Micro-volume couette flow sample orientation for absorbance and fluorescence linear dichroism

Linear dichroism (LD) can be used to study the alignment of absorbing chromophores within long molecules. In particular, Couette flow LD has been used to good effect in probing ligand binding to DNA and to fibrous proteins. This technique has been previously limited by large sample requirements. Here we report the design and application of a new micro-volume Couette flow cell that significantly enhances the potential applications of flow LD spectroscopy by reducing the sample requirements for flow linear dichroism to 25 microL (with concentrations such that the absorbance maximum of the sample in a 1-cm pathlength cuvette is approximately 1). The micro-volume Couette cell has also enabled the measurement of fluorescence-detected Couette flow linear dichroism. This new technique enables the orientation of fluorescent ligands to be probed even when their electronic transitions overlap with those of the macromolecule and conversely. The potential of flow-oriented fluorescence dichroism and application of the micro-volume Couette LD cell are illustrated by the collection of data for DNA with minor groove and intercalating ligands: DAPI, Hoechst, and ethidium bromide. As with conventional fluorescence, improved sensitivity compared with absorbance LD is to be expected after instrumentation optimization.     

Protein Fiber Linear Dichroism for Structure Determination and Kinetics in a Low-Volume, Low-Wavelength Couette Flow Cell

High-resolution structure determination of soluble globular proteins relies heavily on x-ray crystallography techniques. Such an approach is often ineffective for investigations into the structure of fibrous proteins as these proteins generally do not crystallize. Thus investigations into fibrous protein structure have relied on less direct methods such as x-ray fiber diffraction and circular dichroism. Ultraviolet linear dichroism has the potential to provide additional information on the structure of such biomolecular systems. However, existing systems are not optimized for the requirements of fibrous proteins. We have designed and built a low-volume (200 μL), low-wavelength (down to 180 nm), low-pathlength (100 μm), high-alignment flow-alignment system (couette) to perform ultraviolet linear dichroism studies on the fibers formed by a range of biomolecules. The apparatus has been tested using a number of proteins for which longer wavelength linear dichroism spectra had already been measured. The new couette cell has also been used to obtain data on two medically important protein fibers, the all-β-sheet amyloid fibers of the Alzheimer's derived protein Aβ and the long-chain assemblies of α₁-antitrypsin polymers.     

The structure of DNA-DOPC aggregates formed in presence of calcium and magnesium ions: A small-angle synchrotron X-ray diffraction study

The structure of aggregates formed due to DNA interaction with dioleoylphosphatidylcholine (DOPC) vesicles in presence of Ca²⁺ and Mg²⁺ cations was investigated using synchrotron small-angle X-ray diffraction. For DOPC/DNA = 1:1 mol/base and in the range of concentration of the cation²⁺ 0-76.5 mM, the diffractograms show the coexistence of two lamellar phases: L^x phase with repeat distance d_Lx ∼ 8.26-7.39 nm identified as a phase where the DNA strands are intercalated in water layers between adjacent lipid bilayers, and L_DOPC phase with repeat distance d_DOPC ∼ 6.45-5.65 nm identified as a phase of partially dehydrated DOPC bilayers without any divalent cations and DNA strands. The coexistence of these phases was investigated as a function of DOPC/DNA molar ratio, length of DNA fragments and temperature. If the amount of lipid increases, the fraction of partially dehydrated L_DOPC phase is limited, depends on the portion of DNA in the sample and also on the length of DNA fragments. Thermal behaviour of DOPC + DNA + Ca²⁺ aggregates was investigated in the range 20-80 °C. The transversal thermal expansivities of both phases were evaluated.     

Direct observation of unstained wet biological samples by scanning-electron generation X-ray microscopy

Analytical tools of nanometre-scale resolution are indispensable in the fields of biology, physics and chemistry. One suitable tool, the soft X-ray microscope, provides high spatial resolution of visible light for wet specimens. For biological specimens, X-rays of water-window wavelength between carbon (284 eV; 4.3 nm) and oxygen (540 eV; 2.3 nm) absorption edges provide high-contrast imaging of biological samples in water. Among types of X-ray microscope, the transmission X-ray microscope using a synchrotron radiation source with diffractive zone plates offers the highest spatial resolution, approaching 15-10 nm. However, even higher resolution is required to measure proteins and protein complexes in biological specimens; therefore, a new type of X-ray microscope with higher resolution that uses a simple light source is desirable. Here we report a novel scanning-electron generation X-ray microscope (SGXM) that demonstrates direct imaging of unstained wet biological specimens. We deposited wet yeasts in the space between two silicon nitride (Si₃N₄) films. A scanning electron beam of accelerating voltage 5 keV and current 1.6 nA irradiates the titanium (Ti)-coated Si₃N₄ film, and the soft X-ray signal from it is detected by an X-ray photodiode (PD) placed below the sample. The SGXM can theoretically achieve better than 5 nm resolution. Our method can be utilized easily for various wet biological samples of bacteria, viruses, and protein complexes.     

Biological applications of synchrotron radiation infrared spectromicroscopy

Extremely brilliant infrared (IR) beams provided by synchrotron radiation sources are now routinely used in many facilities with available commercial spectrometers coupled to IR microscopes. Using these intense non-thermal sources, a brilliance two or three order of magnitude higher than a conventional source is achievable through small pinholes (< 10 μm) with a high signal to-noise ratio. IR spectroscopy is a powerful technique to investigate biological systems and offers many new imaging opportunities. The field of infrared biological imaging covers a wide range of fundamental issues and applied researches such as cell imaging or tissue imaging. Molecular maps with a spatial resolution down to the diffraction limit may be now obtained with a synchrotron radiation IR source also on thick samples. Moreover, changes of the protein structure are detectable in an IR spectrum and cellular molecular markers can be identified and used to recognize a pathological status of a tissue. Molecular structure and functions are strongly correlated and this aspect is particularly relevant for imaging. We will show that the brilliance of synchrotron radiation IR sources may enhance the sensitivity of a molecular signal obtained from small biosamples, e.g., a single cell, containing extremely small amounts of organic matter. We will also show that SR IR sources allow to study chemical composition and to identify the distribution of organic molecules in cells at submicron resolution is possible with a high signal-to-noise ratio. Moreover, the recent availability of two-dimensional IR detectors promises to push forward imaging capabilities in the time domain. Indeed, with a high current synchrotron radiation facility and a Focal Plane Array the chemical imaging of individual cells can be obtained in a few minutes. Within this framework important results are expected in the next years using synchrotron radiation and Free Electron Laser (FEL) sources for spectro-microscopy and spectral-imaging, alone or in combination with Scanning Near-field Optical Microscopy methods to study the molecular composition and dynamic changes in samples of biomedical interest at micrometric and submicrometric scales, respectively.     

Restriction enzyme kinetics monitored by UV linear dichroism

The use of linear dichroism (LD) spectroscopy for biological applications has been brought to the forefront recently by our development of thermostated microvolume Couette cells. We present a method for following the digestion of DNA by restriction endonucleases in real time without the use of any extrinsic dyes or labels. This is accomplished using linear dichroism spectroscopy (the differential absorbance of light polarized parallel and perpendicular to the sample orientation axis). The differential absorbance signal depends on the degree of alignment of the molecules. In this case the DNA is aligned by Couette flow (flowing the solution in the annular gap between two concentric cylinders), and we monitor the increase in alignment upon linearization of a circular DNA molecule. In addition, we observe a decrease in alignment upon further digestion and subsequent shortening of the DNA. Ten enzymes were investigated: seven enzymes with a single cut site (EcoRI, KpnI, NdeI, NotI, NruI, SmaI, XbaI), two enzymes with two cut sites (BstZ17I, EagI), and one enzyme with no cut site (ClaI). LD, as implemented in this new assay, is broadly applicable across a wide range of DNA-modifying enzymes and compounds and, as such, is a useful addition to the toolbox of biological characterization.     

The reversible depletion and reconstitution of a copper ion in Coprinus cinereus laccase followed by spectroscopic techniques

The specific activities of crude and purified Coprinus cinereus laccase preparations could be enhanced by a factor of 10-12 by activation with copper ions. The copper to protein contents of purified non-activated laccase were 2.3 ± 0.1 compared to 3.3 ± 0.1 in purified activated laccase indicating that only a fraction of the laccase can be activated. Purified laccase not activated with copper ions shows in isoelectric focusing four bands in order of decreasing pI in a ratio 1/5/3/1 where only bands I and II had laccase activity. Purified activated laccase showed only three bands (I, II and III) in the ratio 5/4/1 all with some laccase activity. The pH profile of the activity for activated and non-activated laccase showed identical behavior indicating that the active forms were the same. The change in UV-Vis around 330 nm following the depletion and reconstitution of the enzyme combined with activity measurements supports the reversibility of the selective removal and insertion of copper ions at the type 2 site. The circular dichroism spectrum of activated purified laccase has characteristic changes around 350 nm relative to non-activated laccase indicative of changes at the type 2/type 3 sites. The difference between the electron paramagnetic resonance spectra of non-activated and activated C. cinereus laccase indicates that a fraction of the non-activated purified laccase contained a copper(II) signal with a coupling constant between a type 1 and a type 2 copper(II). This electron paramagnetic resonance signal could be explained by an induced asymmetry in the type 3 site due to a missing type 2 copper ion.     

Efficiency of two-stage combinations of subsurface vertical down-flow and up-flow constructed wetland systems for treating variation in influent C/N ratios of domestic wastewater

The performance and temporal variation of four hybrid, intermittent loading, pilot-scale vertical flow constructed wetlands (VFCWs) were tested for treating domestic wastewater of three different C/N ratios (2.5:1, 5:1, and 10:1, respectively). Two hybrid systems each consisted of the two identical VFCWs in-series, with up-up or down-down flow. The other two hybrid systems consisted of the first VFCWs (up or down flow) followed by a second VFCWs (down or up flow, respectively). The effects of combination mode, season, load level, and interactions on nutrient removal were studied in synthetic wastewater in the two-stage VFCW systems. With varying C/N ratios for influent water (from 2.5:1, 5:1 to 10:1) average removal efficiencies for the two-bed two-stage systems were as follows: COD 73-93%, TN 46-87%, TP 75-90%, and TOC 40-66%, respectively. All two-bed hybrid VFCWs were efficient in removing organics and total phosphorus, and reached the highest removal rates when the C/N ratios were 10 and 5, respectively. The hybrid systems for different flow direction beds had significantly higher performance (P < 0.05) during the wetlands operational period. Compared to the four types of hybrid VFCWs, the two-stage combination with different flow directions achieved significantly higher TN and TOC reductions (P < 0.05). The highest total nitrogen (P < 0.05) and total phosphorus reductions in down-up flow VFCWs were observed at C/N 5:1. However, for organic matter and total organic carbon, the highest COD and TOC removal rates occurred when C/N ratios were 5-10 for the down-up flow VFCWs. With appropriate control of combined mechanisms in series, the concentrations of carbon and nitrogen sources in the influent can achieve the optimal effects of nutrient removal.     

A new method for fibrous protein analysis illustrated by application to tubulin microtubule polymerisation and depolymerisation

A thermostatted micro volume Couette cell has been designed to enable linear dichroism (LD) data to be collected at a range of temperatures. The cell is a development of the traditional Couette flow LD cell and includes the recent development of micro-volume LD (20-40 microL) coupled with the addition of a heating element, temperature probe and controller. This new micro volume Couette LD cell opens the way not only to the LD analysis of systems where sample volume is critical, but also for the LD analysis of temperature sensitive samples. The polymerization of the microtubule protein tubulin has been followed in a range of different conditions using the thermostatted micro volume Couette LD cell. The focusing lenses on the cell, which are required for the microvolume cell, have the side benefit of significantly reducing the light-scattering artifacts caused by the large size of tubulin microtubules. It is now possible to monitor real-time polymerization and depolymerization kinetics, and any structural rearrangements of chromophores within the polymer. In the case of tubulin, the LD spectra revealed a greater change in the orientation of tryptophan residues at approximately 290 nm during polymerization compared to other contributing chromophores-guanine, phenylalanine, and tyrosine. The improvements in instrumental design have also allowed LD spectra of tubulin to be collected down to approximately 230 nm (previous data have only been available from the near UV region), which means that some indication of protein backbone-orientation changes are now available. It was observed during this work that apparent LD intensity maxima are in fact artifacts when the high-tension voltage is high. The onset of such artifacts has been observed at much lower voltages with light-scattering fibrous proteins (including tubulin) than with nonscattering samples. Therefore, caution must be used when interpreting LD data collected with medium to high photomultiplier tube voltages.     

The ADP-ribosylation of Sulfolobus solfataricus Sso7 modulates protein/DNA interactions in vitro

The 7 kDa Sso7 is a basic protein particularly abundant in Sulfolobus solfataricus and is involved in DNA assembly. This protein undergoes in vitro ADP-ribosylation by an endogenous poly(ADP-ribose) polymerase-like enzyme. The circular dichroism spectrum of purified ADP-ribosylated Sso7 shows that this modification stabilizes the prevalent protein β-conformation, as suggested by shifting of negative ellipticity minimum to 220 nm. Moreover, a short ADP-ribose chain (up to 6-mers) bound to Sso7 is able to reduce drastically the thermoprotective and DNA condensing ability of the protein, suggesting a possible regulatory role of ADP-ribosylation in sulfolobal DNA organization.     

A simple and sensitive HPLC fluorescence method for determination of tadalafil in mouse plasma

A simple and sensitive high-performance liquid chromatographic (HPLC) method utilizing fluorescence detection was developed for the determination of the phosphodiesterase type 5 inhibitor tadalafil in mouse plasma. This method utilizes a simple sample preparation (protein precipitation) with high recovery of tadalafil (∼98%), which eliminates the need for an internal standard. For constituent separation, the method utilized a monolithic C₁₈ column and a flow rate of 1.0 mL/min with a mobile phase gradient consisting of aqueous trifluoroacetic acid (0.1% TFA in deionized water pH 2.2, v/v) and acetonitrile. The method calibration was linear for tadalafil in mouse plasma from 100 to 2000 ng/mL (r > 0.999) with a detection limit of approximately 40 ng/mL. Component fluorescence detection was achieved using an excitation wavelength of 275 nm with monitoring of the emission wavelength at 335 nm. The intra-day and inter-day precision (relative standard deviation, RSD) values for tadalafil in mouse plasma were less than 14%, and the accuracy (percent error) was within −14% of the nominal concentration. The method was utilized on mouse plasma samples from research evaluating the potential cardioprotective effects of tadalafil on mouse heart tissue exposed to doxorubicin, a chemotherapeutic drug with reported cardiotoxic effects.     

Electrochemical DNA biosensor based on silver nanoparticles/poly(3-(3-pyridyl) acrylic acid)/carbon nanotubes modified electrode

In this work, we present an electrochemical DNA sensor based on silver nanoparticles/poly(trans-3-(3-pyridyl) acrylic acid) (PPAA)/multiwalled carbon nanotubes with carboxyl groups (MWCNTs-COOH) modified glassy carbon electrode (GCE). The polymer film was electropolymerized onto MWCNTs-COOH modified electrode by cyclic voltammetry (CV), and then silver nanoparticles were electrodeposited on the surface of PPAA/MWCNTs-COOH composite film. Thiol group end single-stranded DNA (HS-ssDNA) probe was easily covalently linked onto the surface of silver nanoparticles through a 5′ thiol linker. The DNA hybridization events were monitored based on the signal of the intercalated adriamycin by differential pulse voltammetry (DPV). Based on the response of adriamycin, only the complementary oligonucleotides gave an obvious current signal compared with the three-base mismatched and noncomplementary oligonucleotides. Under the optimal conditions, the increase of reduction peak current of adriamycin was linear with the logarithm of the concentration of the complementary oligonucleotides from 9.0 × 10⁻¹² to 9.0 × 10⁻⁹ M with a detection limit of 3.2 × 10⁻¹² M. In addition, this DNA sensor exhibited an excellent reproducibility and stability during DNA hybridization assay.     

Mutual structural effect of bilirubin and model membranes by vibrational circular dichroism

In this study, vibrational circular dichroism (VCD) spectroscopy was employed for the first time to study the bilirubin (BR) interaction with model membranes and models for membrane proteins. An enantioselective interaction of BR with zwitterionic 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and sphingomyelin (SPM) liposomes was observed by VCD and electronic circular dichroism (ECD) complemented by absorption and fluorescence spectroscopy. The M-form of BR was preferentially recognized in the BR/DMPC system at concentration above 1 × 10^− 4 M, for lower concentrations the P-form of BR was recognized by the DMPC liposomes. The VCD spectra also showed that the SPM liposomes, which represent the main component of nerve cell membrane, were significantly more disturbed by the presence of BR than the DMPC liposomes—a stable association with a strong VCD signal was observed providing the explanations for the supposed BR neurotoxicity. The effect of time and pH on the BR/DMPC or SPM liposome systems was shown to be essential while the effect of temperature in the range of 15–70 °C was negligible demonstrating the surprisingly high temperature stability of BR when interacting with the studied membranes. The influence of a membrane protein was tested on a model consisting of poly-l-arginine (PLAG) bound in the α-helical form to the surface of 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol) liposomes and sodium dodecyl sulfate micelles. VCD and also ECD spectra showed that a variety of BR diastereoisomers interacted with PLAG in such systems. In a system of PLAG with micelles composed of sodium dodecyl sulfate, the M-form of bound BR was observed.     

Is photocleavage of DNA by YOYO-1 using a synchrotron radiation light source sequence dependent?

The photocleavage of double-stranded and single-stranded DNA by the fluorescent dye YOYO-1 was investigated in real time by using the synchrotron radiation light source ASTRID (ISA, Denmark) both to initiate the reaction and to monitor its progress using Couette flow linear dichroism (LD) throughout the irradiation period. The dependence of LD signals on DNA sequences and on time in the intense light beam was explored and quantified for single-stranded poly(dA), poly[(dA-dT)₂], calf thymus DNA (ctDNA) and Micrococcus luteus DNA (mlDNA). The DNA and ligand regions of the spectrum showed different LD kinetic behaviors, and there was significant sequence dependence of the kinetics. However, in contrast to expectations from the literature, we found that poly(dA), mlDNA, low salt ctDNA and low salt poly[(dA-dT)₂] all had significant populations of groove-bound YOYO. It seems that this mode was predominantly responsible for the catalysis of DNA cleavage. In homopolymeric DNAs, intercalated YOYO was unable to cleave DNA. In mixed-sequence DNAs the data suggest that YOYO in some but not all intercalated binding sites can cause cleavage. It is also likely that cleavage occurs at transient single-stranded regions. The reaction rates for a 100 mA beam current of 0.5-μW power varied from 0.6 h⁻¹ for single-stranded poly(dA) to essentially zero for low salt poly[(dG-dC)₂] and high salt poly[(dA-dT)₂]. At the conclusion of the experiments with each kind of DNA, uncleaved DNA with intercalated YOYO remained.     

Interaction of Topotecan,DNA Topoisomerase I Inhibitor,with Double-stranded Polydeoxyribonucleotides. 3. Binding at the Minor Groove

Interaction of topotecan (TPT) with calf thymus DNA, coliphage T4 DNA, and poly(dGdC) · poly(dG-dC) was studied by optical (linear flow dichroism, UV-vis spectroscopy) and quantum chemical methods. The linear dichroism signal of TPT bound to DNA was shown to have positive sign in the range 260–295 nm. This means that the plane of quinoline fragment (rings A and B) of TPT forms an angle less than 54° with the long axis of DNA, and hence the TPT molecule cannot intercalate between DNA base pairs. TPT was established to bind to calf thymus DNA as readily as to coliphage T4 DNA whose cytosines in the major groove were all glycosylated at the 5th position. Consequently, the DNA major groove does not participate in TPT binding. TPT molecule was shown to compete with distamycin for binding sites in the minor groove of DNA and poly(dG-dC) · poly(dG-dC). Thus, it was demonstrated for the first time that TPT binds to DNA at its minor groove.     

Linear dichroism characteristics of ethidium-and proflavine-supercoiled DNA complexes

A flow linear dichroism technique is utilized to study the unwinding of supercoiled DNA induced by the binding of ethidium bromide (EB) and proflavine (PF) at different ratios r (drug added/DNA base). In the case of either EB or PF bound to linear calf thymus DNA, the reduced linear dichroism signals LD/A (LD: linear dichroism; A: absorbance, both measured at the same wavelength), determined at 258, and 520 or 462 nm (corresponding to contributions predominantly from the partially oriented DNA bases, intercalated EB, or PF, respectively) are nearly independent of drug concentration. In the case of supercoiled DNA, the magnitude of LD/A at 258 nm first increases to a maximum value near r = 0.04-0.05, and then decreases as r is increased further, mimicking the behavior of the sedimentation coefficients, viscosities, and gel electrophoresis patterns measured by other workers at similar values of r. However, LD/A at 520 nm, which is due to DNA-bound EB molecules, is constant within the range of r values of 0.02-0.06 in which the magnitude of LD/A determined at 258 nm due to the DNA bases exhibits a pronounced maximum. In contrast, in the case of PF, the magnitudes of LD/A determined at 258 or 462 nm are characterized by similar dependencies on r, both exhibiting pronounced maxima at r = 0.05; this parallel behavior is expected according to a simple intercalation model in which the DNA bases and drug molecules are stacked on top of one another, and in which both are oriented to similar extents in the flow gradient. The unexpected differences in the dependencies of (LD/A)258 and (LD/A)520 on r in the case of EB bound to supercoiled DNA, are attributed to differences in the net overall alignment of the EB molecules and DNA bases in the flow gradient. The magnitude of the LD signal at 258 nm reflects the overall degree of orientation of the supercoiled DNA molecules that, in turn, depends on their hydrodynamic shapes and sizes; the LD signals characterizing the bound EB molecules may reflect this orientation also, as well as the partial alignment of individual DNA segments containing bound EB molecules.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effectiveness of gender determination using polymerase chain reaction and radioimmunoassay methods in cattle

The aim of this study was to use polymerase chain reaction (PCR) by amplifying DNA from bovine (Bos taurus) fetal cells recovered through uterine puncture and subsequent amniotic fluid aspiration and to compare the effectiveness of the PCR method with amniotic dihydrotestosterone (DHT) levels in gender determination. Amniotic DHT levels between sexes were significantly higher in males than in females in all periods except the period 91 to 120 d. The differences among the amniotic DHT levels at different gestation periods (61 to 90, 91 to 120, 121 to 150, 151 to 180, 181 to 210 d) were not significant in females but were significant in males in the period 61 to 90 d compared with three other periods. Sensitivity was equal to 97.8% (95% CI = 88.2% to 99.6%), and specificity was equal to 85.4% (95% CI = 80.0% to 97.6%). These two values correspond with a cutoff of DHT in amniotic fluid. Distributions of the two sex groups were classified according to the 192.1 pg/mL cutoff value. A total of 93 amniotic fluid samples were examined by PCR analysis. The sex determination of 91 samples by PCR and electrophoresis was in agreement with the visual sexes of the fetuses. In two amniotic fluid samples, DNA was not isolated, and thus no sex determination was made. Fetal gender was correctly identified by PCR in 44 of 45 males and in 47 of 48 females. In PCR, one band (at the length of 102 bp) and two bands (at the lengths of 102 and 226 bp) were observed respectively for female and male fetuses. It may be concluded that the levels of amniotic DHT and PCR might be used for embryo sexing in pregnant cows.     

Novel type of red-shifted chlorophyll a antenna complex from Chromera velia: II. Biochemistry and spectroscopy

A novel chlorophyll a containing pigment–protein complex expressed by cells of Chromera velia adapted to growth under red/far-red illumination [1]. Purification of the complex was achieved by means of anion-exchange chromatography and gel-filtration. The antenna is shown to be an aggregate of ~ 20 kDa proteins of the light–harvesting complex (LHC) family, unstable in the isolated form. The complex possesses an absorption maximum at 705 nm at room temperature in addition to the main chlorophyll a maximum at 677 nm producing the major emission band at 714 nm at room temperature. The far-red absorption is shown to be the property of the isolated aggregate in the intact form and lost upon dissociation. The purified complex was further characterized by circular dichroism spectroscopy and fluorescence spectroscopy. This work thus identified the third different class of antenna complex in C. velia after the recently described FCP-like and LHCr-like antennas. Possible candidates for red antennas are identified in other taxonomic groups, such as eustigmatophytes and the relevance of the present results to other known examples of red-shifted antenna from other organisms is discussed. This work appears to be the first successful isolation of a chlorophyll a-based far-red antenna complex absorbing above 700 nm unrelated to LHCI.     

2-Aminopurine/cytosine base pair containing oligonucleotides: fluorescence spectroscopy studies on DNA-polyamide binding

Studies on the binding of a triamide f-IPI (1) to its cognate sequence labeled with a 2-aminopurine (2AP or G^∗) group are described. ITC studies showed that f-IPI (1) bound to the cognate site (ACG^∗CGT) with only 3.5-fold lower affinity than binding to the unlabeled DNA (ACGCGT) (K_eq = 2 × 10⁷ and 7 × 10⁷ M⁻¹, respectively). Titration of f-IPI (1) to both sequences gave strong induced bands at 330 nm via circular dichroism studies. The compound also gave comparable ΔT_m values of 5.0 and 7.8 °C, respectively. These techniques also proved that the sequence selectivity of f-IPI (1) was uncompromised, as only limited binding to the non-cognate sequence ACCG^∗GT was observed. Fluorescence studies demonstrated a 2:1 ligand:DNA binding motif as anticipated, and indicated that the limit of detection for this technique was 20 μM DNA concentration. The results demonstrate that 2-aminopurine is a sufficient substitute for guanine in a G·C base pair useful in DNA binding studies.     

Sizing the Bacillus anthracis PA63 channel with nonelectrolyte poly(ethylene glycols)

Nonelectrolyte polymers of poly(ethylene glycol) (PEG) were used to estimate the diameter of the ion channel formed by the Bacillus anthracis protective antigen 63 (PA₆₃). Based on the ability of different molecular weight PEGs to partition into the pore and reduce channel conductance, the pore appears to be narrower than the one formed by Staphylococcus aureus α-hemolysin. Numerical integration of the PEG sample mass spectra and the channel conductance data were used to refine the estimate of the pore's PEG molecular mass cutoff (∼1400 g/mol). The results suggest that the limiting diameter of the PA₆₃ pore is <2 nm, which is consistent with an all-atom model of the PA₆₃ channel and previous experiments using large ions.