Combining structure-based design with phage display to create new Cys(2)His(2) zinc finger dimers.

BACKGROUND: Several strategies have been reported for the design and selection of novel DNA-binding proteins. Most of these studies have used Cys(2)His(2) zinc finger proteins as a framework, and have focused on constructs that bind DNA in a manner similar to Zif268, with neighboring fingers connected by a canonical (Krüppel-type) linker. This linker does not seem ideal for larger constructs because only modest improvements in affinity are observed when more than three fingers are connected in this manner. Two strategies have been described that allow the productive assembly of more than three canonically linked fingers on a DNA site: connecting sets of fingers using linkers (covalent), or assembling sets of fingers using dimerization domains (non-covalent). RESULTS: Using a combination of structure-based design and phage display, we have developed a new dimerization system for Cys(2)His(2) zinc fingers that allows the assembly of more than three fingers on a desired target site. Zinc finger constructs employing this new dimerization system have high affinity and good specificity for their target sites both in vitro and in vivo. Constructs that recognize an asymmetric binding site as heterodimers can be obtained through substitutions in the zinc finger and dimerization regions. CONCLUSIONS: Our modular zinc finger dimerization system allows more than three Cys(2)His(2) zinc fingers to be productively assembled on a DNA-binding site. Dimerization may offer certain advantages over covalent linkage for the recognition of large DNA sequences. Our results also illustrate the power of combining structure-based design with phage display in a strategy that assimilates the best features of each method.

Analysis of zinc fingers optimized via phage display: evaluating the utility of a recognition code.

Cys2His2 zinc finger proteins are composed of modular DNA-binding domains and provide an excellent framework for the design and selection of proteins with novel site specificity. Crystal structures of zinc finger-DNA complexes have shown that many Cys2His2 zinc fingers use a conserved docking arrangement that juxtaposes residues at key positions in the "recognition helix" with corresponding base positions in the three to four base-pair subsite. Several groups have proposed that specificity can be explained with a zinc finger-DNA recognition code that correlates specific amino acids at these key positions in the alpha-helix with specific bases in each position of the corresponding subsite. Here, we explore the utility of such a code through detailed studies of zinc finger variants selected via phage display. These proteins provide interesting systems for detailed analysis since they have affinities and specificities for their sites similar to those of naturally occurring DNA-binding proteins. Comparisons are facilitated by the fact that only key DNA-binding residues are varied in each finger while leaving all other regions of the structure unchanged. We study these proteins in detail by (1) selecting their optimal binding sites and comparing these binding sites with sites that might have been predicted from a code; (2) by examining the "evolutionary history" of these proteins during the phage display protocol to look for evidence of context-dependent effects; and (3) by reselecting finger 1 in the presence of the optimized finger 2/finger 3 domains to obtain further data on finger modularity. Our data for optimized fingers and binding sites demonstrate a clear correlation with contacts that would be predicted from a code. However, there are enough examples of context-dependent effects (not explained by any existing code) that selection is the most reliable method for maximizing the affinity and specificity of new zinc finger proteins.

Imipramine and brief therapists-aided exposure in agoraphobics having self-exposure homework.

Forty-five chronic agoraphobics were randomly assigned to treatment by placebo or imipramine in doses up to 200 mg/day for 28 weeks. All patients also had systematic self-exposure homework with an instruction manual. In addition, half of each drug group had therapist-aided exposure and half had therapist-aided relaxation, each totalling three hours. Patients in both drug groups improved substantially and maintained their gains for one year of follow-up. Imipramine had no significant therapeutic effect despite satisfactory plasma levels and significant drug side effects. Patients' low initial Hamilton depression scores might explain the absence of any drug effect. Antidepressants may be ineffective for agoraphobics who have normal mood. Brief therapist-aided exposure improved phobias and panics to a significant but limited extent, and is a useful adjuvant to self-exposure homework, which can be a powerful therapeutic agency by itself.

Structural differences between histone H1 molecules from sea urchin (Strongylocentrotus intermedius) sperm and calf thymus: hydrodynamic and c.d. studies.

Comparative sedimentation, diffusion and circular dichroism (c.d.) measurements have been performed on two histones H1 from sperm of the sea urchin Strongylocentrotus intermedius (H1S) and from calf thymus (H1T), at a high salt concentration of M NaCl. Both the Stokes radius and the frictional ratio derived from the hydrodynamic parameters were found to be somewhat smaller for H1S than the corresponding values for H1T. In view of the considerably higher molar mass of H1S compared with that of H1T, this result indicates that H+S in 2 M NaCl has a more compact conformation than H1T, probably due to a higher degree of secondary structure in the flanking domains of H1S. The c.d. measurements likewise show that H1S has a higher content of ordered structures than H1T. Model considerations indicate that the C-terminal tail of H1S is the main candidate for accommodation of these additional secondary structure regions.

[Structure of chromatin from the pigeon brain. I. Composition, electrophoretic motility and circular dichroism spectra of nucleosome particles]. / Struktura khromatina mozga golubia. I. Sostav, élektroforeticheskaia podvizhnost' i spektry krugovogo dikhroizma nukleosomnykh chastits.

Composition and structural properties of pigeon brain hemisphere neuron chromatin have been studied. In these cells nucleosomal DNA repeat length is about 165 nucleotide pairs. The content of H1 and H2A histones was found to decrease by 18 and 30% respectively in comparison with the chromatin possessing the normal quantity of those histones. At the same time the content of protein uH2A (A-24), being the conjugate of H2A histone and ubiquitine, is increased. Mononucleosomes isolated from neuron chromatin was found to have relatively low electrophoretic mobility in polyacrylamide gel taking into account the size of their DNA fragment. Circular dichroism spectra of nucleosome particles show that the neuron mononucleosomes are more unfolded than the rat thymus ones. Data obtained allow to suggest that the short DNA repeat and accumulation of protein uH2A in neurons are the factors influencing the compactization of neuron chromatin.

[Conformational properties of protamines]. / Konformatsionnye osobennosti protaminov.

Conformational peculiarities of protamines in a wide range of various conditions have been studied by the CD and X-ray analysis. Characteristic peculiarities of CD-spectra and position of diffraction maximum in X-ray diagrams of protamines argue in favor that their conformation is similar to that of the extended left handed helix of poly(L)-proline II. Though conformation parameters of this structure are very sensitive to temperature or pH variations, to specific action of different ions, alcohols, and detergents the structure itself is rather stable and is destroyed under extreme conditions only. This conformation seems to be optimal for protamines to interact with DNA and necessary for cross-linking of DNA molecules and for the more dense packing of chromatin.

[Optical and hydrodynamic properties of DNA complexes with histones H1 from the calf thymus and sea urchin sperm]. / Issledovanie opticheskikh i gidrodinamicheskikh svo'istv kompleksov DNK s gistonami H1 is timusa telenka i spermiev morskogo ezha.

A comparative study of the complexes between DNA and H1 histones from calf thymus (H1-T) and sea urchin sperm (H1-S) has been performed using methods of flow birefringence, viscometry and circular dichroism (CD). Both nucleohistone complexes undergo a conformational transition with the increase of protein content. A two-fold drop of intrinsic viscosity was observed when the histone content in the complex increased up to 9-12%. The transition is not accompanied by essential changes in the nucleohistone anisotropy, the latter coincides with the anisotropy of DNA in H1-T containing complexes and is close to that in H1-S containing complexes. CD spectra of the two types of complexes are not the same. For the H1-S containing complexes the decrease of the amplitude of the CD positive band and the shift of its maximum to the longwave region are observed while the spectrum of H1-T containing complex coincides with that of DNA. The spectral changes are partly due to slight aggregation of the H1-S containing complexes. It is also possible that changes in the local structure of DNA within the H1-S containing nucleohistone complexes take place.

[Comparative study of nucleosome particles in chromatin from normal and tumor cells. I. Structural parameters]. / Sravnitel'noe issledovanie nukleosomnykh chastits khromatina normal'nykh i opukholevykh kletok. I. Strukturnye parametry.

The composition and structure of nucleosomic fragments isolated from the ascitic hepatoma 22A cells, liver and from cells of C3HA mice in norm and after partial hepatectomy were investigated. Via electrophoresis in 1.5% agarose gel with the emplogment of reperic restrictive DNA fragments and with the help of mathematical processing, the value of the nucleosomic DNA repeat in ascitic hepatoma 22A was calculated to be 187 b.p., and in regenerating liver--196 b.p. The absence of the H1 degree subfraction in chromatin of ascitic hepatoma 22A cells was found. Lower electrophoretic mobility in 5% polyacrylamid gel of nucleosomic chromatin fragments of ascitic hepatoma 22A as compared with their counterparts from healthy mice liver was established. The method of circular dichroism allowed to reveal differences in the RNA and protein structural state in nucleosomes of normal and tumour cells. The structure of nucleosomes of regenerating mice liver of the C3HA strain did not differ from that of normal liver of the same mice.

[Comparative study of nucleosome particles in chromatin from normal and tumor cells. II. Reconstitution, compaction and association induced by ionic strength of a solution]. / Sravnitel'noe issledovanie nukleosomnykh chastits khromatina normal'nykh i opukholevykh kletok. II. Rekonstruktsiia, kompaktizatsiia i assotsiiatsiia pod deistviem ionnoi sily rastvora.

The method of circular dichroism (CD) has been used to investigate the reconstitution of mononucleosomes from C3HA mice liver and ascitic hepatoma 22A cells chromatin. It has been revealed that the more unfolding state of DNA in ascitic nucleosomes (discovered earlier) is determined by the peculiarities of the interactions between DNA and the dimers H2A-H2B, as well as by the linker histones of the H1 group. The investigation of the DNA folding in the oligonucleosome chains with increasing ionic strength has shown complete invariability of the DNA compactness in the ascitic chromatin up to 100 mM NaCl, while in liver nucleosomes an additional folding of the linker portion of the DNA was observed within the range of 20-40 mM NaCl. Oligonucleosomes from ascitic chromatin are less inclined to association upon increasing ionic strength, as compared with those from liver chromatin.

[Study of DNA complexes with a synthetic fragment of histone H4 and polypeptide (Gly-Orn-Gly)n]. / Issledovanie kompleksov DNK s sinteticheskim fragmentom gistona H4 i polipeptidom (gly-Orn-Gly)n.

Complexes of DNA with synthetic N-terminal peptide of histone H4 (A-13) and with poly(Gly-Orn-Gly) in 0.015 M NaCl solution have been studied by means of viscometry, light scattering and circular dichroism. The process of intramolecular compactization begins to occur at a low content of poly(Gly-Orn-Gly) in complexes, while in the case of A-13 compactization takes place only when the content of A-13 in complexes attains 13--14%. Intermolecular aggregation of both complexes takes place with the increase of peptide content in the complexes. Intramolecular compactization and aggregation do not influence the local conformation of DNA as revealed by CD spectra. The possible mechanism of these peptide-DNA interactions suggested.

[Structural transformations of oligonucleosomes from pigeon erythrocyte chromatin]. / Strukturnye prevrashcheniia oligonukleosom khromatina éritrotsitov golubia.

The methods of velocity sedimentation and circular dichroism have been used to investigate structural rearrangements of pigeon erythrocyte oligonucleosomes isolated after digestion with micrococcal nuclease (oligonucleosomes-M) or pancreatic DNase I (oligonucleosomes-D), in the wide range of ionic strength (mu from 0.005 to 0.5). The electrophoretic analysis of DNA isolated from the oligonucleosomes has revealed internal cuts in the DNA chain of oligonucleosomes-D. In spite of this fact the conformational parameters of DNA in both types of oligonucleosomes are practically indistinguishable, and their optical and hydrodynamic properties vary in a similar way with increasing ionic strength of the solution. The specificity of DNase I action results in the ability of oligonucleosomes-D to form homogeneous associates at mu = 0.065, which seems to be due to the existence of elongated intact ends of linker DNA in oligonucleosomes-D. It has been shown that the integrity of oligonucleosomes-D in a wide range of ionic strength is maintained by histones H1 and H5, because after their dissociation the sedimentation coefficient sharply decreases. The results obtained reveal the multifunctional role of lysine-rich histones and intact linker in the processes of compaction and association of oligonucleosomes.

[Structure of illexine I2 and its complexes with DNA]. / Issledovanie struktury illeksina I2 i ego kompleksov s DNK.

Conformational peculiarities of illexine I2 both in the solution and in the complexes with DNA were studied by circular dichroism, UV-spectroscopy and spectrophotometric melting. IIlexine I2 is shown to have an extended left-handed helical conformation of poly-L-proline II type, that are stable in a wide range of experimental conditions. Upon interaction of illexine I2 with DNA, the parameters of conformation are somewhat distorted but the main peculiarities remain. The DNA double helix changes from B- to the divection of C-form at its interaction with illexine I2. The interaction of illexine I2 with DNA at low ionic strength is non-cooperative and is characterized by some specificity to A--T sequences of DNA. Illexine I2 strongly affects the DNA stability by increasing the melting temperature of DNA.

[Chromatin compactification using a model system of DNA-protein complexes]. / Issledovanie kompaktizatsii khromatina s ispol'zovaniem model'noi sistemy DNK-belkovykh kompleksov.

Conformational peculiarities of DNA complexes with histones of the H1 family have been studied by the method of circular dichroism (CD). The H1 histones were isolated from spermatozoa of the sea urchin Strongylocentrotus intermedius, starfish Aphelasterias japonica, and bivalve mollusc Chlamis islandicus and also from the rat thymus. It is shown that these sperm-specific histones do not compact DNA in low ionic strength solution. At physiologic conditions H1 from the sea urchin and starfish sperms compact DNA more intensively than other histones. The H1 from rat thymus has a minimum ability to compact DNA. This histone does not change the structure of DNA double helix. It was supposed that it could be associated with interactions of this histone with DNA in the major groove of its helix. At the same time sperm-specific H1 can interact with DNA not only in the major groove but also in the minor groove, and this induces changes in DNA structure. This DNA-protein interaction is specific for the sperm chromatin and may support the supercompact organization of the sperm chromatin.

[Conformational peculiarity of H1 histones of calf thymus and sea urchin sperm]. / Konformatsionnye osobennosti gistonov H1 iz timusa telenka i spermiev morskogo ezha.

The secondary structure, sizes and form of H1 histone molecules of calf thymus (H1-T) and sea urchin sperm (H1-S) were studied by circular dichroism, sedimentation and diffusion. An increase in the ionic strength of solution is shown to result in the formation of alpha-helical parts in H1-T and both alpha-helical and beta-structural parts of H1-S. An analysis of amino acid composition of histones and their compaction under these conditions enables a conclusion that the resulting B-structure is antiparallel and intramolecular. The above properties of H1-S can result in a more dense compactness of the sea urchin sperm chromatin.

[Nucleosome histones isolated from normal and tumor cells and the effect of bivalent metals on their conformation]. / Issledovanie nukleosomnykh gistonov, vydelennykh iz normal'nykh i opukholevykh kletok, i vliianie dvukhvalentnykh metallov na ikh konformatsiiu.

A change of the secondary structure of histones isolated from tumor cells was observed. This changed structure showed increased percentage of elongated left helix of poly-l-proline II type. It is concerned with an increased content of bivalent Ca++ and Mg++ ions.

[Processes of reconstruction and association by nucleosomes with various formations of histones]. / Issledovanie protsessov rekonstruktsii i assotsiatsii nukleosom s razlichnym sostavom gistonov.

The experiments on reconstruction of chromatin (without H1) from DNA and histone octamer containing either H2B from sea urchin sperm (H2B-S) or H2B from calf thymus are reported. It has been shown that H2B-S affects the mode of interaction of histones with DNA during the reconstitution of nucleosomal particles on one hand and on the other hand H2B-S plays a major role in the interactions of reconstituted mononucleosomes. These interactions result in supranucleosomal structures.

[Comparative study of synthetic peptide fragments of histones H2B]. / Sravnitel'noe issledovanie sinteticheskikh peptidnykh fragmentov gistona H2B.

Oligopeptides of the N-terminal end of H2B histone with different molecular weights and amino acid sequences are studied by the CD method and X-ray analysis. Characteristic peculiarities of CD-spectra and position of diffraction maximums in X-ray diagrams in all the studied oligopeptides show that their conformation by the structure is similar to the extended left--handed helix of polyproline II. The investigation of CD-spectra under different conditions showed the dependence of the stability of "proII" conformation on the oligopeptide chain length. Their spectral and conformational properties may be due to peculiarities of their amino acid composition.

[Comparison of conformational possibilities of polypeptides representing the terminal segments of different histones]. / Sravnenie konformatsionnykh vozmozhnostei polipeptidov, modeliruiushchikh kontsevye uchastki razlichnykh gistonov.

Regular polypeptides--models of the N-terminal fragments of histones H2A and H4 and the C-terminal half of histone H1 were synthesized. Conformations of these polypeptides were investigated by using the methods of circular dichroism and optical rotatory dispersion. It was shown that all polypeptides studied in aqueous solutions at neutral pH and at low temperature (+2 degrees C) had the conformation of left-handed helix (LHH) or poly-L-proline type. The neutralization of positive charges of side groups at alkaline pH of screening of charged groups at a high ionic strength (up to 1 M NaF) results in increase of the degree of defectness of this conformation. There occur no transition of LHH to such an ordered conformation as alpha-helix or beta-sheet structure or complete disappearance of LHH. The influence of temperature, 80% ethanol and 1% sodium dodecylsulphate on the structure of these polypeptides was also studied. Differences in conformational potencies of two studied groups of polypeptides which are the models of the terminal fragments of various histones were discovered and associated with different biological functions of these histones in chromatin.

[Structure of histone fragments and their models. I. Structural features of N-terminal oligopeptides of histone H4]. / Issledovanie struktury fragmentov gistonov i ikh modelei. I. Strukturnye osobennosti N-kontsevykh oligopeptidov gistona H4

Circular dichroism (CD) and infrared spectroscopy (IRS) studies of aqueous solutions of fourth N-terminal peptides of histone H4 with different chain length were carried out under various conditions. It was shown that all studied peptides had conformation of extended left-handed helix as well as poly-1-proline II at the acidic and neutral pH, in moderate ionic strength (0,15), in 80% ethanol, 0,2 M sodium dodecylsulphate, in 8 M urea and 5 M guanidinum hydrochloride. This conformation was changed by raising temperature, under transition to the range of basic pH and in the concentrated solutions of CaCl2 (5M).

[Simultaneous calculation of the secondary structure and one of the reference spectra according to the protein circular dichroism]. / O sovmestnom vychislenii vtorichnoi struktury i odnogo iz repernykh spektrov po krugovomu dikhroizmu belka.

The method of simultaneous calculation of protein structure and one of the references from the set of CD-spectra of this protein under different conditions suggested by Markussen and Volund have been considered. It has been shown that the organization of this problemin [6] contains an error and ther is no simple solution. Possible methods for reducing indeterminacy of this problem have been discussed.