Thermodynamic basis of the α-helix and DNA duplex.

Analysis of calorimetric and crystallographic information shows that the α-helix is maintained not only by the hydrogen bonds between its polar peptide groups, as originally supposed, but also by van der Waals interactions between tightly packed apolar groups in the interior of the helix. These apolar contacts are responsible for about 60% of the forces stabilizing the folded conformation of the α-helix and their exposure to water on unfolding results in the observed heat capacity increment, i.e. the temperature dependence of the melting enthalpy. The folding process is also favoured by an entropy increase resulting from the release of water from the peptide groups. A similar situation holds for the DNA double helix: calorimetry shows that the hydrogen bonding between conjugate base pairs provides a purely entropic contribution of about 40% to the Gibbs energy while the enthalpic van der Waals interactions between the tightly packed apolar parts of the base pairs provide the remaining 60%. Despite very different structures, the thermodynamic basis of α-helix and B-form duplex stability are strikingly similar. The general conclusion follows that the stability of protein folds is primarily dependent on internal atomic close contacts rather than the hydrogen bonds they contain.

Linker histones: History and current perspectives.

Although the overall structure of the fifth histone (linker histone, H1) is understood, its location on the nucleosome is only partially defined. Whilst it is clear that H1 helps condense the chromatin fibre, precisely how this is achieved remains to be determined. H1 is not a general gene repressor in that although it must be displaced from transcription start sites for activity to occur, there is only partial loss along the body of genes. How the deposition and removal of H1 occurs in particular need of further study. Linker histones are highly abundant nuclear proteins about which we know too little.

Forces for Folding.

Understanding the nature of the forces driving the folding of proteins, nucleic acids and the formation of their complexes absolutely requires thermodynamic data, in addition to structural information. In practical terms, this means the use of super-sensitive scanning and titration calorimeters for experimental determination of the heats (enthalpies) characterising these processes. Peter Privalov was both an experimental thermodynamicist and a calorimeter designer/manufacturer who followed and propagated this credo. The sum total of his many publications, every one of which addresses a fundamental question, is his lasting epitaph.

DNA looping in the RNA polymerase I enhancesome is the result of non-cooperative in-phase bending by two UBF molecules.

The so-called upstream binding factor (UBF) is required for the initial step in formation of an RNA polymerase I initiation complex. This function of UBF correlates with its ability to induce the ribosomal enhancesome, a structure which resembles in its mass and DNA content the nucleosome of chromatin. DNA looping in the enhancesome is probably the result of six in-phase bends induced by the HMG boxes of a UBF dimer. Here we show that insertion/deletion mutations in the basic peptide linker lying between the N-terminal dimerisation domain and the first HMG box of Xenopus UBF prevent the DNA looping characteristic of the enhancesome. Using these mutants we demonstrate that (i) the enhancesome structure does not depend on tethering of the entering and exiting DNA duplexes, (ii) UBF monomers induce hemi-enhancesomes, bending the DNA by 175 +/- 24 degrees and (iii) two hemi-enhancesomes are precisely phased by UBF dimerisation. We use this and previous data to refine the existing enhancesome model and show that HMG boxes 1 and 2 of UBF lie head-to-head along the DNA.

Changes in nuclear proteins on transformation of rat epithelial thyroid cells by a murine sarcoma retrovirus.

Two-dimensional electrophoresis has been used to document changes in nuclear proteins following viral transformation of an epithelial cell line exhibiting differentiation markers. After transformation, these markers are lost, and the cells become tumorigenic and capable of growth in soft agar. A sharp rise in the phosphorylation of histones H1, H2A, and ubiquitinated H2A is seen on transformation, together with the appearance of three phosphorylated proteins that are extractable by perchloric acid and appear related to high mobility group Protein 14, a constituent of active chromatin. Since comparison is made between normal and transformed cells that are each grown to confluence and since there is little difference between their observed growth rates, the changes seen represent intrinsic differences between the cell lines and are thus a direct reflection of the process of transformation.

Baculovirus expression of human basic fibroblast growth factor from a synthetic gene: role of the Kozak consensus and comparison with bacterial expression.

Synthetic genes encoding the 146 and 155 amino acid forms of human basic fibroblast growth factor (bFGF) were constructed with codon usage biased towards the polyhedrin-encoding gene of Autographa californica nuclear polyhedrosis virus (AcNPV). Expression of both bFGF genes in Spodoptera frugiperda (SF-21) suspension cell culture using a recombinant baculovirus yielded approximately 2.5 mg of mitogenically fully active protein per 10(9) cells following heparin-affinity chromatography. To improve translational efficiency, the Kozak consensus sequence was introduced and it was found that neither the replacement of a pyrimidine by a purine at position -3, nor the nature of the base at position +4 had any noticeable effect on the final levels of bFGF expression in SF-21 cells. The bases at these critical points in the consensus do not therefore play a major role in expression levels of the bFGF synthetic genes. The two synthetic genes were also expressed in Escherichia coli as native proteins using the T7 expression system. 5 mg of mitogenically fully active bFGF were obtained from 1 l of bacterial culture. Both insect cell- and E. coli-derived bFGF were equally mitogenic for Swiss 3T3 fibroblasts.

Molluscan sperm proteins: Ensis minor.

The three major proteins, EM1, EM5 and EM6, from the mature sperm of the bivalve mollusc Ensis minor have been partially sequenced in order to establish which category they belong to and their potential for phosphorylation. Protein EM1 is protamine-like with about 50% basic amino acids, some of which are included in SK(R) repeats. Three SPXX potential phosphorylation sites were observed in the N-terminal domain. EM1 does not fold (Giancotti et al. (1983) Eur. J. Biochem. 136, 509-516). Protein EM6 (approx. 270 residues) is histone H1-like, having a globular domain homologous to other H1 family proteins. The N-domain of EM6 contains SK(R) repeats like EM1, but there are few, if any, SPXX sites in the chain. Proteins EM1 and EM6 are the two proteins specific for mature sperm. Protein EM5, of about 150 residues and present at lower levels than EM1 and EM6, is also an H1-family molecule. A sequence from its globular domain shows close homology to chicken H5 and to sea urchin somatic H1. Its presence may relate to the existence of a low level of nucleosomal structure.

Fluorescence of buried tyrosine residues in proteins.

Histone H1 contains only one tyrosine and no tryptophan. The intrinsic fluorescence of the tyrosine rises by about 400% as the protein folds from a random coil to a globular structure (Giancotti, V., Fonda, M. and Crane-Robinson, C. (1977) Biophys. Chem. 6, 379-383). Measurements of external quenching by a large variety of quenchers shows very much reduced quenching in the folded state as compared to the disordered. It is concluded that the tyrosine is a buried residue. This is supported by the observation that the fluorescence of modified amino-tyrosyl H1 is similar to that of buried tyrosines in ribonuclease. The classification of tyrosine fluorescence in tryptophan-free proteins (Cowgill, R.W. (1976) in Biochemical Fluorescence Concepts, Vol. 2 to include the case of residues buried in a hydrophobic environment and having a relative quantum yield RTyr, greater than unity.

Location of the globular region in chicken erythrocyte histone H5.

Chicken erythrocyte histone H5 has been cleaved by acetic acid hydrolysis at the two aspartic acid residues 65 and 99 and the 4 peptides (1-65), (66-185) (1-99) (100-185) recovered in a pure form. 270 MHz magnetic resonance and circular dichroic studies show that the two C-terminal peptides are unable to form secondary or tertiary structure. The N-terminal peptides however, form both secondary and tertiary structure. In particular, the peptide (1-99) at high ionic strength possesses a similar number of helical residues to intact histone H5 and also had a closely related nuclear magnetic resonance spectrum. It is concluded that the peptide (1-99) contains most, but not quite all of the residues that are included in the globular segment of histone H5.

The chromatin of sea urchin sperm.

Digestion of sea urchin sperm nuclei with micrococcal nuclease yields nucleosomal monomer fragments of 151 and 164 base pairs. Prior trypsin treatment of the sperm chromatin does not alter the size of these monomer DNA fragments despite the fact that the H1 histone is reduced to a limit globular peptide of about 83 residues. Heterologous reconstitution experiments show that this peptide is capable of protecting an extra 22 base pairs beyond the core particle in a chromatosome. Nuclease digestion of reconstitutes from DNA and sperm core histones yields a core monomer of about 141 base pairs. It is concluded that this sperm chromatin contains a chromatosome of 164 bp essentially similar to that observed in the more usual chromatins. Edman degradation of the H1 limit peptide shows its sequence to be closely analogous to the corresponding peptide of calf H1 and chicken H5. Circular dichroism studies of histone H1 from the sperm of three sea urchin species demonstrate the presence of trypsin-sensitive helical regions outside the globular domain that are absent in calf H1 and chicken H5.

The conformation of histone H5 bound to DNA. Maintenance of the globular structure after binding.

Trypsin digestion is used to investigate the conformation of histone H5 when bound to DNA. A central region of H5 comprising residues (22--100) is found to be resistant to digestion and it is concluded that this region is compacted whilst the remaining N- and C-terminal regions are more extended. Since this is the same result found previously for the free solution conformation of histone H5 it follows that a 3-domain structure is preserved on DNA binding. The binding of H5 and the central region (22--100) to DNA is also studied using proton magnetic resonance (270 MHz) and a precipitation approach. It is concluded that all 3 domains of H5 bind to DNA at low ionic strengths. The central domain (residues 22--100) is released at 0.3--0.4 M NaCl, but 0.7 M NaCl is required to release the N- and C-terminal regions. Comparison is made of H5 binding to DNA with that of the related histone H1.

The energetics of HMG box interactions with DNA: thermodynamic description of the target DNA duplexes.

The thermal properties and energetics of formation of 10, 12 and 16 bp DNA duplexes, specifically interacting with the HMG box of Sox-5, have been studied by isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC). DSC studies show that the partial heat capacity of these short duplexes increases considerably prior to the cooperative process of strand separation. Direct extrapolation of the pre and post-transition heat capacity functions into the cooperative transition zone suggests that unfolding/dissociation of strands results in no apparent heat capacity increment. In contrast, ITC measurements show that the negative enthalpy of complementary strand association increases in magnitude with temperature rise, implying that strand association proceeds with significant decrease of heat capacity. Furthermore, the ITC-measured enthalpy of strand association is significantly smaller in magnitude than the enthalpy of cooperative unfolding measured by DSC. To resolve this paradox, the heat effects upon heating and cooling of the separate DNA strands have been measured by DSC. This showed that cooling of the strands from 100 degrees C to -10 degrees C proceeds with significant heat release associated with the formation of intra and inter-molecular interactions. When the enthalpy of residual structure in the strands and the temperature dependence of the heat capacity of the duplexes and of their unfolded strands have been taken into account, the ITC and DSC results are brought into agreement. The analysis shows that the considerable increase in heat capacity of the duplexes with temperature rise is due to increasing fluctuations of their structure (e.g. end fraying and twisting) and this effect obscures the heat capacity increment resulting from the cooperative separation of strands, which in fact amounts to 200(+/-40) JK(-1) (mol bp)(-1). Using this heat capacity increment, the averaged standard enthalpy, entropy and Gibbs energy of formation of fully folded duplexes from fully unfolded strands have been determined at 25 degrees C as -33(+/-2) kJ (mol bp)(-1), -93(+/-4) J K(-1) (mol bp)(-1) and -5.0(+/-0.5) kJ (mol bp)(-1), respectively.

Roles of H1 domains in determining higher order chromatin structure and H1 location.

Peptides derived from calf thymus H1 and rat liver H1, comprising only the globular and COOH-terminal domains of the intact molecule and therefore lacking NH2-terminal domains, have been shown by reconstitution to be as effective as the complete H1 molecule in inducing higher-order-chromatin structure. As the globular domain of H1 alone cannot induce chromatin folding, our results demonstrate that this function is primarily controlled by the COOH-terminal domain of the molecule. Surprisingly, these peptides do not locate correctly with respect to the nucleosome. This is demonstrated by their failure to confer upon reconstitutes the ability to protect DNA fragments of chromatosome length when digested with micrococcal nuclease. The precise placement of the H1 molecule (globular domain) with respect to the nucleosome is shown to be influenced by the "tail" domains of both H1 and the core histones.

The energetics of HMG box interactions with DNA: thermodynamics of the DNA binding of the HMG box from mouse sox-5.

The energetics of the Sox-5 HMG box interaction with DNA duplexes, containing the recognition sequence AACAAT, were studied by fluorescence spectroscopy, isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC). Fluorescence titration showed that the association constant of this HMG box with the duplexes is of the order 4x10(7) M(-1), increasing somewhat with temperature rise, i.e. the Gibbs energy is -40 kJ mol(-1) at 5 degrees C, decreasing to -48 kJ mol(-1) at 32 degrees C. ITC measurements of the enthalpy of association over this temperature range showed an endothermic effect below 17 degrees C and an exothermic effect above, suggesting a heat capacity change on binding of about -4 kJ K(-1) mol(-1), a value twice larger than expected from structural considerations. A straightforward interpretation of ITC data in heat capacity terms assumes, however, that the heat capacities of all participants in the association reaction do not change over the considered temperature range. Our previous studies showed that over the temperature range of the ITC experiments the HMG box of Sox-5 starts to unfold, absorbing heat and the heat capacities of the DNA duplexes also increase significantly. These heat capacity effects differ from that of the DNA/Sox-5 complex. Correcting the ITC measured binding enthalpies for the heat capacity changes of the components and complex yielded the net enthalpies which exhibit a temperature dependence of about -2 kJ K(-1) mol(-1), in good agreement with that predicted on the basis of dehydration of the protein-DNA interface. Using the derived heat capacity change and the enthalpy and Gibbs energy of association measured at 5 degrees C, the net enthalpy and entropy of association of the fully folded HMG box with the target DNA duplexes was determined over a broad temperature range. These functions were compared with those for other known cases of sequence specific DNA/protein association. It appears that the enthalpy and entropy of association of minor groove binding proteins are more positive than for proteins binding in the major groove. The observed thermodynamic characteristics of protein binding to the A+T-rich minor groove of DNA might result from dehydration of both polar and non-polar groups at the interface and release of counterions. The expected entropy of dehydration was calculated and found to be too large to be compensated by the negative entropy of reduction of translational/rotational freedom. This implies that DNA/HMG box association proceeds with significant decrease of conformational entropy, i.e. reduction in conformational mobility.

Redefinition of the cleavage sites of DNase I on the nucleosome core particle.

DNase I has been widely used for the footprinting of DNA-protein interactions including analyses of nucleosome core particle (NCP) structure. Our understanding of the relationship between the footprint and the structure of the nucleosome complex comes mainly from digestion studies of NCPs, since they have a well-defined quasi-symmetrical structure and have been widely investigated. However, several recent results suggest that the established consensus of opinion regarding the mode of digestion of NCPs by DNase I may be based on erroneous interpretation of results concerning the relationship between the NCP ends and the dyad axis. Here, we have used reconstituted NCPs with defined ends, bulk NCPs prepared with micrococcal nuclease and molecular modelling to reassess the mode of DNase I digestion. Our results indicate that DNase I cuts the two strands of the nucleosomal DNA independently with an average stagger of 4 nt with the 3'-ends protruding. The previously accepted value of 2 nt stagger is explained by the finding that micrococcal nuclease produces NCPs not with flush ends, but with approximately 1 nt 5'-recessed ends. Furthermore we explain why the DNA stagger is an even and not an odd number of nucleotides. These results are important for studies using DNase I to probe nucleosome structure in complex with other proteins or any DNA-protein complex containing B-form DNA. We also determine the origin of the 10n +/- 5 nt periodicity found in the internucleosomal ladder of DNase I digests of chromatin from various species. The explanation of the 10n +/- 5 nt ladder may have implications for the structure of the 30 nm fibre.

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.

A "minimal epitope" anti-protein antibody that recognises a single modified amino acid.

Antibodies that recognise proteins bind to epitopes of varying size, but a grouping of the order of six amino acids, contiguous or not, is regarded as a typical number. By using as immunogen a highly abundant and universal eukaryotic nuclear protein (histone H4) modified in a manner not typical of secreted proteins (acetylation of lysine side chains), antiserum has been raised in rabbits having the single amino acid epsilon-N-acetyl lysine as the recognition epitope. The affinity-purified antibody should be useful for studying the functional role of this modification. The methodology has potential for raising antibodies to other types of post-translationally modified proteins.

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.

Characterization of an SRY-like gene, DSox14, from Drosophila.

We have characterized the DSox14 gene, a new member of the family of transcription factors related to the mammalian sex determining factor, SRY. It contains two exons and the intron is large for Drosophila at 2.8 kb. The encoded protein consists of 691 amino acids (72 kDa) and includes an HMG box domain, which is closely related to the mouse Sox4 DNA binding domain. Expression of the DSox14 HMG box domain in vitro shows that it binds the sequence AACAAT with a K(d) of 190 nM, generating a bend angle of 48.6 degrees. At higher protein concentrations, a second HMG box binds at the recognition sequence, increasing the bend angle by 5 degrees. DSox14 is variably expressed throughout development as three alternative transcripts but not at all during the 1st and 2nd larval instars. The several mRNA transcripts are produced primarily from different transcriptional start sites. Analysis of the expression of DSox14 mRNAs during early development shows that they are maternally contributed at a low level and ubiquitously expressed during embryogenesis. The widespread pattern of expression suggests that DSox14 affects a large number of target genes.

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.