Circular dichroism for the analysis of protein-DNA interactions.

Circular dichroism (CD) is a well-established technique for the analysis of both protein and DNA structure. The analysis of protein-nucleic acid complexes presents greater challenges, but at wavelengths above 250 nm, the circular dichroism signal from the DNA predominates. Examples are given of the use of CD to examine structural changes to DNA induced by protein binding.

DNA structural deformations in the interaction of the controller protein C.AhdI with its operator sequence.

Controller proteins such as C.AhdI regulate the expression of bacterial restriction-modification genes, and ensure that methylation of the host DNA precedes restriction by delaying transcription of the endonuclease. The operator DNA sequence to which C.AhdI binds consists of two adjacent binding sites, O(L) and O(R). Binding of C.AhdI to O(L) and to O(L) + O(R) has been investigated by circular permutation DNA-bending assays and by circular dichroism (CD) spectroscopy. CD indicates considerable distortion to the DNA when bound by C.AhdI. Binding to one or two sites to form dimeric and tetrameric complexes increases the CD signal at 278 nm by 40 and 80% respectively, showing identical local distortion at both sites. In contrast, DNA-bending assays gave similar bend angles for both dimeric and tetrameric complexes (47 and 38 degrees, respectively). The relative orientation of C.AhdI dimers in the tetrameric complex and the structural role of the conserved Py-A-T sequences found at the centre of C-protein-binding sites are discussed.

Solution structure and backbone dynamics of the DNA-binding domain of mouse Sox-5.

The fold of the murine Sox-5 (mSox-5) HMG box in free solution has been determined by multidimensional NMR using (15)N-labeled protein and has been found to adopt the characteristic twisted L-shape made up of two wings: the major wing comprising helix 1 (F10--F25) and helix 2 (N32--A43), the minor wing comprising helix 3 (P51--Y67) in weak antiparallel association with the N-terminal extended segment. (15)N relaxation measurements show considerable mobility (reduced order parameter, S(2)) in the minor wing that increases toward the amino and carboxy termini of the chain. The mobility of residues C-terminal to Q62 is significantly greater than the equivalent residues of non-sequence-specific boxes, and these residues show a weaker association with the extended N-terminal segment than in non-sequence boxes. Comparison with previously determined structures of HMG boxes both in free solution and complexed with DNA shows close similarity in the packing of the hydrophobic cores and the relative disposition of the three helices. Only in hSRY/DNA does the arrangement of aromatic sidechains differ significantly from that of mSox-5, and only in rHMG1 box 1 bound to cisplatinated DNA does helix 1 have no kink. Helix 3 in mSox-5 is terminated by P68, a conserved residue in DNA sequence-specific HMG boxes, which results in the chain turning through approximately 90 degrees.

Conformational studies of the C-terminal domain of bacteriophage Pf1 gene 5 protein.

The gene 5 protein (g5p) of the bacteriophage Pf1 is a 144 residue single-stranded (ss) DNA binding protein involved in replication and packaging of the viral DNA. Compared to the gene 5 proteins of other filamentous bacteriophages, such as fd, the Pf1 g5p has an additional C-terminal sequence ( approximately 40 residues) with an unusual amino acid composition, being particularly rich in proline, glutamine and alanine. This C-terminal sequence is susceptible to limited proteolysis, in contrast to the globular N-terminal domain of the protein. The C-terminal sequence has been shown to play a role in the stabilisation of the protein-ssDNA complex. In the present study, the DNA sequence corresponding to the 38 amino acid residue C-terminal peptide has been cloned and expressed. A variety of biophysical techniques suggest that this peptide has a largely irregular conformation in solution, in contrast to the N-terminal globular domain that is principally beta-sheet. However, circular dichroism (CD) spectroscopy indicates that the peptide can be induced to form a structure that resembles a left-handed polyproline-like (P(II)) helix, suggesting that the C-terminal tail of the protein may adopt a more structured conformation in the appropriate physiological environment.

The energetics of HMG box interactions with DNA. Thermodynamic description of the box from mouse Sox-5.

The structural energetics of the HMG box from the DNA-binding protein mouse Sox-5 were examined calorimetrically. It was found that this box, notwithstanding its small size (molecular mass about 10 kDa), does not behave as a single cooperative unit and, on heating, the box reversibly unfolds in two separate stages. The first transition (tt approximately 34 degrees C) involves about 40% of the total enthalpy and the second (tt approximately 46 degrees C) the remainder. Both transitions proceed with significant heat capacity increment, showing that they are associated with the unfolding of two sub-domains having non-polar cores. According to heat capacity, ellipticity, fluorescence and NMR criteria, this HMG box is in a fully compact native state only below 5 degrees C. HMG boxes consist of two approximately orthogonal wings: the minor wing comprises helix 3 and its associated antiparallel N-terminal strand, whilst the major wing is composed of helices I and II. Analysis of the fluorescence and NMR spectra for this box obtained at different temperatures shows that the lower melting transition can be assigned to the minor wing and the upper transition to the major wing. Under physiological conditions (37 degrees C), the minor wing is considerably unfolded, whilst the major wing is essentially fully folded. DNA binding in vivo therefore involves refolding of the minor wing.

The DNA sequence specificity of HMG boxes lies in the minor wing of the structure.

To establish the basis of sequence-specific DNA recognition by HMG boxes we separately transferred the minor and major wings from the sequence-specific HMG box of TCF1 alpha into their equivalent position in the non-sequence-specific box 2 of HMG1. Thus chimera THT1 contains the minor wing (of 11 N-terminal and 25 C-terminal residues) from the HMG box of TCF1 alpha and the major wing (the 45 residue central section) from HMG1 box 2, whilst the situation is reversed in chimera HTH1. The structural integrity of the two chimeric proteins was established by CD, NMR and their binding to four-way junction DNA. Gel retardation and circular permutation assays showed that only chimera THT1, containing the TCF1 alpha minor wing, formed a sequence-specific complex and bent the DNA. The bend angle was estimated to be 59 degrees for chimera THT1 and 52 degrees for the HMG box of TCF1 alpha. Our results, in combination with mutagenesis and other data, suggests a model for the DNA binding of HMG boxes in which the N-terminal residues and part of helix 1 contact the minor groove on the outside of a bent DNA duplex.

DNA binding and bending properties of the post-meiotically expressed Sry-related protein Sox-5.

Sox-5 is one of a family of genes which show homology to the HMG box region of the testis determining gene SRY. We have used indirect immunofluorescence to show that Sox-5 protein is localized to the nucleus of post-meiotic round spermatids in the mouse testis. In vitro footprinting and gel retardation assays demonstrate that Sox-5 binds specifically to the sequence AACAAT with moderately high affinity (Kd of approximately 10(-9) M). Moreover, interaction of Sox-5 with its target DNA induces a significant bend in the DNA, characteristic of HMG box proteins. Circular dichroism spectroscopy of the Sox-5 HMG box and its specific complex with DNA shows an alteration in the DNA spectrum, perhaps as a consequence of DNA bending, but none in the protein spectrum on complex formation. The dependence of the change in the CD spectrum with protein to DNA ratio demonstrates the formation of a 1:1 complex. Analysis of the structure of the Sox-5 HMG box by 2D NMR suggests that both the location of helical secondary structure as well as the tertiary structure is similar to that of HMG1 box 2.

Solution structure of a DNA-binding domain from HMG1.

We have determined the tertiary structure of box 2 from hamster HMG1 using bacterial expression and 3D NMR. The all alpha-helical fold is in the form of a V-shaped arrowhead with helices along two edges and one rather flat face. This architecture is not related to any of the known DNA binding motifs. Inspection of the fold shows that the majority of conserved residue positions in the HMG box family are those involved in maintaining the tertiary structure and thus all homologous HMG boxes probably have essentially the same fold. Knowledge of the tertiary structure permits an interpretation of the mutations in HMG boxes known to abrogate DNA binding and suggests a mode of interaction with bent and 4-way junction DNA.

Thymosins: both nuclear and cytoplasmic proteins.

A simple procedure based on perchloric acid extraction has been developed for the preparation and purification of bovine prothymosin alpha and thymosins beta 4 and beta 9 in high yields. Spectroscopic observations show these proteins to be non-folding at neural pH. The cellular locations of human prothymosin alpha, rat parathymosin and calf thymosin beta 4, all so-called 'thymic hormones', have been studied by injection into the cytoplasm of Xenopus oocytes, followed by separate monitoring of nuclear and cytoplasmic concentrations. It is shown that human prothymosin alpha and rat parathymosin both migrate to the nucleus whilst thymosin beta 4 remains in the cytoplasm. The peptide (1-88) of calf prothymosin alpha is shown not to accumulate in the Xenopus nucleus, demonstrating that the C-terminal 21 residues, which include a KKQK sequence, are required for nuclear migration. The present data, in association with existing evidence of wide tissue distribution and the lack of signal peptides, indicate that these proteins do not behave as hormones in the usual sense of the word. It is suggested that thymosin beta 4 should be grouped separately from the pro- and parathymosins.

Prothymosin alpha is a nuclear protein.

The cellular location of the so-called 'thymic hormone' prothymosin alpha has been studied by microinjection into the cytoplasm of Xenopus oocytes, followed by separate monitoring of nuclear and cytoplasmic concentrations. It is shown that prothymosin alpha migrates to the nucleus at a rate comparable to that of histone H1. Prothymosin alpha cannot therefore be a hormone in the usual sense of the word.

Histone H1 structure probed by Staphylococcus aureus V8-proteinase.

Proteolytic digestion of calf thymus histone H1 with Staphylococcus aureus V8-proteinase under structuring conditions generates one major limit peptide P1 which consists of approx. 170 residues. Edman degradation establishes the N-terminal sequence as: Leu-Ile-Thr-Lys-Ala-Val-Ala-Ala-Ser-Lys. Chymotryptic fingerprinting shows that the C-terminal part of the H1 molecule is fully preserved. The peptide therefore comprises the residues H1 (42-210). The Glu-41 cleavage is extremely unusual as it occurs in the structured G-domain which is known to be resistant to proteinases (Hartman, P. G., Chapman, G. E., Moss, T. and Bradbury, E. M. (1977) Eur. J. Biochem. 77, 45-71; Böhm, L., Sautière, P., Cary, P. D. and Crane-Robinson, C. (1982) Biochem. J. 203, 577-582). The V8-proteinase cleavage product H1 (42-210) shows only 20% folding as compared to 95-99% folding shown by the peptides H1 (34-121), H1 (31-210) and H1 (33-210). Folding of the G-domain thus critically depends upon the presence of the eight residues 33-41 amongst which the Gly-Pro-Pro sequence at position 36-38 and a beta-turn predicted at position 35 are considered to be particularly important. The location of the cleavage site in the G-domain renders Staphylococcus aureus V8-proteinase suitable as a structural probe.

The metabolism of 7-ethylbenz[a]anthracene by rat liver microsomal preparations.

7-Ethylbenz[a]anthracene (7-EBA) is a much weaker carcinogen than the 7-methyl analogue (7-MBA), and this difference may be based upon differences in the pathways by which the two compounds are metabolized and activated. In the present work, 7-EBA and the related 7 alpha- and 7 beta-hydroxyethyl derivatives (7-OHEBA and 7-beta-OHEBA) have been incubated with microsomes prepared from the livers of rats pretreated with 3-methylcholanthrene, the metabolites were extracted and purified by TLC, and the products present in the dihydrodiol band were examined by analytical HPLC. Metabolites were identified by comparison with authentic reference standards and by their chromatographic, UV, fluorescence, mass, and NMR spectral characteristics. The 7-EBA metabolites included the 1,2- 3,4, 5,6- 8,9- and 10,11-dihydrodiols, the 3,4- 8,9- and 10,11-dihydrodiols of 7-alpha-OHEBA, and three phenolic dihydrodiols that were not completely characterized. No 7-beta-OHEBA derivatives were detected as metabolites of 7-EBA. The results obtained so far have not revealed any qualitative differences in the routes by which 7-EBA and 7-MBA are metabolized in rat liver microsomal preparations.

The metabolism of 9-methylanthracene by rat-liver microsomal preparations.

The metabolism of 9-methylanthracene by liver microsomal preparations from 3-methylcholanthrene-treated rats was examined. The principal metabolites identified were 9-hydroxymethylanthracene, the 1,2- and 3,4-dihydrodiols of the parent hydrocarbon and the 3,4-dihydrodiol of the 9-hydroxymethyl derivative. A small amount of a product that appeared to be a phenolic derivative of the hydrocarbon was also formed. The structures of the major metabolites were confirmed by u.v., n.m.r., and mass-spectral analysis and, wherever possible, by direct comparison of their chromatographic properties with those of the authentic compounds. 9-Hydroxymethylanthracene was further metabolized by rat-liver microsomal preparations to the 3,4-dihydrodiol but not to the related 1,2-dihydrodiol.

Preparation of chromosomal protein A24 (uH2A) by denaturing gel filtration and preparation of its free nonhistone component ubiquitin by ion-exchange chromatography.

Chromosomal protein A24 (uH2A) is unique in that it comprises the nucleosomal core histone H2A in isopeptide linkage with the highly conserved, globular, and stable nonhistone protein ubiquitin. Some 10% of the chromatin complement of H2A is modified in this way and studies to elucidate a role for this modification have concentrated on observations requiring no purification of A24 due to the difficulty in isolating the protein in large and pure quantities. We describe a method for isolating A24 by chromatography on Pharmacia G-100 gel filtration medium under urea denaturing conditions. A24 prepared by this method is structurally intact and is available in the quantities required for studies of the behavior and influence of the protein on histone-histone, histone-DNA, and enzymatic interactions. In conjunction with this method we describe a procedure for the isolation of large quantities of free ubiquitin of far greater purity than previously reported.

Antibodies from patients with drug-induced and idiopathic lupus erythematosus react with epitopes restricted to the amino and carboxyl termini of histone.

The sera of patients with systemic lupus erythematosus (SLE) and drug-induced lupus (DIL) were used to study the antigenic epitopes on nuclear histones that bind antibodies in these sera. ELISA and immunoblotting techniques showed that antibodies from both patient groups bound all classes of intact histone: H1 greater than H2B greater than H2A greater than H3 greater than H4. The different classes of histone were enzymatically or chemically cleaved to produce a series of peptide fragments which were then used to map the reactive epitopes by ELISA and immunoblotting. Ten of 11 DIL sera and 11 of 12 SLE sera bound the carboxy and amino terminal peptides. Only one sera of each group bound to the central hydrophobic polypeptide. The reactivity of DIL sera with fractionated histone polypeptides was similar to that observed with SLE sera. This observation suggests that the histone epitopes reacting with DIL sera are no less restricted than those reacting with SLE.

Physical studies by NMR and circular dichroism determining three structurally different domains in Physarum polycephalum histone H1.

Combined studies which include, NMR spectroscopy, circular dichroism, amino acid analysis and polyacrylamide gel electrophoresis together show that the protein designated as histone H1 from Physarum polycephalum has many of the features of histone H1 derived from other sources. The molecular masses of the globular peptide and the whole molecule were found to be 9000 +/- 1000 Da and 33000 +/- 3000 Da respectively. NMR melting experiments showed that the half-melt temperature was 53 +/- 1 degree C and the enthalpy of melting was 100 kJ . mol-1. Unusual facets of the molecule are the relatively large numbers of histidine residues (6 or 7) and the mono, di and trimethylation of some of the lysines, the major type of modification being trimethylation of 9 +/- 2 residues. The conditions necessary for structuring Physarum H1 are not the same as the histone H1 from calf thymus. It is suggested that titration of the histidine residues is the most decisive step for the development of tertiary folding of the globular unit.

The metabolism of 9,10-dimethylanthracene by rat liver microsomal preparations.

The metabolism of the weakly-carcinogenic hydrocarbon, 9,10-dimethylanthracene (DMA) by rat-liver microsomal preparations has been examined. 9-Hydroxymethyl-10-methylanthracene (9-OHMeMA) and 9,10-dihydroxymethyl-anthracene (9,10-DiOHMeA) were identified as metabolites by comparing their chromatographic and spectral properties with those of the authentic compounds. The trans-1,2-dihydro-1,2-dihydroxy derivative of DMA (DMA 1,2-diol) was the major metabolite formed which was identified by its chromatographic, u.v., n.m.r. and mass spectral properties. The dihydrodiol was also formed in the oxidation of DMA in an ascorbic acid-ferrous sulphate-EDTA system. Two other dihydrodiols that were formed from DMA by metabolism appeared to be the trans-1,2- and 3,4-dihydrodiols of 9-OHMeMA (9-OHMeMA 1,2-diol and 9-OHMeMA 3,4-diol) and the further metabolism of DMA 1,2-diol yielded both of these dihydrodiols. When 9-OHMeMA was further metabolized, two main metabolites were formed; one was identified as 9,10-DiOHMeA and the other appeared to be 9-OHMeMA 3,4-diol. No metabolites were detected when 9,10-DiOHMeA was incubated with rat-liver microsomal fractions.

Conformation and domain structure of the non-histone chromosomal proteins HMG 1 and 2. Domain interactions.

The sequence of the 224 residues of HMG 1 suggests it consists of three domains. We have previously proposed [Cary et al. (1980 Eur. J. Biochem. 131, 367-374] that the A and B domains can fold autonomously and that there is also a small N domain. Several proteases are now found to cut at the end of the B domain (at or close to residue 184). It is shown that the A + B-domain fragment also folds and probably contains all the helix of intact HMG 1. The stability of the B domain is enhanced by the presence of the A domain. The acidic C domain undergoes a coil----helix transition on lowering the pH. Several peptides have been prepared by cleavage at tryptophan. Peptide 57--C-terminus contains complete B and C domains but does not fold. In the absence of the A domain the C domain is thus able to destabilise the B domain. It is concluded that the stability of the B domain in HMG 1 is due to interaction with the A domain and the C domain has a separate function from the other domains.

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.

Metabolic activation of chrysene by hamster embryo cells: evidence for the formation of a 'bay-region' diol-epoxide-N2-guanine adduct in RNA.

Ribonucleoside-hydrocarbon adducts present in hydrolysates of RNA isolated from hamster embryo cells treated with 3H-labelled chrysene were examined by chromatography on Sephadex LH20 and by h.p.l.c. on Zorbax ODS. Two adducts formed in cells had chromatographic properties identical to those of two synthetic adducts formed when r-1,t-2-dihydroxy-t-3,4-oxy-1,2,3,4-tetrahydrochrysene (antichrysene 1,2-diol 3,4-oxide) reacted with poly G in vitro. Another adduct formed in cells had chromatographic properties identical to those of a synthetic adduct formed when antichrysene 1,2-diol 3,4-oxide reacted with poly A. In addition to the characterized adducts, other minor adducts were detected whose structures are not known. The structure of the more abundant guanosine--hydrocarbon adduct formed in cells was investigated by determining its pK values and stability in 1 M KOH. The structures of the synthetic guanosine--hydrocarbon adducts were investigated by 1H-n.m.r. spectroscopy. The data show that, in the hydrocarbon--guanosine adducts studied, the hydrocarbon moiety is attached to the exocyclic amino group of guanine.