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PubMed2006

Influence of water, loading rate and bonder on the adhesion of porcelain to titanium.

OBJECTIVES: The aim of this study was to measure the influence of water on the adhesion between titanium and porcelain by means of the strain energy release rate (G-value or interfacial toughness). METHODS: Rectangular specimens of grade II titanium and a reference gold-palladium porcelain fused to metal (PFM) alloy were veneered with feldspathic porcelains. In the case of the titanium, two gold and one ceramic bonder systems were used to enhance the adhesion of the porcelain. The G-value was measured with a four-point bending configuration developed by Charalambides et al. (Journal of Applied Mechanics (Trans ASME). 1989;56:77-82), at four different loading rates (from 0.1 to 15 N/s) in water and at one loading rate (1.5 N/s) in kerosine. RESULTS: There was a significant difference (ANOVA, p<0.01) in the interfacial toughness between the different bonding systems, with the two goldbonder systems exhibiting higher toughness in kerosine and water than the ceramic bonder. The two goldbonders achieved the best results across all loading rates. Their interfacial toughness, tested in kerosine, varied between 45.83+/-6.72 and 73.54+/-17.01 J/m(2) and tested in water from 40.86+/-2.23 to 69.17+/-12.33 J/m(2), respectively. The ceramic Ti bonder achieved the lowest G-values throughout (30.15+/-5.52 J/m(2) in kerosine to 11.08+/-2.18 J/m(2) in water). The gold-palladium PFM alloy showed more constant behaviour (33.48+/-2.24 J/m(2) to 23.07+/-0.53 J/m(2) in kerosine and water, respectively). CONCLUSION: Compared to the gold-palladium PFM alloy, the goldbonders achieved much higher G-values whereas the ceramic Ti bonder G-values were lower when tested in water and in kerosine.

Adhesiveness↗
PubMed2006

The influence of montmorillonite clay reinforcement on the performance of a glass ionomer restorative.

OBJECTIVES: A pristine calcium montmorillonite (Ca-MMT) and an organically modified 12-amino-dodecanoicacid treated montmorillonite (ADA-MMT) clay were evaluated to determine the reinforcement effect on the performance of a glass ionomer (GI) restorative ChemFil Superior (Dentsply DeTrey, Kanstanz, Germany) to assess the potential as a posterior filling material. METHODS: Mean compressive fracture strengths and standard deviations were determined for groups of 30 cylindrical specimens with 0.5-2.5 wt.% Ca-MMT and ADA-MMT additions to either the powder or the liquid elements of the GI. Working characteristics were assessed using an oscillating rheometer, the interlayer d-spacings (d(001)) of each MMT clay was determined using X-ray diffraction (XRD) and the set cement structure was determined by scanning electron microscopy (SEM). RESULTS: The addition of up to 1.0 wt.% ADA-MMT resulted in a significant increase in the mean compressive fracture strength of the GI utilising the one-way ANOVA and Tukey test comparisons at P<0.05. When Ca-MMT clay was added to the GI a significant reduction in mean strength was recorded (P<0.05). MMT clay addition to the powder and liquid element of the GI in excess of 1.0 wt.% resulted in no significant differences in working characteristics although increases in setting times were evident for MMT addition up to 1.0 wt.%. XRD patterns identified the d(001) peaks for Ca- and ADA-MMT at 2 theta angles of 8.44 degrees and 5.07 degrees and d(001) spacings of 1.04 and 1.74 nm, respectively. CONCLUSIONS: The increased interlayer d-spacings recorded for ADA-MMT clay demonstrates that the clay had expanded layers which may have provided an increased opportunity for the polyacrylic acid chains of the GI restorative to diffuse into the MMT galleries. It is postulated that the increased interlayer d-spacings for the ADA-MMT clay had a positive reinforcing effect on the GI compared with the Ca-MMT clay where no increased performance was identified. The study has highlighted the potential for increasing the performance of GI restoratives when using an organically modified clay as a reinforcement which could increase the potential of GIs as posterior filling materials.

Aluminum Silicates↗
PubMed2007

Resin strengthening of dental ceramics- the impact of surface texture and silane.

OBJECTIVES: To examine the effect of porcelain surface texture achieved utilising three clinically relevant surface preparation methods prior to silane and unfilled resin application on the flexure strength of a low fusing feldspathic porcelain. MATERIALS AND METHODS: Five hundred and forty dentine disc-shaped specimens (15.0mm diameter and 0.9mm thickness) were condensed, fired and allocated to 18 groups. Six groups were stored as-fired, six were alumina abraded and six were acid-etched. Samples were coated with silane, unfilled resin or both prior to bi-axial flexure testing. Group means were compared utilising a three factor design general linear model and post hoc all paired Tukey tests at P<0.05. Weibull analysis was employed to examine the reliability of the strength data. Profilometry was used to characterise the surface texture of the specimen surfaces. RESULTS: Alumina abraded and acid-etched specimens had significantly lower flexure strengths than the as-fired specimens. A significant difference between the degree of strengthening observed following unfilled resin application on the three surface texture investigated was observed (P<0.001). Silane application prior to resin coating resulted in no further significant increases in the mean bi-axial flexure strengths of the three surface textures. The as-fired surfaces had a low frequency of irregular amplitude defects, alumina abraded surfaces had an increased frequency of regular amplitude defects whilst the acid-etched surface consisted of an increased frequency of irregular amplitude defects. CONCLUSION: The strengthening mechanism whilst dependent on surface texture was independent of defect severity. No significant strengthening occurred following silane priming suggesting that, for the unfilled resin utilised, the strengthening mechanism was not enhanced by improved resin-ceramic adhesion.

Acid Etching, Dental↗
PubMed2004

A thermodynamic basis of DNA sequence selectivity by the ETS domain of murine PU.1.

The ETS domain of the transcription factor PU.1 tolerates a large number of DNA cognate variants that differ exclusively in the sequences flanking a critical central consensus, 5'-GGAA-3'. We investigated the thermodynamics of site selection by the DNA-binding domain by following the PU.1 ETS/DNA equilibrium with a large set of cognate variants under various temperature and salt conditions by filter binding. Our results indicate that the stability of the ETS/DNA complex is quantitatively tied to variations in the change in heat capacity. Thermodynamic effects induced by changing Na(+) concentrations from 150 mM to 250 mM are complex and not readily interpreted by polyelectrolyte theory. We also extended our understanding of data from our previous investigation on energetic base-neighbour coupling, by dissecting the thermodynamic contributions underlying the observed free-energy coupling. In conjunction with available structural and biochemical data, we propose that site selectivity by the PU.1 ETS domain arises from differential protein/DNA contacts in the flanking sequences that modulate the orientation of the ETS recognition helix and trigger a coupled reduction in the flexibility observed in the unbound ETS domain.

Animals↗
PubMed2004

Structure of the integrin alpha2beta1-binding collagen peptide.

We have determined the 1.8A crystal structure of a triple helical integrin-binding collagen peptide (IBP) with sequence (Gly-Pro-Hyp)(2)-Gly-Phe-Hyp-Gly-Glu-Arg-(Gly-Pro-Hyp)(3). The central GFOGER hexapeptide is recognised specifically by the integrins alpha2beta1, alpha1beta1, alpha10beta1 and alpha11beta1. These integrin/collagen interactions are implicated in a number of key physiological processes including cell adhesion, cell growth and differentiation, and pathological states such as thrombosis and tumour metastasis. Comparison of the IBP structure with the previously determined structure of an identical collagen peptide in complex with the integrin alpha2-I domain (IBP(c)) allows the first detailed examination of collagen in a bound and an unbound state. The IBP structure shows a direct and a water-mediated electrostatic interaction between Glu and Arg side-chains from adjacent strands, but no intra-strand interactions. The interactions between IBP Glu and Arg side-chains are disrupted upon integrin binding. A comparison of IBP and IBP(c) main-chain conformation reveals the flexible nature of the triple helix backbone in the imino-poor GFOGER region. This flexibility could be important to the integrin-collagen interaction and provides a possible explanation for the unique orientation of the three GFOGER strands observed in the integrin-IBP(c) complex crystal structure.

Amino Acid Motifs↗
PubMed2004

Assessing the flexibility of intermediate filaments by atomic force microscopy.

Eukaryotic cells contain three cytoskeletal filament systems that exhibit very distinct assembly properties, supramolecular architectures, dynamic behaviour and mechanical properties. Microtubules and microfilaments are relatively stiff polar structures whose assembly is modulated by the state of hydrolysis of the bound nucleotide. In contrast, intermediate filaments (IFs) are more flexible apolar structures assembled from a approximately 45 nm long coiled-coil dimer as the elementary building block. The differences in flexibility that exist among the three filament systems have been described qualitatively by comparing electron micrographs of negatively stained dehydrated filaments and by directly measuring the persistence length of F-actin filaments (approximately 3-10 microm) and microtubules (approximately 1-8 mm) by various physical methods. However, quantitative data on the persistence length of IFs are still missing. Toward this goal, we have carried out atomic force microscopy (AFM) in physiological buffer to characterise the morphology of individual vimentin IFs adsorbed to different solid supports. In addition, we compared these images with those obtained by transmission electron microscopy (TEM) of negatively stained dehydrated filaments. For each support, we could accurately measure the apparent persistence length of the filaments, yielding values ranging between 0.3 microm and 1 microm. Making simple assumptions concerning the adsorption mechanism, we could estimate the persistence length of an IF in a dilute solution to be approximately 1 microm, indicating that the lower measured values reflect constraints induced by the adsorption process of the filaments on the corresponding support. Based on our knowledge of the structural organisation and mechanical properties of IFs, we reason that the lower persistence length of IFs compared to that of F-actin filaments is caused by the presence of flexible linker regions within the coiled-coil dimer and by postulating the occurrence of axial slipping between dimers within IFs.

Actin Cytoskeleton↗
PubMed2004

Mechanics of DNA flexibility visualized by selective 2'-amine acylation at nucleotide bulges.

We used selective acylation of 2'-amine-substituted nucleotides to visualize local backbone conformations that occur preferentially at bulged sites in DNA duplexes. 2'-Amine acylation reports local nucleotide flexibility because unconstrained 2'-amino nucleotides more readily reach a reactive conformation in which the amide-forming transition state is stabilized by interactions between the amine nucleophile and the adjacent 3'-phosphodiester group. Bulged 2'-amine-substituted cytidine nucleotides react approximately 20-fold more rapidly than nucleotides constrained by base-pairing at 35 degrees C. In contrast, base-paired 2'-amine-substituted nucleotides flanked by a 5' or 3' bulge react two- or six-fold more rapidly, respectively, than the perfectly paired duplex. The relative lack of 2'-amine reactivity for nucleotides adjacent to a DNA bulge emphasizes, first, that structural perturbations do not extend significantly into the flanking duplex structure. Second, the exquisite sensitivity towards very local perturbations in nucleic acid structure suggests that 2'-amine acylation can be used to chemically interrogate deletion mutations in DNA. Finally, these data support the mechanical interpretation that the reactive ribose conformation for 2'-amine acylation requires that the base lies out of the helix and in the major groove, a mechanistic insight useful for designing 2'-amine-based sensors.

Acylation↗
PubMed2004

Testing a flexible-receptor docking algorithm in a model binding site.

Sampling receptor flexibility is challenging for database docking. We consider a method that treats multiple flexible regions of the binding site independently, recombining them to generate different discrete conformations. This algorithm scales linearly rather than exponentially with the receptor's degrees of freedom. The method was first evaluated for its ability to identify known ligands of a hydrophobic cavity mutant of T4 lysozyme (L99A). Some 200000 molecules of the Available Chemical Directory (ACD) were docked against an ensemble of cavity conformations. Surprisingly, the enrichment of known ligands from among a much larger number of decoys in the ACD was worse than simply docking to the apo conformation alone. Large decoys, accommodated in the larger cavity conformations sampled in the ensemble, were ranked better than known small ligands. The calculation was redone with an energy correction term that considered the cost of forming the larger cavity conformations. Enrichment improved, as did the balance between high-ranking large and small ligands. In a second retrospective test, the ACD was docked against a conformational ensemble of thymidylate synthase. Compared to docking against individual enzyme conformations, the flexible receptor docking approach improved enrichment of known ligands. Including a receptor conformational energy weighting term improved enrichment further. To test the method prospectively, the ACD database was docked against another cavity mutant of lysozyme (L99A/M102Q). A total of 18 new compounds predicted to bind this polar cavity and to change its conformation were tested experimentally; 14 were found to bind. The bound structures for seven ligands were determined by X-ray crystallography. The predicted geometries of these ligands all corresponded to the observed geometries to within 0.7A RMSD or better. Significant conformational changes of the cavity were observed in all seven complexes. In five structures, part of the observed accommodations were correctly predicted; in two structures, the receptor conformational changes were unanticipated and thus never sampled. These results suggest that although sampling receptor flexibility can lead to novel ligands that would have been missed when docking a rigid structure, it is also important to consider receptor conformational energy.

Algorithms↗
PubMed2004

Biochemical and physiological properties of the DNA binding domain of AraC protein.

Intact AraC protein is poorly soluble and difficult to purify, whereas its dimerization domain is the opposite. Unexpectedly, the DNA binding domain of AraC proved also to be soluble in cells when overproduced and is easily purified to homogeneity. The DNA binding affinity of the DNA binding domain for its binding site could not be measured by electrophoretic mobility shift because of its rapid association and dissociation rates, but its affinity could be measured with a fluorescence assay and was found to have a dissociation constant of 1 x 10(-8)M in 100 mM KCl. The binding of monomers of the DNA binding domain to adjacent half-sites occurs without substantial positive or negative cooperativity. A simple analysis relates the DNA binding affinities of monomers of DNA binding domain and normal dimeric AraC protein.

AraC Transcription Factor↗
PubMed2004

Modulation of S6 fibrillation by unfolding rates and gatekeeper residues.

We present a protein engineering analysis of the fibrillation of a protein from a thermophilic organism, the 101 residue S6 from Thermus thermophilus. When agitated, S6 fibrillates at pH 2.0 in 0.4 M NaCl. Under these solvent conditions, S6 has native-like secondary structure and also unfolds and refolds cooperatively. However, its tertiary structure appears to be more plastic than at neutral pH, and some regions of the protein may be partially unstructured. At 42 degrees C, there is a lag phase of several days after which fibrillation takes place over several hours. Data from the fibrillation behaviour of a comprehensive series of single and double mutants of S6 suggests that several factors control the onset of fibrillation. Firstly, there appears to be a contiguous region of "gatekeeper" residues that inhibit fibrillation, since their truncation significantly reduces the duration of the lag phase. This region overlaps extensively with the partially unstructured region of the protein, suggesting that residues with enhanced flexibility and solvent-accessibility are important for the initiation of fibrillation. Secondly, longer lag phases correlate with faster rates of unfolding. We interpret this to mean that kinetic stability also controls fibrillation but in the sense that the quasi-native state, rather than the denatured state, is the species that participates in nucleation. This implies that fibrillation can also occur from a quasi-native state as opposed to an ensemble of highly fluctuating structures, and highlights the delicate balance between flexibility and structure required to form organized assemblies of polypeptide chains.

Amino Acids↗
PubMed2004

Relative flexibility of DNA and RNA: a molecular dynamics study.

State of the art molecular dynamics simulations are used to study the structure, dynamics, molecular interaction properties and flexibility of DNA and RNA duplexes in aqueous solution. Special attention is paid to the deformability of both types of structures, revisiting concepts on the relative flexibility of DNA and RNA duplexes. Our simulations strongly suggest that the concepts of flexibility, rigidity and deformability are much more complex than usually believed, and that it is not always true that DNA is more flexible than RNA.

Computer Simulation↗
PubMed2004

Independent movement, dimerization and stability of tandem repeats of chicken brain alpha-spectrin.

Previous X-ray crystal structures have shown that linkers of five amino acid residues connecting pairs of chicken brain alpha-spectrin and human erythroid beta-spectrin repeats can undergo bending without losing their alpha-helical structure. To test whether bending at one linker can influence bending at an adjacent linker, the structures of two and three repeat fragments of chicken brain alpha-spectrin have been determined by X-ray crystallography. The structure of the three-repeat fragment clearly shows that bending at one linker can occur independently of bending at an adjacent linker. This observation increases the possible trajectories of modeled chains of spectrin repeats. Furthermore, the three-repeat molecule crystallized as an antiparallel dimer with a significantly smaller buried interfacial area than that of alpha-actinin, a spectrin-related molecule, but large enough and of a type indicating biological specificity. Comparison of the structures of the spectrin and alpha-actinin dimers supports weak association of the former, which could not be detected by analytical ultracentrifugation, versus strong association of the latter, which has been observed by others. To correlate features of the structure with solution properties and to test a previous model of stable spectrin and dystrophin repeats, the number of inter-helical interactions in each repeat of several spectrin structures were counted and compared to their thermal stabilities. Inter-helical interactions, but not all interactions, increased in parallel with measured thermal stabilities of each repeat and in agreement with the thermal stabilities of two and three repeats and also partial repeats of spectrin.

Actinin↗
PubMed2004

Structural bases of the redox-dependent conformational switch in the serpin PAI-2.

Depending on the redox-status, the serpin plasminogen activator inhibitor type 2 (PAI-2) can exist in either a stable monomeric or polymerogenic form. The latter form, which spontaneously forms loop-sheet polymers, has an open beta-sheet A and is stabilized by a disulfide bond between C79 (in the CD-loop) and C161 (at the bottom of PAI-2). Reduction of this bond results in a closing of the beta-sheet A and converts PAI-2 to a stable monomeric form. Here we show that the stable monomeric and polymerogenic forms of PAI-2 are fully interconvertible, depending on redox-status of the environment. Our intramolecular distance measurements indicate that the CD-loop folds mainly on one side of the stable monomeric form of the inhibitor. However, the loop can translocate about 54A to the bottom of PAI-2 so that the C79-C161 disulfide bond can form under oxidizing conditions. We show also that the redox-active C79 can form a disulfide-link to the matrix protein vitronectin, suggesting that vitronectin can stabilize active PAI-2 in extracellular compartments. PAI-2 is therefore a rare example of a redox-sensitive protein for which the activity and polymerization ability are regulated by reversible disulfide bond formation leading to major translocation of a loop and significant conformational changes in the molecule.

Cysteine↗
PubMed2005

A survey of flexible protein binding mechanisms and their transition states using native topology based energy landscapes.

Many cellular functions rely on interactions between protein pairs and higher oligomers. We have recently shown that binding mechanisms are robust and owing to the minimal frustration principle, just as for protein folding, are governed primarily by the protein's native topology, which is characterized by the network of non-covalent residue-residue interactions. The detailed binding mechanisms of nine dimers, a trimer, and a tetramer, each involving different degrees of flexibility and plasticity during assembly, are surveyed here using a model that is based solely on the protein topology, having a perfectly funneled energy landscape. The importance of flexibility in binding reactions is manifested by the fly-casting effect, which is diminished in magnitude when protein flexibility is removed. Many of the grosser and finer structural aspects of the various binding mechanisms (including binding of pre-folded monomers, binding of intrinsically unfolded monomers, and binding by domain-swapping) predicted by the native topology based landscape model are consistent with the mechanisms found in the laboratory. An asymmetric binding mechanism is often observed for the formation of the symmetric homodimers where one monomer is more structured at the binding transition state and serves as a template for the folding of the other monomer. Phi values were calculated to show how the structure of the binding transition state ensemble would be manifested in protein engineering studies. For most systems, the simulated Phi values are reasonably correlated with the available experimental values. This agreement suggests that the overall binding mechanism and the nature of the binding transition state ensemble can be understood from the network of interactions that stabilize the native fold. The Phi values for the formation of an antibody-antigen complex indicate a possible role for solvation of the interface in biomolecular association of large rigid proteins.

Antigen-Antibody Complex↗
PubMed2005

The essential role of the flexible termini in the temperature-responsiveness of the oligomeric state and chaperone-like activity for the polydisperse small heat shock protein IbpB from Escherichia coli.

Small heat shock proteins (sHSPs) represent an abundant and ubiquitous family of molecular chaperones that are believed to prevent irreversible aggregation of other cellular proteins under stress conditions. One of the most prominent features of sHSPs is that they exist as homo-oligomers. Examples of both monodisperse and polydisperse oligomers are found within this family. The small heat shock inclusion-body binding protein B (IbpB) of Escherichia coli, originally discovered as a component of inclusion bodies, exhibits a pronounced polydispersity in its oligomeric state. This research was performed to elucidate the temperature effect on the oligomeric state and chaperone-like activity of the polydisperse IbpB oligomers, as well as the structural basis for such a temperature effect. The data presented here demonstrate that the large oligomers of IbpB progressively dissociate into smaller ones at increasing heat-shock temperatures, accompanied by a notable enhancement of chaperone-like activities. The secondary structure, enriched mainly by beta-strands, is slightly changed with such temperature increases. The dimeric building blocks, which seem to be highly stable, act as the functional unit of IbpB. Limited proteolysis was used to identify the susceptible sites in IbpB that may compose the subunit interfaces, which indicated that the 11 residues at both the N and the C terminus are highly flexible and the removal of each will lead to the formation of dimers, as well as the disappearance of chaperone-like activities. Truncation of 11 residues from either end, using recombinant DNA technology, also led to the formation of dimeric mutant IbpB proteins lacking chaperone-like activities. Taken together, the flexible termini appear to be essential for small heat shock protein IbpB to generate various temperature-responsive oligomers, which exhibit various levels of chaperone-like activities, by interlinking or separating the dimer building blocks.

Circular Dichroism↗
PubMed2005

The relationship between the flexibility of proteins and their conformational states on forming protein-protein complexes with an application to protein-protein docking.

We investigate the extent to which the conformational fluctuations of proteins in solution reflect the conformational changes that they undergo when they form binary protein-protein complexes. To do this, we study a set of 41 proteins that form such complexes and whose three-dimensional structures are known, both bound in the complex and unbound. We carry out molecular dynamics simulations of each protein, starting from the unbound structure, and analyze the resulting conformational fluctuations in trajectories of 5 ns in length, comparing with the structure in the complex. It is found that fluctuations take some parts of the molecules into regions of conformational space close to the bound state (or give information about it), but at no point in the simulation does each protein as whole sample the complete bound state. Subsequent use of conformations from a clustered MD ensemble in rigid-body docking is nevertheless partially successful when compared to docking the unbound conformations, as long as the unbound conformations are themselves included with the MD conformations and the whole globally rescored. For one key example where sub-domain motion is present, a ribonuclease inhibitor, principal components analysis of the MD was applied and was also able to produce conformations for docking that gave enhanced results compared to the unbound. The most significant finding is that core interface residues show a tendency to be less mobile (by size of fluctuation or entropy) than the rest of the surface even when the other binding partner is absent, and conversely the peripheral interface residues are more mobile. This surprising result, consistent across up to 40 of the 41 proteins, suggests different roles for these regions in protein recognition and binding, and suggests ways that docking algorithms could be improved by treating these regions differently in the docking process.

Cluster Analysis↗
PubMed2005

Crystal structures of human DcpS in ligand-free and m7GDP-bound forms suggest a dynamic mechanism for scavenger mRNA decapping.

Eukaryotic cells utilize DcpS, a scavenger decapping enzyme, to degrade the residual cap structure following 3'-5' mRNA decay, thereby preventing the premature decapping of the capped long mRNA and misincorporation of methylated nucleotides in nucleic acids. We report the structures of DcpS in ligand-free form and in a complex with m7GDP. apo-DcpS is a symmetric dimer, strikingly different from the asymmetric dimer observed in the structures of DcpS with bound cap analogues. In contrast, and similar to the m7GpppG-DcpS complex, DcpS with bound m7GDP is an asymmetric dimer in which the closed state appears to be the substrate-bound complex, whereas the open state mimics the product-bound complex. Comparisons of these structures revealed conformational changes of both the N-terminal swapped-dimeric domain and the cap-binding pocket upon cap binding. Moreover, Tyr273 in the cap-binding pocket displays remarkable conformational changes upon cap binding. Mutagenesis and biochemical analysis suggest that Tyr273 seems to play an important role in cap binding and product release. Examination of the crystallographic B-factors indicates that the N-terminal domain in apo-DcpS is inherently flexible, and in a dynamic state ready for substrate binding and product release.

Apoproteins↗
PubMed2005

Bacterial repression loops require enhanced DNA flexibility.

The Escherichia coli lac operon provides a classic paradigm for understanding regulation of gene transcription. It is now appreciated that lac promoter repression involves cooperative binding of the bidentate lac repressor tetramer to pairs of lac operators via DNA looping. We have adapted components of this system to create an artificial assay of DNA flexibility in E.coli. This approach allows for systematic study of endogenous and exogenous proteins as architectural factors that enhance apparent DNA flexibility in vivo. We show that inducer binding does not completely remove repression loops but it does alter their geometries. Deletion of the E.coli HU protein drastically destabilizes small repression loops, an effect that can be partially overcome by expression of a heterologous mammalian HMG protein. These results emphasize that the inherent torsional inflexibility of DNA restrains looping and must be modulated in vivo.

Animals↗