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The velocities and conformations of single DNA chains were probed as they electromigrated at varying electric field strength through a crystalline array of silica colloids. An optically transparent film consisting of 300-nm silica colloids was formed on glass as a transparent crystalline layer of 7 mum thickness with an effective pore size of 45 nm. The behaviors of individual lambda-DNA molecules (48,502 base pairs) electromigrating through this material were observed to be analogous to the behaviors of long DNA chains in electrophoresis gels, including chain extension, hooking of chains around the matrix, and hernia formation. The electrophoretic mobility of lambda-DNA in this dense, narrow-pore material is surprisingly high: 1.8 cm2/Vs at 10 V/cm, which is at least as high as for much wider-pore gels. Imaging of the single molecules revealed that higher field strength caused increased chain extension and increased mobility, which reached an apparent plateau just above 2.0 cm2/Vs at 200 V/cm. Pulsed, crossed electric fields of 200 V/cm at 120 degrees to one another were applied to the material. The DNA chains were observed by imaging to electromigrate in an orderly fashion, and the migration rate was found to be length-dependent. The results indicate that these thin, robust, self-assembling inorganic materials are interesting as possible alternatives to polymeric gels for higher speed electrophoresis.

The equilibrium unfolding transitions of Cro repressor variants, dimeric variant Cro F58W and monomer Cro K56[DGEVK]F58W, have been studied by urea and guanidine hydrochloride to probe the folding mechanism. The unfolding transitions of a dimeric variant are well described by a two state process involving native dimer and unfolded monomer with a free energy of unfolding, DeltaG(0,un)(0), of approximately 10-11 kcal/mol. The midpoint of transition curves is dependent on total protein concentration and DeltaG(0,un)(0) is independent of protein concentration, as expected for this model. Unfolding of Cro monomer is well described by the standard two state model. The stability of both forms of protein increases in the presence of salt but decreases with the decrease in pH. Because of the suggested importance of a N2<-->2F dimerization process in DNA binding, we have also studied the effect of sodium perchlorate, containing the chaotropic perchlorate anion, on the conformational transition of Cro dimer by CD, fluorescence and NMR (in addition to urea and guanidine hydrochloride) in an attempt both to characterize the thermodynamics of the process and to identify conditions that lead to an increase in the population of the folded monomers. Data suggest that sodium perchlorate stabilizes the protein at low concentration (<1.5 M) and destabilizes the protein at higher perchlorate concentration with the formation of a "significantly folded" monomer. The tryptophan residue in the "significantly folded" monomer induced by perchlorate is more exposed to the solvent than in native dimer.

The loss of biological activity of phage lambda DNA was much greater when the DNA was sheared using a ceramic-coated needle attached to a syringe compared with a conventional stainless steel needle. Inactivation of the biological activity was due to breakage at the middle of the molecule. The thickness of the ceramic-coating was a crucial factor for the breakage. Because approximately the same level of inactivation was observed with a non-coated needle as with thin glass and quartz tubes, it was concluded that the unknown characteristic(s) of the silicon nitride (SiNx) coating itself resulted in the effective breakage of lambda DNA molecules by shearing force.

Bioprocessors for concentrating bioparticles, such as cells and molecules, are commonly needed in bioanalysis systems. In this microfluidic processor, a global flow field generated by ac electroosmosis transports the embedded particles to the regions near the electrode surface. The processor then utilizes electrophoretic and dielectrophoretic forces, which are effective in short range, to trap the target cells and molecules on the electrode surface. By optimizing the operating parameters, we have concentrated various biological objects in a large range of sizes, including Escherichia coli bacteria, lambda phage DNA, and single-stranded DNA fragments as small as 20 bases that have a radius of gyration of only 3 nm.

SHP, the capsid-stabilizing protein of lambdoid phage 21, is highly resistant against denaturant-induced unfolding. We demonstrate that this high functional stability of SHP is due to a high kinetic stability with a half-life for unfolding of 25 days at zero denaturant, while the thermodynamic stability is not unusually high. Unfolding experiments demonstrated that the trimeric state (also observed in crystals and present on the phage capsid) of SHP is kinetically stable in solution, while the monomer intermediate unfolds very rapidly. We also determined the crystal structure of trimeric SHP at 1.5A resolution, which was compared to that of its functional homolog gpD. This explains how a tight network of H-bonds rigidifies crucial interpenetrating residues, leading to the observed extremely slow trimer dissociation or denaturation. Taken as a whole, our results provide molecular-level insights into natural strategies to achieve kinetic stability by taking advantage of protein oligomerization. Kinetic stability may be especially needed in phage capsids to allow survival in harsh environments.

Complexes of anionic DNA and cationic liposomes self-assemble into a multilamellar structure where two-dimensional lipid sheets confine a periodic one-dimensional lattice of parallel DNA chains, between which Cd(2+) ions can condense, and be subsequently reacted with H(2)S to form CdS nanorods. In this work, we identify the synergistic roles of the anionic and cationic components within the DNA-membrane template; DNA is highly anionic and condenses the Cd(2+) ions, while the cationic membrane modulates the concentration of condensed Cd(2+) ions to control the final CdS nanorod dimensions. Due to the strong electrostatic interactions between the DNA sugar-phosphate backbone and the Cd(2+) ions, crystallographic control of CdS nanostructures is possible using these simple DNA-membrane templates, which we demonstrate using nanobeam electron diffraction experiments on individual templated CdS nanorods.

The mechanical response generated by binding of the nonspecific DNA-bending proteins HMGB1, NHP6A, and HU to single tethered 48.5 kb lambda-DNA molecules is investigated using DNA micromanipulation. As protein concentration is increased, the force needed to extend the DNA molecule increases, due to its compaction by protein-generated bending. Most significantly, we find that for each of HMGB1, NHP6A, and HU there is a well-defined protein concentration, not far above the binding threshold, above which the proteins do not spontaneously dissociate. In this regime, the amount of protein bound to the DNA, as assayed by the degree to which the DNA is compacted, is unperturbed either by replacing the surrounding protein solution with protein-free buffer or by straightening of the molecule by applied force. Thus, the stability of the protein-DNA complexes formed is dependent on the protein concentration during the binding. HU is distinguished by a switch to a DNA-stiffening function at the protein concentration where the formation of highly stable complexes occurs. Finally, introduction of competitor DNA fragments into the surrounding solution disassembles the stable DNA complexes with HMGB1, NHP6A, and HU within seconds. Since spontaneous dissociation of protein does not occur on a time scale of hours, we conclude that this rapid protein exchange in the presence of competitor DNA must occur only via "direct" DNA-DNA contact. We therefore observe that protein transport along DNA by direct transfers occurs even for proteins such as NHP6A and HU that have only one DNA-binding domain.

This paper presents a model which is based on biological research using the movable finite automata (MFA) on a self-assembly of T4 phage, and exhibits the results of artificial life simulation. In the previous work, Thompson and Goel [Artificial Life, Addison Weley, 1989, pp. 317-340; Biosystems 18 (1985) 23; J. Theor. Biol. 131 (1988) 351] presented the movable finite automata (MFA) which has a capability of moving on finite automata, and simulated on a computer. They were represented individual rectangular boxes, however, the results of simulation was different from real T4 phage. We propose the sphere model as a protein structure, and simulate the self-assembly of the entire structure of the T4 phage on a computer.

We describe a microfluidic all-optical technique to measure the thermophoresis of molecules. Within micrometer-thick chambers, we heat aqueous solutions with a micrometer-sized focus of infrared light. The temperature increase of about 1 K is monitored with temperature-sensitive fluorescent dyes. We test the approach in measuring the thermophoresis of DNA. We image the concentration of DNA in a second fluorescence-color channel. DNA is depleted away from the heated spot. The profile of depletion is fitted by the thermophoretic theory to reveal the Soret coefficient. We evaluate the method with numerical 3D calculations of temperature profiles, drift, convection and thermophoretic depletion using finite element methods. The approach opens new ways to monitor thermophoresis at the single molecule level, near boundaries and in complex mixtures. The flexible microfluidic setting is a good step towards microfluidic applications of thermophoresis in biotechnology.

Two classes of lambda(6-85) mutants (those richer in alanine, and those richer in glycine) have very similar slopes in an Arrhenius plot of the unfolding rates but very different temperature dependencies of the folding rates. Temperature-dependent interactions (e.g., hydrophobicity) play a large role in the initial stages of folding but not in the initial stages of unfolding of lambda(6-85). Placement of the transition state in terms of its surface exposure and entropy shows that at least two reaction coordinates are required to describe folding of all mutants over the full temperature range. The unusual Arrhenius plots of the very fastest mutant provide an additional kinetic signature for downhill folding.

The water-soluble (> 200 mg/mL) antibiotics tobramycin, kanamycin, and neomycin spontaneously produce rigid fibers on negatively charged surfaces (mica, graphite, DNA). Atomic force microscopy showed single strands of tobramycin on mica at pH 7 with a length of several hundred nanometers and a diameter of 0.5 nm and double helices with a diameter of 1.0 nm and a helical pitch of 7 nm. At pH 13 (NaOH) up to 15 microm long, rigid fibers with a uniform height of 2.4 nm and an apparent helical pitch of 30 nm were formed along the sodium silicate channels on the surface of mica. Kanamycin and neomycin behaved similarly. Fibers of similar length and width, but without secondary structure, were obtained from aqueous solutions at pH 7 on amorphous, hydrophilized carbon and characterized by transmission electron microscopy. Overstretched phage lambda-DNA strands with a height of 1.0 nm on mica did not interact with tobramycin coils at pH 7. After treatment with EDTA, however, the height of the magnesium-free lambda-DNA strands grew from 1.0 to 3.8 nm after treatment with tobramycin, which suggests a wrapping by the supramolecular fibers. Such fibers may interact with F-actin fibers in biological cells, which would explain the known aggressiveness of aminoglycosides toward bacterial cell membranes and their ototoxicity., (Copyright 2004 American Chemical Society)

We investigate multilayered helical packaging of double-stranded DNA, or of a general polymer chain with persistence length lb, into an ideal, inert cylindrical container, reaching densities slightly below close packaging. We calculate the free energy as a function of the packaged length, based on the energies for bending, twisting, the suffered entropy loss, and the electrostatic energy in a Debye-Hückel model. In the absence of charges on the packaged polymer, a critical packaging force can be determined, similar to the mechanism involved in DNA unzipping models. When charges are taken into consideration, in the final packaging state the charges which are chemically distant become geometrically close, and therefore a steep rise is seen in the free energy. We argue that due to the extremely ordered and almost closely packaged final state the actual packaging geometry does not influence the behaviour of the free energy, pointing towards a certain universality of this state of the polymer. Our findings are compared to a recent simulations study, showing that the model is sensitive to the screening length.

Molecular combing is a powerful and simple method for aligning DNA molecules onto a surface. Using this technique combined with fluorescence microscopy, we observed that the length of lambda-DNA molecules was extended to about 1.6 times their contour length (unextended length, 16.2 microm) by the combing method on hydrophobic polymethylmetacrylate coated surfaces. The effects of sodium and magnesium ions and pH of the DNA solution were investigated. Interestingly, we observed force-induced melting of single DNA molecules., ((c) 2004 American Institute of Physics)

Background: The measurement of physical properties from single molecules has been demonstrated. However, the majority of single-molecule studies report values based on relatively large data sets (e.g., N > 50). While there are studies that report physical quantities based on small sample sets, there has not been a detailed statistical analysis relating sample size to the reliability of derived parameters., Methods: Monte Carlo simulations and multinomial analysis, dependent on quantifiable experimental parameters, were used to determine the minimum number of single-molecule measurements required to produce an accurate estimate of a population mean. Simulation results were applied to the fluorescence-based sizing of DNA fragments by ultrasensitive flow cytometry (FCM)., Results: Our simulations show, for an analytical technique with a 10% CV, that the average of as few as five single-molecule measurements would provide a mean value within one SD of the population mean. Additional simulations determined the number of measurements required to obtain the desired number of replicates for each subpopulation within a mixture. Application of these results to flow cytometry data for lambda/HindIII and S. aureus Mu50/SmaI DNA digests produced accurate DNA fingerprints from as few as 98 single-molecule measurements., Conclusions: A surprisingly small number of single-molecule measurements are required to obtain a mean measurement descriptive of a normally-distributed parent population., (Copyright 2004 Wiley-Liss, Inc.)

An efficient cell-free protein synthesis system has been developed using a novel energy-regenerating source. Using the new energy source, 3-phosphoglycerate (3-PGA), protein synthesis continues beyond 2 h. In contrast, the reaction rate slowed down considerably within 30-45 min using a conventional energy source, phosphoenol pyruvate (PEP) under identical reaction conditions. This improvement results in the production of twice the amount of protein obtained with PEP as an energy source. We have also shown that Gam protein of phage lambda, an inhibitor of RecBCD (ExoV), protects linear PCR DNA templates from degradation in vitro. Furthermore, addition of purified Gam protein in extracts of Escherichia coli BL21 improves protein synthesis from PCR templates to a level comparable to plasmid DNA template. Therefore, combination of these improvements should be amenable to rapid expression of proteins in a high-throughput manner for proteomics and structural genomics applications., (Copyright 2004 Elsevier B.V.)

Secondary structure punctuation through specific backbone and side chain interactions at the beginning and end of alpha-helices has been proposed to play a key role in hierarchical protein folding mechanisms [Baldwin, R. L., and Rose, G. D. (1999) Trends Biochem. Sci. 24, 26-33; Presta, L. G., and Rose, G. D. (1988) Science 240, 1632-1641]. We have made site-specific substitutions in the N- and C-cap motifs of the 5-helix protein monomeric lambda repressor (lambda(6-85)) and have measured the rate constants for folding and unfolding of each variant. The consequences of C-cap changes are strongly context-dependent. When the C-cap was located at the chain terminus, changes had little energetic and no kinetic effect. However, substitutions in a C-cap at the boundary between helix 4 and the subsequent interhelical loop resulted in large changes to the stability and rate constants of the variant, showing a substantial kinetic role for this interior C-cap and suggesting a general kinetic role for interior helix C-caps. Statistical preferences tabulated separately for internal and terminal C-caps also show only weak residue preferences in terminal C-caps. This kinetic distinction between interior and terminal C-caps can explain the discrepancy between the near-absence of stability and kinetic effects seen for C-caps of isolated peptides versus the very strong C-cap effects seen for proteins in statistical sequence preferences and mutational energetics. Introduction of consensus, in-register N-capping motifs resulted in increased stability, accelerated folding, and slower unfolding. The kinetic measurements indicate that some of the new native-state capping interactions remain unformed in the transition state. The accelerated folding rates could result from helix stabilization without invoking a specific role for N-caps in the folding reaction.

We report the solution structure of the Cro protein from bacteriophage P22. Comparisons of its sequence and structure to those of lambda Cro strongly suggest an alpha-to-beta secondary structure switching event during Cro evolution. The folds of P22 Cro and lambda Cro share a three alpha helix fragment comprising the N-terminal half of the domain. However, P22 Cro's C terminus folds as two helices, while lambda Cro's folds as a beta hairpin. The all-alpha fold found for P22 Cro appears to be ancestral, since it also occurs in cI proteins, which are anciently duplicated paralogues of Cro. PSI-BLAST and transitive homology analyses strongly suggest that the sequences of P22 Cro and lambda Cro are globally homologous despite encoding different folds. The alpha+beta fold of lambda Cro therefore likely evolved from its all-alpha ancestor by homologous secondary structure switching, rather than by nonhomologous replacement of both sequence and structure.

An analytical method to determine the genome-wide DNA methylation in only 100 ng DNA is presented. The analysis is based on DNA isolation and hydrolysis followed by derivatization of the 2'-desoxyribonucleoside-3'-monophosphates with a fluorescence dye (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylene diamine hydrochloride, Bodipy FL EDA). The separation of the derivatives was carried out by micellar electrokinetic chromatography, and laser-induced fluorescence was used for detection. To calculate the methylation level, the derivatization factor and the quantum yields of the Bodipy conjugates of 2'-desoxycytidine-3'-monophosphate (dCMP) and 2'-desoxy-5-methylcytidine-3'-monophosphate (5m-dCMP) were determined by measurement of methylated Lambda DNA. The assignment was made by cochromatography with the synthesized and characterized standard compound 5m-dCMP. After optimization of the method it was possible to determine the methylation level in 100-ng DNA samples with a standard deviation of less than 5%.

Terminase enzymes are common to both eukaryotic and prokaryotic double-stranded DNA viruses. These enzymes possess ATPase and nuclease activities that work in concert to "package" a viral genome into an empty procapsid, and it is likely that terminase enzymes from disparate viruses utilize a common packaging mechanism. Bacteriophage lambda terminase possesses a site-specific nuclease activity, a so-called helicase activity, a DNA translocase activity, and multiple ATPase catalytic sites that function to package viral DNA. Allosteric interactions between the multiple catalytic sites have been reported. This study probes these catalytic interactions using enzyme kinetic, photoaffinity labeling, and vanadate inhibition studies. The ensemble of data forms the basis for a minimal kinetic model for lambda terminase. The model incorporates an ADP-driven conformational reorganization of the terminase subunits assembled on viral DNA, which is central to the activation of a catalytically competent packaging machine. The proposed model provides a unifying mechanism for allosteric interaction between the multiple catalytic sites of the holoenzyme and explains much of the kinetic data in the literature. Given that similar packaging mechanisms have been proposed for viruses as dissimilar as lambda and the herpes viruses, the model may find general utility in our global understanding of the enzymology of virus assembly.

lambda-Cro repressor protein incorporated with a 2-anthraquinonylalanine (anqAla) at the 64th position was chemically synthesized by solid-phase method. The 64th position was selected according to previous information on various mutants of Cro incorporated with a single anqAla unit, that were synthesized through an Escherichia coli in vitro protein-synthesizing system. The 64anqAla mutant bound to a dsDNA of consensus operator sequence and underwent strand- and position-specific photocleavage of the dsDNA at the GG sequence after treatment with piperidine. The mutant also underwent position-specific self-photoscission. The self-photoscission was retarded in the presence of the dsDNA., (Copyright 2004 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2004)

We develop a mathematical model of the phage lambda lysis/lysogeny switch, taking into account recent experimental evidence demonstrating enhanced cooperativity between the left and right operator regions. Model parameters are estimated from available experimental data. The model is shown to have a single stable steady state for these estimated parameter values, and this steady state corresponds to the lysogenic state. When the CI degradation rate (gammacI) is slightly increased from its normal value (gammacI approximately 0.0 min(-1)), two additional steady states appear (through a saddle-node bifurcation) in addition to the lysogenic state. One of these new steady states is stable and corresponds to the lytic state. The other steady state is an (unstable) saddle node. The coexistence these two globally stable steady states (the lytic and lysogenic states) is maintained with further increases of gammacI until gammacI approximately 0.35 min(-1), when the lysogenic steady state and the saddle node collide and vanish (through a reverse saddle node bifurcation) leaving only the lytic state surviving. These results allow us to understand the high degree of stability of the lysogenic state because, normally, it is the only steady state. Further implications of these results for the stability of the phage lambda switch are discussed, as well as possible experimental tests of the model.

We investigate the sensitivity of the right operator in bacteriophage lambda. In particular, the system is probed in the three different regulatory protein concentration-regimes: 1), lysogen (CI dominates); 2), during induction (CI and Cro at comparable concentrations); and 3), after induction (Cro dominates). Systematic perturbations of the protein-operator binding energies show in a lysogen that the activity (production rate) at promoter PRM is robust to variations, in contrast to PR, where the sensitivity is high. Both promoters, however, show large sensitivity in regimes 2 and 3. In all regimes we identify several suppressors, meaning that for a given large perturbation (+/-2 kcal/mol) of one binding energy, there exist compensating perturbation(s) that restore the wild-type activity.

We used optical tweezers to tie individual DNA molecules in knots. Although these knots become highly localized under tension, they remain surprisingly mobile and undergo thermal diffusion with classical random walk statistics. The diffusion constants of knots with different complexities correlate with theoretical calculations of knot sizes. We show that this correlation can be explained by a simple hydrodynamical model of "self-reptation" of the knot along a polymer.

N proteins from bacteriophages lambda, P22, and phi21 modulate transcription elongation by binding nascent "boxB" mRNA hairpins. This RNA recognition is mediated by N-terminal arginine-rich peptide sequences capable of interacting with their cognate boxB RNA targets. Here, we have analyzed the affinity and specificity of the peptide-RNA interactions that modulate this transcriptional switch. To do this, we constructed a series of peptides based on the wild-type lambda, P22, and phi21 N protein binding domains ranging from 11 to 22 residues and analyzed their interactions with the leftward and rightward boxB RNA hairpin targets for all three phage. Binding constant (K(d)) values were determined using RNA hairpins labeled with 2-aminopurine (2AP) and monitoring the fluorescence change as peptide was added. K(d)'s demonstrate that lambda and P22 N peptides bind to their cognate boxB targets with high specificity and show equal affinities for their leftward and rightward hairpins. Surprisingly, phi21 shows very little specificity for its cognate targets. Lambda and P22 N peptides exhibit differential modes of recognition with specificity conferred by their amino- and carboxy-terminal modules, respectively. We have generated a reciprocal matrix of substituted peptides to examine the contributions of individual residues to specificity. Amino acid coupling analysis supports a binding model where the Arg8 residue of lambda peptide acts as a conformational hot spot, anchoring the induced loop fold of its boxB hairpin target.

A microfluidic device has been designed, fabricated and tested for its ability to purify bacteriophage lambda DNA and bacterial chromosomal DNA, a necessary prerequisite for its incorporation into a biosensor. This device consists of a microfabricated channel in which silica-coated pillars were etched to increase the surface area within the channel by 300-600%, when the etch depth is varied from 20 to 50 microm. DNA was selectively bound to these pillars in the presence of the chaotropic salt guanidinium isothiocyanate, followed by washing with ethanol and elution with low-ionic strength buffer. Positive pressure was used to move solutions through the device, removing the need for centrifugation steps. The binding capacity for DNA in the device was approximately 82 ng/cm2 and on average, 10% of the bound DNA could be purified and recovered in the first 50 microl of elution buffer. Additionally, the device removed approximately 87% of the protein from a cell lysate. Nucleic acids recovered from the device were efficiently amplified by the polymerase chain reaction suggesting the utility of these components in an integrated, DNA amplification-based biosensor. The miniaturized format of this purification device, along with its excellent purification characteristics make it an ideal component for nucleic acid-based biosensors, especially those in which nucleic acid amplification is a critical step.

The Nun protein of lambdoid phage HK022 excludes lambda-phage superinfection by blocking expression of genes downstream from the lambda nut sequences. Heteronuclear NMR studies have been performed on a Nun peptide comprising residues 1-49 bound to the nutR BoxB RNA. The pattern of (13)C chemical shifts indicates that the arginine-rich motif of Nun forms an induced alpha-helix, consisting of residues 23-43, when bound to BoxB RNA, consistent with the structure of a shorter (residues 22-44) Nun peptide/BoxB RNA complex [Faber, C., Schärpf, M., Becker, T., Sticht, H. and Rösch (2001) J. Biol. Chem. 276, 32064-32070]. The N-terminal extension, residues 1-22, does not show chemical shifts or nuclear Overhauser effects characteristic of stable secondary structure. Nonetheless, (15)N relaxation measurements indicate that this region is not completely disordered, as expected for a random coil peptide. Restriction of conformation flexibility in the N-terminal extension of Nun may be important for binding to other target molecules involved in transcription termination.

We report on coherent charge transport studies in periodic Poly(dG)-Poly(dC) and aperiodic lambda-phage DNA sequences. The extent and efficiency of charge transfer is discussed as a function of sequence dependent energetics, of temperature dependent base-base couplings, and in relation with experiments.

Fluorescence correlation spectroscopy was used to probe the dynamics of lambda-phage DNA in aqueous solution labeled with the randomly intercalating dye TOTO. The linear macromolecules (i). carry more than one chromophore and (ii). are larger than the waist of the focal volume. The correlation function decays significantly faster than expected for a stiff globule of corresponding size but is in good agreement with the dynamic model of semiflexible chains including hydrodynamic interactions. As the chromophore density is lowered the correlation time decreases in accordance with this model.

Histonelike nucleoid structuring protein (H-NS) is an abundant prokaryotic protein participating in nucleoid structure, gene regulation, and silencing. It plays a key role in cell response to changes in temperature and osmolarity. Force-extension measurements of single, twist-relaxed lambda-DNA-H-NS complexes show that these adopt more extended configurations compared to the naked DNA substrates. Crosslinking indicates that H-NS can decorate DNA molecules at one H-NS dimer per 15-20 bp. These results suggest that H-NS polymerizes along DNA, forming a complex of higher bending rigidity. These effects are not observed above 32 degrees C or at high osmolarity, supporting the hypothesis that a direct H-NS-DNA interaction plays a key role in gene silencing. Thus, we propose that H-NS plays a unique structural role, different from that of HU and IHF, and functions as one of the environmental sensors of the cell.

In a previous communication (Kindt et al., 2001) we reported preliminary results of Brownian dynamics simulation and analytical theory which address the packaging and ejection forces involving DNA in bacteriophage capsids. In the present work we provide a systematic formulation of the underlying theory, featuring the energetic and structural aspects of the strongly confined DNA. The free energy of the DNA chain is expressed as a sum of contributions from its encapsidated and released portions, each expressed as a sum of bending and interstrand energies but subjected to different boundary conditions. The equilibrium structure and energy of the capsid-confined and free chain portions are determined, for each ejected length, by variational minimization of the free energy with respect to their shape profiles and interaxial spacings. Numerical results are derived for a model system mimicking the lambda-phage. We find that the fully encapsidated genome is highly compressed and strongly bent, forming a spool-like condensate, storing enormous elastic energy. The elastic stress is rapidly released during the first stage of DNA injection, indicating the large force (tens of pico Newtons) needed to complete the (inverse) loading process. The second injection stage sets in when approximately 1/3 of the genome has been released, and the interaxial distance has nearly reached its equilibrium value (corresponding to that of a relaxed torus in solution); concomitantly the encapsidated genome begins a gradual morphological transformation from a spool to a torus. We also calculate the loading force, the average pressure on the capsid's walls, and the anisotropic pressure profile within the capsid. The results are interpreted in terms of the (competing) bending and interaction components of the packing energy, and are shown to be in good agreement with available experimental data.

Using molecular dynamics simulations in explicit solvent, we investigated the behavior of a 50-bp DNA sequence containing the 434 bacteriophage operators OR1 and OR2 separated by an 8-bp spacer. Two simulations of 1 ns each were carried out, with DNA alone and with DNA complexed to dimers of the R1-69 DNA binding domain of the phage 434 cI repressor protein at the OR1 and OR2 sites. Strong correlations among average structural parameters are observed between our simulations and available experimental data for the bound OR1/OR2 subsites. In the free state, some differences appear between the three relevant fragments (OR1, the spacer, and OR2). Unbound OR1 exhibits a large, shallow major groove into which the base atoms protrude and is also bent toward the major groove. This structure is maintained because structural fluctuations are weak. Unbound OR2 resembles canonical B-DNA although the structural parameters show greater fluctuations, essentially due to a malleable step (the innermost CpA/TpG), absent in OR1. Complexation with the proteins slightly alters the base positions but strongly modifies the sugar and backbone motions. The most crucial repressor effects are changes in the flexibility of the OR1/OR2 sites. Structural fluctuations are enhanced for OR1, conferring a favorable energetic contribution to the OR1 binding, whereas they are reduced for OR2. Therefore, both structural and dynamic properties of DNA suggest OR1 is the most attractive site for the repressor, which may explain the different binding association constants observed for the OR1 and OR2 sites. Finally, we also investigated the impact of the protein on the DNA backbone dynamics and find that direct or indirect interactions facilitate the DNA structural variations required for achieving complementarity with the protein., (Copyright 2003 Wiley Periodicals, Inc.)

A novel development has allowed for the direct observation of single, pairwise interactions of linear DNA with cationic vesicles and of DNA-cationic lipid complexes with anionic vesicles. A new cationic phospholipid derivative, l,2-dioleoyl-sn-glycero-3-ethylphosphocholine, was used to prepare giant bilayer vesicles and to form DNA-cationic lipid complexes (lipoplexes). The cationic vesicles were electrophoretically maneuvered into contact with DNA, and similarly, complexes were brought into contact with anionic phospholipid vesicles composed of dioleoylphosphatidylglycerol (DOPG; 100%), DOPG/dioleoylphosphatidylethanolamine (DOPE; 1:1) or DOPG/dioleoylphosphatidylcholine (DOPC; 1:1). Video fluorescence microscopy revealed that upon contact with phospholipid anionic vesicles, lipoplexes exhibited four different types of behavior: adhesion, vesicle rupture, membrane perforation (manifested as vesicle shrinkage and/or content loss), and expansion of DNA (which was always concomitant with membrane perforation.) In one instance, the lipoplex was injected into the target vesicle just prior to DNA expansion. In all other instances, the DNA expanded over the outer surface of the vesicle, and expansion was faster, the larger the area of vesicle over which it expanded. Given the likelihood of incorporation of cellular anionic lipids into lipoplexes, the expansion of the DNA could be important in DNA release during cell transfection. Upon contact with naked DNA, giant cationic vesicles usually ruptured and condensed the DNA into a small particle. Contact of cationic vesicles that were partially coated with DNA usually caused the DNA to wrap around the vesicle, leading to vesicle rupture, vesicle fusion (with other attached vesicles or lipid aggregates), or simply cessation of movement. These behaviors clearly indicated that both DNA and vesicles could be partly or fully covered by the other, thus modifying surface charges, which, among others, allowed adhesion of DNA-coated vesicles with uncoated vesicles and of lipid-coated DNA with uncoated DNA.

We show the importance of finite particle size in microfluidic asymmetric continuous-flow diffusion arrays, specifically the critical nature of the particle size with respect to the barrier gaps. We show that particles much smaller than the barrier gap follow individual field lines through narrow gaps and are poorly fractionated. In contrast, particles comparable to the gap size lose memory of their incoming field line and can be fractionated with high resolution. We demonstrate this effect using a new technological approach to create very straight and narrow injection bands in such arrays, and completely resolve bands of DNA of lengths 48,500 and 16,7000 base pairs.

Extensive atomic force and electron microscopy reveal a new, generic DNA-colloid complex with a fixed number of DNA bases per colloid. The fiber shaped complex is stable in the presence of excess colloids in the solution. As more DNA is added to the solution and the ratio between colloids and DNA approaches the fiber's stoichiometry, the system undergoes a sharp coagulation transition. The system is restabilized at even higher DNA concentrations through localization of small colloid clusters on extensive DNA networks.

Turbulent drag reduction induced by lambda-DNA is studied. The double-stranded DNA is found to be a good drag reducer when compared with the other normal linear polymers. However, this drag reducing power disappears when the DNA denatures to form two single-strand molecules. Mechanical degradation of DNA is also different from that of the normal linear-chain polymers: DNA is always cut in half by the turbulence. Our results suggest that the mechanism for turbulent degradation of DNA is different from that of the normal flexible long-chain polymers.

The bacteriophage lambda FII protein (gpFII) is a 117 residue structural protein found in the phage particle that is required for the joining of phage heads and tails at the last step of morphogenesis. We have performed biophysical experiments to show that gpFII is stable, monomeric, and reversibly folded. We have also determined the atomic resolution structure of gpFII using NMR spectroscopy. gpFII is shown to possess a novel fold consisting of seven beta-strands and a short alpha-helix. It also displays two large unstructured regions at the N terminus (residues 1-24) and in a large loop near the middle of the protein (residues 46-62). We speculate that these unstructured regions become structured when gpFII assembles into the phage particle, and that these conformational changes play an important role in regulating the assembly pathway. Alignment of the gpFII sequence with those of homologues from other lambdoid phages has allowed us to putatively identify distinct surfaces on the gpFII structure that mediate binding to the phage head and tail.

Drugs may interact with double stranded DNA via a variety of binding modes, each mode giving rise to a specific pharmacological function. Here we demonstrate the ability of single molecule force spectroscopy to discriminate between different interaction modes by measuring the mechanical properties of DNA and their modulation upon the binding of small molecules. Due to the unique topology of double stranded DNA and due to its base pair stacking pattern, DNA undergoes several well-characterised structural transitions upon stretching. We show that small molecule binding markedly affects these transitions in ways characteristic to the binding mode and that these effects can be detected at the level of an individual molecule. The minor groove binder berenil, the crosslinker cisplatin and the intercalator ethidium bromide are compared.

The three-dimensional structure of the lambda repressor C-terminal domain (CTD) has been determined at atomic resolution. In the crystal, the CTD forms a 2-fold symmetric tetramer that mediates cooperative binding of two repressor dimers to pairs of operator sites. Based upon this structure, a model was proposed for the structure of an octameric repressor that forms both in the presence and absence of DNA. Here, we have determined the structure of the lambda repressor CTD in three new crystal forms, under a wide variety of conditions. All crystals have essentially the same tetramer, confirming the results of the earlier study. One crystal form has two tetramers bound to form an octamer, which has the same overall architecture as the previously proposed model. An unexpected feature of the octamer in the crystal structure is a unique interaction at the tetramer-tetramer interface, formed by residues Gln209, Tyr210 and Pro211, which contact symmetry-equivalent residues from other subunits of the octamer. Interestingly, these residues are also located at the dimer-dimer interface, where the specific interactions are different. The structures thus indicate specific amino acid residues that, at least in principle, when altered could result in repressors that form tetramers but not octamers., (Copyright 2001 Academic Press.)

Excreted secreted antigens of the protozoan parasite Toxoplasma gondii play a key role in stimulating the host immune system during acute and chronic infection. With the aim of identifying the immunodominant epitopes of T. gondii antigens involved in the human B-cell response against the parasite, we employed a novel immunological approach. A library of cDNA fragments from T. gondii tachyzoites was displayed as fusion proteins to the amino-terminus of lambda bacteriophage capsid protein D. The lambda D-tachyzoite library was then affinity-selected by using a panel of sera of pregnant women, all infected with the parasite. Some of the clones identified through this procedure matched the sequence of the dense granule GRA1 protein (p24), allowing us to identify its antigenic regions. In particular, the analysis of human antibody response against the recombinant GRA1 antigen fragments revealed the existence of an immunodominant epitope (epi-24 peptide).

The CII protein of the temperate bacteriophage lambda is a transcriptional activator involved in the lysis-lysogeny switch of the phage. It is an unstable protein of 97 amino acids and is known to exist as a tetramer in the native state. The cII gene has been cloned and expressed in Escherichia coli using a T7 promoter based over-expression system. The recombinant CII protein has been purified to homogeneity by ammonium sulfate fractionation followed by two steps of ion-exchange chromatography. The purified protein crystallized at pH 8.2 in hanging-drop vapor diffusion method at 293 K. The crystals diffract to a resolution of 2.8 A and belong to the space group C222 with unit-cell parameters a = 64.10, b = 106.95 and c = 120.16 A., (Copyright 2001 Academic Press.)

We have carried out molecular dynamics simulation of the lambda OL1 DNA operator on the free and the protein-bound forms. Our results lead us to conclude that the binding of the repressor actually makes the N-7 atom of Gua8' more solvent exposed, thereby enhancing its reactivity to chemical methylation. This increase in solvent accessibility surface area occurs simultaneously with the formation of hydrogen bonds between Lys-4 of the nonconsensus flexible N-terminal arm and Gua6' of the nonconsensus half-site operator DNA. Calculations of protein--DNA interaction energies reveal that among the residues of the arm, Lys-4 contributes the most favorably to the interaction energies. This result is consistent with mutagenesis studies that established that lysine at position 4 is absolutely required for tight binding. We find that the nonconsensus arm and the nonconsensus monomer interacts less favorably with DNA than do their respective counterparts of the consensus monomer. Moreover, the six-residue flexible arm accounts for at least half the total protein--DNA interactions energy. These results are in agreement with previous experimental studies. In accord with the diffuse electron density map observed in crystallographic studies of the nonconsensus flexible arm, we find that our model built for this region is more flexible and exhibits more conformations than its consensus counterpart. The simulation also reveals that DNA bending observed near the outer edge of the operator site is an intrinsic sequence-dependent property. By contrast, the DNA-bending features observed toward the center of the operator are induced by the protein. On the whole, stepwise protein-induced bending is more pronounced in the consensus half-site operator. We also find that the unusually large helical twist (49 degrees ) observed in the protein-bound form near the center of the operator results from the binding of the protein at a base step with some propensity for high twists., (Copyright 2001 John Wiley & Sons, Inc. Biopolymers 59: 205-225, 2001)

Single photon burst techniques were used to detect double-stranded DNA molecules in poly(methylmethacrylate) (PM MA) and polycarbonate (PC) microfluidic devices. A confocal epi-illumination detection system was constructed to monitor the fluorescence signature from single DNA molecules that were multiply labeled with the mono-intercalating dye, TOPRO-5, which possessed an absorption maximum at 765 nm allowing excitation with a solid-state diode laser and fluorescence monitoring in the near-infrared (IR). Near-IR excitation minimized autofluorescence produced from the polymer substrate, which was found to be significantly greater when excitation was provided in the visible range (488 nm). A solution containing lambda-DNA (48.5 kbp) was electrokinetically transported through the microfluidic devices at different applied voltages and solution pH values to investigate the effects of polymer substrate on the transport rate and detection efficiency of single molecular events. By applying an autocorrelation analysis to the data, we were able to obtain the molecular transit time of the individual molecules as they passed through the 7 microm laser beam. It was observed that the applied voltage for both devices affected the transport rate. However, solution pH did not alter the transit time for PM MA-based devices since the electroosmotic flow of PMMA was independent of solution pH. In addition, efforts were directed toward optimizing the sampling efficiency (number of molecules passing through the probe volume) by using either hydrodynamically focused flows from a sheath generated by electrokinetic pumping from side channels or reducing the channel width of the microfluidic device. Due to the low electroosmotic flows generated by both PMMA and PC, tight focusing of the sample stream was not possible. However, in PMMA devices, flow gating was observed by applying field strengths > -120 V/cm to the sheath flow channels. By narrowing the microchannel width, the number of molecular events detected per unit time was found to be four times higher in channels with 10 microm widths compared to those of 50 microm, indicating improved sampling efficiency for the narrower channels without significantly deteriorating detection efficiency. Attempts were made to do single molecule sizing of lambda-DNA, M13 (7.2 kbp) and pUC19 (2.7 kbp) using photon burst detection. While the average number of photons for each DNA type were different, the standard deviations were large due to the Gaussian intensity profile of the excitation beam. To demonstrate the sensitivity of single molecule analysis in the near-IR using polymer microfluidic devices, the near-IR chromophore, NN382, wasanalyzed using ourconfocal imager. A detection efficiency of 94% for single NN382 molecules was observed in the PC devices.

Nun protein from coliphage HK022 binds to phage boxB RNA and functions, in contrast to phage lambda N protein, as a transcriptional terminator. The basic Nun-(10-44) peptide contains the boxB RNA binding arginine rich motif, ARM. The peptide binds boxB RNA and competes with the phage lambda ARM peptide N-(1-36) as indicated by nuclear magnetic resonance (NMR) spectroscopy titrations. In two-dimensional nuclear Overhauser enhancement spectroscopy experiments boxB RNA in complex with Nun-(20-44) exhibits the same pattern of resonances as it does in complex with N peptides containing the ARM, and we could show that Nun-(20-44) forms a bent alpha-helix upon binding to the boxB RNA. The structure of the boxB RNA-bound Nun-(20-44) was determined on the basis of 191 intra- and 30 intermolecular distance restraints. Ser-24 is anchored to the lower RNA stem, and stacking of Tyr-39 and A7 is clearly experimentally indicated. Arg-28 shows numerous contacts to the RNA stem. Leu-22, Ile-30, Trp-33, Ile-37, and Leu-41 form a hydrophobic surface, which could be a recognition site for additional host factors such as NusG. Such a hydrophobic surface area is not present in N-(1-36) bound to boxB RNA.