calorimetric measurements on a central tryptic fragment from histones H1 and H5 show these fragments to have the same molar melting enthalpy as the parent histones. The folded structures of H1 and H5 are therefore located only within these fragments. For histone H1 this conclusion is supported by the finding that the C-terminal fragment (residues 122-213) shows no peak in the specific heat curve.
Calorimetry, Differential Scanning↗
Effect of acetylation on the binding of N-terminal peptides of histone H4 to DNA.
The trypsin-sensitive N-terminal domain of histone H4 (residues 1-19) contains four acetylation sites at residues 5, 8, 12 and 16 and may play a separate role in chromatin structure from the remainder of the H4 chain. High-resolution proton NMR has been used to probe the DNA-binding of this H4 domain using the peptides (4-17), (1-23) and (1-37). Binding strength is in the order (1-37) greater than (1-23) greater than (4-17) but is weak even for (1-37). The observed weak binding correlates with arginine rather than lysine content and marked changes in the glycine resonance indicate the involvement of the peptide backbone in binding. When peptides (1-23) and (4-17) are fully acetylated with acetic anhydride, this weak binding is totally abolished. Circular dichroism indicates that neither acetylated nor unacetylated peptides take up any secondary structure. The results are consistent with the view that acetylation of H4 in vivo lifts the N-terminal domain off the DNA and thereby promulgates a major structural change in the chromatin.
Acetylation↗
Secondary and tertiary structural differences between histone H1 molecules from calf thymus and sea-urchin (Sphaerechinus granularis) sperm.
Tryptic digestion of histone H1 from the sperm of the sea urchin Sphaerechinus granularis leaves a limiting peptide of approx. 80 residues that is of similar size to the limit peptide from calf thymus H1 or chicken erythrocyte H5. The S. granularis limit peptide folds to form tertiary structure similar to that of the intact parent histone H1 (shown by n.m.r. spectra), but the helical content is decreased by the digestion from 64 residues to 28. In contrast, intact calf thymus H1 and chicken erythrocyte H5 histones have only about 28 helical residues, which are preserved in their limit peptides. The extra helix in S. granularis is shown to be rapidly digested away by trypsin, and its location in histone H1 is discussed. A possible relationship of this structural feature to the length of linker DNA is proposed.
Amino Acids↗
Preparation and characterization of histone H1 from the sperm of the sea-urchin Sphaerechinus granularis.
The separation and purification of histone H1 from the sperm of the sea-urchin Sphaerechinus granularis is described. Physical studies were used to compare this histone H1 molecule with H1 histones from other species. C.d. and 270 MHz n.m.r. spectroscopy indicate that, despite significant compositional differences from other sea-urchin sperm H1 histones, their secondary and tertiary structures are very similar. A large difference in helicity was, however, found between S. granularis histone H1 and calf thymus histone H1, and their n.m.r. and fluorescence spectra also differ considerably. It is concluded that secondary structure and tertiary structure have not been conserved in the evolution of the H1 histone family.
Amino Acids↗
The 20,000 molecular weight variant of human growth hormone. Preparation and some physical and chemical properties.
Extracts of frozen human pituitary glands contain, in addition to normal growth hormone (hGH (Mr = 22,005), a variant of lower molecular weight (approximately 20,000) (Singh, R. N. P., Seavey, B. K. and Lewis, U. J. (1974) Endocrinol. Res. Commun. 1, 449-464). This material, designated 20K hGH, occurs in various forms with the majority present as a heterologous dimer of Mr = 20,000 hGH and hGH. The purification scheme for obtaining the variant from these fractions is described. Analysis of Mr = 20,000 hGH in the sequenator has confirmed that residues 32-46 of hGH are absent in Mr = 20,000 hGH. The structures of hGH and Mr = 20,000 hGH have been compared by 1H nuclear magnetic resonance spectroscopy, circular dichroism, spectrophotometric titration, and nitration of the tyrosine residues by tetranitromethane. The results indicate that the folding of the polypeptide chain of the two proteins is similar but not identical.
Amino Acid Sequence↗
The sea urchin sperm histone H2B readily forms a complex with heterologous H2A despite having an elongated N-terminal domain.
A test has been made of the postulate that interaction between histones H2A and H2B occurs only in the C-terminal domain of H2B and is independent of the N-terminal domain in which the sites of chemical modification occur. Sea urchin sperm H2B's have much extended N-terminal domains when compared to other studied H2B's and the interactions of Sphaerechinus granularis H2B1 with homologous H2A and with calf thymus H2A have been studied. Continuous variation analysis of circular dichroism results indicates that both homologous and heterologous H2A/H2B pairs can cooperatively form complexes, which in all cases involve the same number of helical residues (approximately equal to 80). It is concluded that although the sequence of the N-terminal domain of H2B can indirectly influence the free solution association constant with histone H2A, it does not take part in the interaction.
Amino Acid Sequence↗
Proteolytic digestion studies of chromatin core-histone structure. Identification of the limit peptides of histones H3 and H4.
Trypsin digestion of chromatin results in a well-defined set of limit peptides (P1--P5) derived from the four core histones. Those from histones H3 and H4 have been identified. P1 is H3 residues 27--129; P4 is H4 residues 18--102 and P5 is H4 residues 20--102. The N-terminal sequences removed correlate well with the regions that undergo post-synthetic acetylation and which show the greatest degree of sequence conservation. Autolytic digestion of chromatin releases a peptide (P1') from H3 representing residues 21--135. The implications of protease digestion for the higher order structure of chromatin are discussed.
Amino Acid Sequence↗
Structural studies of the non-histone chromosomal proteins HMG-T and H6 from trout testis.
Trout testis contains two proteins of the high mobility group H6 and HMG-T that have been implicated in the structure of active chromatin. Protein H6 was studied by high resolution proton NMR and by circular dichroism and showed no evidence of secondary or tertiary structure formation in free solution. At low ionic strength protein H6 binds to DNA by a weak interaction in the N-terminal region between residues 10 and 35. Proteins H6 therefore behaves structurally like the homologous calf-thymus high-mobility-group proteins HMG-14 and HMG-17. Salt addition to solutions of protein HMG-T results in secondary and tertiary structure formation that is accompanied by aggregation. Circular dichroism shows that the helical content of protein HMG-T (approximately equal to 9%) is very much less than that of the homologous calf thymus proteins HMG-1 and HMG-2. At low ionic strength protein HMG-T precipitates DNA due to the formation of large scale aggregates that disperse only when the protein is released at approximately equal to 0.35 M NaCl. The NMR spectrum of the aggregated state does not show the presence of a large number of free acidic residues, in contrast to spectra of soluble complexes of HMG-1 and DNA at the same ionic strengths. It is concluded that HMG-T lacks the highly acidic domain found in HMG-1 (and HMG-2) that remains free from DNA under these conditions. It is concluded that the entire chain of HMG-T binds to DNA. There are therefore major structural differences between HMG-T and the homologous calf thymus proteins.
Animals↗
The structure of histone H1 and its location in chromatin.
On the basis of their primary structure, the lysine-rich histones are a unified family of proteins. Each has an amino acid chain which falls into three distinct domains. Only the central domain (approximately 80 residues) is in a folded conformation. It is protected from trypsin digestion in chromatin and corresponds to the segment of highest sequence conservation. Without the flanking domains it is able to close two full turns of DNA in the nucleosome and can thus locate the H1 molecule.
Amino Acid Sequence↗