Linker histone interaction shows divalent character with both supercoiled and linear DNA.

The interaction of linker histone H1 with both linear and superhelical double-stranded DNA has been investigated at low ionic strengths. Gel mobility retardation experiments demonstrate strikingly different behavior for the two forms of DNA. First, the experiments strongly suggest that linker histone binds to superhelical DNA in a negatively cooperative mode. In contrast, binding of linker histone to linear DNA under the conditions employed here shows no cooperativity. Second, binding of linker histone to linear DNA results in aggregation of histone-DNA complexes, even at very low levels of input histone H1. Because H1 has been shown to interact as a monomer, this aggregation is evidence of the divalent character of the linker histone, for without H1's ability to bind to two duplex strands of DNA, aggregation could not occur. Although aggregation can be made to occur with superhelical DNA, it can do so only at near-saturation levels of input histone H1. Finally, in direct competition, linker histone binds to superhelical DNA to the complete exclusion of linear DNA, indicating that the linker histone's function is related to the crossover structures that differentiate superhelical DNA from linear DNA. We develop a model that explains the observed behavior of binding of linker histone to superhelical DNA that is consistent with both the divalent character of the linker histone and the negative cooperativity by which linker histone and superhelical DNA interact.

Models for chromatin remodeling: a critical comparison.

Nucleosome remodeling has been shown, in many cases, to involve cis displacement of nucleosomes on the DNA. This process seems similar to the long-recognized random diffusion of nucleosomes along DNA, but the remodeling process is unidirectional and ATP dependent. Several years ago, we developed a model for nucleosome migration, based on the diffusion of "twist-defects" within the nucleosomal DNA. This has been modified into a model that incorporates ATP-dependent defect generation, and can account for many observations concerning remodeling. However, certain experimental studies in recent years have cast doubt on the applicability of the twist-diffusion model for remodeling, and seem to favor instead a "reptation" model. We discuss herein these problems and propose a resolution.

Reflections on a century of protein chemistry.

The development of protein structural chemistry during the twentieth century is briefly reviewed. Emphasis is placed on certain major problems that have defined the field, and how they have been resolved, often as a consequence of technological advances. The ways in which incorrect hypotheses have affected the development of the field are also discussed.

A hypothesis concerning diffusion-limited protein-ligand interactions.

A simple assumption allows the prediction of the numerical value for a 'universal' limiting kinetic rate for wholly diffusion-limited reactions between small neutral molecules and macromolecules. This prediction is compared with appropriate experimental data for binding of ligands to myoglobin and to enzymes. It is shown that in the absence of electrostatic effects, this limit is approached but not exceeded. The model also makes very specific predictions concerning the viscosity and temperature dependence of such reactions.

Two-photon excitation microscopy of tryptophan-containing proteins.

We have examined the feasibility of observing single protein molecules by means of their intrinsic tryptophan emission after two-photon excitation. A respiratory protein from spiders, the 24-meric hemocyanin, containing 148 tryptophans, was studied in its native state under almost in vivo conditions. In this specific case, the intensity of the tryptophan emission signals the oxygen load, allowing one to investigate molecular cooperativity. As a system with even higher tryptophan content, we also investigated latex spheres covered with the protein avidin, resulting in 340 tryptophans per sphere. The ratio of the fluorescence quantum efficiency to the bleaching efficiency was found to vary between 2 and 180 after two-photon excitation for tryptophan free in buffer solution, in hemocyanin, and in avidin-coated spheres. In the case of hemocyanin, this ratio leads to about four photons detected before photobleaching. Although this number is quite small, the diffusion of individual protein molecules could be detected by fluorescence correlation spectroscopy. In avidin-coated spheres, the tryptophans exhibit a higher photostability, so that even imaging of single spheres becomes possible. As an unexpected result of the measurements, it was discovered that the population of the oxygenated state of hemocyanin can be changed by means of a one-photon process with the same laser source that monitors this population in a two-photon process.

Structures of two molluscan hemocyanin genes: significance for gene evolution.

We present here the description of genes coding for molluscan hemocyanins. Two distantly related mollusks, Haliotis tuberculata and Octopus dofleini, were studied. The typical architecture of a molluscan hemocyanin subunit, which is a string of seven or eight globular functional units (FUs, designated a to h, about 50 kDa each), is reflected by the gene organization: a series of eight structurally related coding regions in Haliotis, corresponding to FU-a to FU-h, with seven highly variable linker introns of 174 to 3,198 bp length (all in phase 1). In Octopus seven coding regions (FU-a to FU-g) are found, separated by phase 1 introns varying in length from 100 bp to 910 bp. Both genes exhibit typical signal (export) sequences, and in both cases these are interrupted by an additional intron. Each gene also contains an intron between signal peptide and FU-a and in the 3' untranslated region. Of special relevance for evolutionary considerations are introns interrupting those regions that encode a discrete functional unit. We found that five of the eight FUs in Haliotis each are encoded by a single exon, whereas FU-f, FU-g, and FU-a are encoded by two, three and four exons, respectively. Similarly, in Octopus four of the FUs each correspond to an uninterrupted exon, whereas FU-b, FU-e, and FU-f each contain a single intron. Although the positioning of the introns between FUs is highly conserved in the two mollusks, the introns within FUs show no relationship either in location nor phase. It is proposed that the introns between FUs were generated as the eight-unit polypeptide evolved from a monomeric precursor, and that the internal introns have been added later. A hypothesis for evolution of the ring-like quaternary structure of molluscan hemocyanins is presented.

Allostery in very large molecular assemblies.

In contrast to small allosteric systems (like hemoglobin) those containing very large numbers (n) of binding sites never exhibit cooperativity (as measured by the Hill coefficient, nH) even approaching the potential limit, n. The reason for this appears to be that in such macromolecules the cooperative unit always represents some sub-structure of the entire structure. On the other hand, it is frequently observed that such sub-structures, when isolated, do not exhibit cooperativity at all. This paper describes studies of some molluscan hemocyanins that explore this apparent anomaly. It is concluded that it is the higher order structure of the molecule that provides a framework within which the sub-structures may exhibit their allosteric behavior.

Linker histone binding and displacement: versatile mechanism for transcriptional regulation.

In recent years, the connection between chromatin structure and its transcriptional activity has attracted considerable experimental effort. The post-translational modifications to both the core histones and the linker histones are finely tuned through interactions with transcriptional regulators and change chromatin structure in a way to allow transcription to occur. Here we review evidence for the involvement of linker histones in transcriptional regulation and suggest a scenario in which the reversible and controllable binding/displacement of proteins of this class to the nucleosome entry/exit point determine the accessibility of the nucleosomal DNA to the transcriptional machinery.

The site of binding of linker histone to the nucleosome does not depend upon the amino termini of core histones.

Using nucleosomes reconstituted on a defined sequence of DNA, we have investigated the question as to whether the N-terminal tails of core histones play a role in determining the site of binding of a linker histone. Reconstitutes used histone cores of three types: intact, lacking the N-terminal H3 tails, or lacking all tails. In each case the same, single defined position for the histone core was observed, using high-resolution mapping. The affinity for binding of linker histone H1(o) was highest for the intact cores, lowest for the tailless cores. However, the location of the linker histone, as judged by micrococcal nuclease protection, was exactly the same in each case, an asymmetric site of about 17 bp to one side of the core particle DNA.

The nucleosome core particle: does it have structural and physiologic relevance?

Although the nucleosomal core particle has been extensively studied as the basic building block of chromatin, the biological significance of a unit carrying exactly 146 bp of DNA remains unclear. Herein, we present data to show that the histone octamer can stably accommodate anywhere from about 100 to 170 bp of DNA. The unfolded structures containing less than 146 bp may well be of greater biological importance than the canonical core particle.

Chromatin structure revisited.

Independently of the enormous progress in our understanding of the structure of the core particle, there remain a multitude of structural questions still to be answered. The main points discussed here can be summarized as follows: (1) The meaning of the term 'core particle' should be widened to reflect the fact that the actual length of DNA wrapped around the histone octamer in the context of the chromatin fiber may vary between approximately 100 and approximately 170 bp. (2) In the chromatosome, the linker histone forms a bridge between one terminus of the chromatosomal DNA and a point close to the dyad axis. (3) The particle that contains one molecule of HMG1 may be classified as a bona fide chromatosome. (4) In the extended fiber, the partition of the nucleosomal DNA into core and linker is a dynamic feature, responding to environmental influences; fiber structure-related constraints demand that linker length be beyond a certain minimal value. (5) The compact fiber structure seems to be rather irregular; the precise nature of this structure is still to be determined. Finally, the term 30-nm fiber should be dropped as a designator of the compact or condensed chromatin fiber structure.

The non-histone chromatin protein HMG1 protects linker DNA on the side opposite to that protected by linker histones.

Linker histones and HMG1/2 constitute the two major proteins that bind to linker DNA in chromatin. While the location of linker histones on the nucleosome has attracted considerable research effort, only a few studies have addressed the location of HMG1 in the particles. In this study, we use a procedure based on micrococcal nuclease digestion of reconstituted nucleosomal particles to which HMG1 has been bound, followed by analysis of the protected DNA by restriction nuclease digestion, to locate the HMG1 binding site. Nucleosomal particles were reconstituted on a 235-base pair DNA fragment, which is known to be a strong nucleosome positioning sequence. The results unequivocally show that HMG1 protects linker DNA on one side of the core particle. Importantly, and possibly of physiological relevance, the linker DNA site protected by HMG1 was located on the side opposite to that already shown to be protected by linker histone binding.

Differential silver-staining sodium dodecyl sulfate-polyacrylamide gel electrophoresis: a nonisotopic method for characterizing gel-separated histone-DNA complexes.

Some nonspecific, DNA-binding proteins, like the linker histones, precipitate DNA upon binding. This is a poorly understood process that limits analysis of such nucleoprotein complexes using standard gel electrophoresis. To circumvent this problem, low concentrations of glutaraldehyde were used to crosslink the linker histones to DNA; then the partially crosslinked complexes were solubilized in SDS2 and separated by SDS-PAGE. Differential detection was accomplished using two different silver staining protocols that preferentially stained either proteins or nucleic acids. A technique was developed which allows the relative proportion of linker histones and DNAs in each detected band to be determined, and is referred to as differential staining SDS-PAGE (DS-SDS-PAGE). DS-SDS-PAGE provides a novel, non-isotopic means for characterizing multiple nucleoprotein bands separated by polyacrylamide gel electrophoresis. In applying this method to a model linker histone-DNA study, we were able to detect both protein-DNA and protein-protein contacts that are important in linker histone assembly onto DNA.

Self-association of linker histone H5 and of its globular domain: evidence for specific self-contacts.

The ability of avian-specific linker histone H5, and the globular domains of H5 (GH5) and H1(0) (GH1(0), to self-associate either free in solution or when bound to DNA was investigated. All three proteins underwent a salt-dependent increase in turbidity that may be indicative of nonspecific interactions. Dithiobis(succinimidyl propionate) cross-linking was used to measure specific contacts for both H5 and GH5 free in solution and bound to DNA. H5 and GH5 each became cross-linked in solution, with GH5 displaying divalent polymerization interactions, which suggests that two specific surfaces were involved in the assembly process. For GH5-DNA complexes, cross-linking appeared to be largely the consequence of aggregation, but under low concentrations of DSP, cross-linking GH5 was observed to assemble preferentially onto DNA before oligomerizing to form massive aggregates. Both linear and supercoiled DNA facilitated GH5 interactions compared to assembly in solution; differences in the distribution of cross-linked polymer sizes indicates that assembly is dependent on both the presence of DNA and the morphology of the DNA. Finally, on the basis of a technique referred to as quantitative proteolysis, GH5 assembly on DNA appears to involve specific protein-protein contacts involving the C terminus of one partner. Overall, the cumulative results reported here support the premise that linker histones assemble specifically both in solution and on DNA.

Linker histones versus HMG1/2: a struggle for dominance?

The linker histones (H1, H1 zero, H5, etc.) and a group of abundant non-histone chromosomal proteins (HMG1/2) bind to linker DNA in chromatin and exhibit both generalized and specific effects on gene transcription. The two classes of proteins share many features of DNA binding behaviour, although they are structurally unrelated. While the linker histones and HMG1/2 exhibit direct competition in binding to such structures as four-way junction DNA, whether they compete for binding to the nucleosome has not been investigated. The possibility for either opposite or synergistic effects on gene regulation must be considered at this point.

Linker histone protection of chromatosomes reconstituted on 5S rDNA from Xenopus borealis:a reinvestigation.

The location of the linker histone (LH) on the nucleosome has been the subject of recent controversy. Although previous evidence had supported a location over the dyad axis, some recent experiments suggest an asymmetric, off-axis position. In this paper we show that the DNA sequence used to reconstitute chromatosomes in these experiments is prone to artifacts in nuclease digestion: results interpreted as 'protection' by LHs can be obtained with either naked DNA or with reconstituted core nucleosomes, in the absence of LHs. Consequently, we feel that general interpretation or extrapolation of such results must be regarded with the utmost caution. In addition, we show that the protection data on an alternative, previously unreported major core position on this same DNA sequence support a model of asymmetric, off-axis position of the LH, with linker DNA protection on only one side of the core particle.

Sequence of the Octopus dofleini hemocyanin subunit: structural and evolutionary implications.

Sequencing of the subunit of the hemocyanin of Octopus dofleini has been completed from a cDNA library. This represents the first molluscan hemocyanin to be completely sequenced. The sequence determined is for one of the two distinguishable cDNAs which have been recognized for this protein. The protein subunit has 2896 amino acids and contains seven functional units, each carrying two sets of three invariant histidine residues constituting the binding sites (A and B) for two copper atoms. The accompanying paper identifies this site in the C-terminal functional unit (Odg). Differences in sequence for the two cDNAs, for the region in which both are available, are concentrated in the "linker regions" between functional units. The sequences of the seven units exhibit high similarity, averaging about 40% identity, with a concentration of conserved sequences in the region surrounding the copper binding sites. The sequences around the B-site show significant homology to the sequences of arthropod hemocyanins. Comparison of the functional unit sequences in terms of hydrophobicity and surface exposure profiles, as well as regions of probable secondary structure, indicate that all functional units probably have a common tertiary folding; the protein subunit is a string of similarly folded beads. A number of putative N-linked carbohydrate binding sites can be recognized in the sequence; one of these corresponds to the carbohydrate observed in the X-ray diffraction study of functional unit Odg as disclosed in the accompying paper. Phylogenetic analysis of the sequences of the O. dofleini functional units, and comparison with other available molluscan sequences indicates that the multi-domain subunit structure must have arisen over a relatively brief period, preceeding the differentiation of major molluscan types.

Crystal structure of a functional unit from Octopus hemocyanin.

Hemocyanins are giant oxygen transport proteins found in many arthropods and molluscs. Freely dissolved in the hemolymph, they are multisubunit proteins that contain many copies of the active site, a copper atom pair that reversibly binds oxygen. Octopus hemocyanin is composed of ten subunits, each of which contain seven oxygen-binding "functional units". The carboxyl-terminal 47 kDa functional unit, Odg, is a proteolytic isolate that binds oxygen reversibly while exhibiting slight Bohr and magnesium ion effects. In this work we present the X-ray structure determination and analysis of Odg at 2.3 A resolution. Odg has two structural domains: a largely alpha-helical copper binding domain, and a five-stranded anti-parallel beta-sandwich with the jelly roll topology found in many viruses. Six histidine residues ligate the copper atoms, one of which is involved in a thioether bridge. The results show that the hemocyanin from the mollusc and that from the arthropod have distinct tertiary folds in addition to the long recognized differences in their quaternary structures. Nonetheless, a comparison of Octopus and horseshoe crab hemocyanin reveals a similar active site, in a striking example of perhaps both convergent and divergent evolution.