Kinetic characterization of small DNA-binding molecules interacting with a DNA strand on a quartz crystal microbalance.

Quantitative studies of the binding of various DNA-binding antibiotics with dsDNA are useful for drug design, not only for effective antibiotics, but also for antitumor drugs. We studied the binding kinetics, association and dissociation rate constants, and association constants (kon, koff, and Ka, respectively) of intercalators and groove binders, including various antibiotics, to double-stranded DNA (dA30·dT30 and dG30·dC30) immobilized on a highly sensitive 27 MHz quartz-crystal microbalance (QCM) in aqueous solution. Although a simple ethidium bromide intercalator bound to both dA30·dT30 and dG30·dC30, antibiotics that are side-binding intercalators, such as daunomycin, aclacinomycin A, and actinomycin D, with sugar or peptide moieties on the intercalator parts selectively bound to dG30·dC30 with high Ka and small koff values. Nogalamycin, a dumbbell-shaped penetrating intercalator, showed low kon and koff values owing to slow duplex unwinding during the penetration process. Groove binders (Hoechst 33258, distamycin A, and mithramycin) had high Ka values owing to the high kon values. Kinetic parameters depended largely on molecular shapes and DNA-binding molecule binding modes.

Real-time monitoring of intermediates reveals the reaction pathway in the thiol-disulfide exchange between disulfide bond formation protein A (DsbA) and B (DsbB) on a membrane-immobilized quartz crystal microbalance (QCM) system.

Disulfide bond formation protein B (DsbBS-S,S-S) is an inner membrane protein in Escherichia coli that has two disulfide bonds (S-S, S-S) that play a role in oxidization of a pair of cysteine residues (SH, SH) in disulfide bond formation protein A (DsbASH,SH). The oxidized DsbAS-S, with one disulfide bond (S-S), can oxidize proteins with SH groups for maturation of a folding preprotein. Here, we have described the transient kinetics of the oxidation reaction between DsbASH,SH and DsbBS-S,S-S. We immobilized DsbBS-S,S-S embedded in lipid bilayers on the surface of a 27-MHz quartz crystal microbalance (QCM) device to detect both formation and degradation of the reaction intermediate (DsbA-DsbB), formed via intermolecular disulfide bonds, as a mass change in real time. The obtained kinetic parameters (intermediate formation, reverse, and oxidation rate constants (kf, kr, and kcat, respectively) indicated that the two pairs of cysteine residues in DsbBS-S,S-S were more important for the stability of the DsbA-DsbB intermediate than ubiquinone, an electron acceptor for DsbBS-S,S-S. Our data suggested that the reaction pathway of almost all DsbASH,SH oxidation processes would proceed through this stable intermediate, avoiding the requirement for ubiquinone.

In situ monitoring of structural changes during formation of 30S translation initiation complex by energy dissipation measurement using 27-MHz quartz-crystal microbalance.

Ribosome is a bionanomachine that facilitates an orderly translation process during protein synthesis in living cells. Real-time monitoring of conformational changes in ribosomal subunits in aqueous solution is important to understand the regulatory mechanism of protein synthesis, because conformational changes in ribosome in E. coli have been predicted to operate the switch from translation initiation to an elongation process during translation. We performed an energy dissipation measurement by using a quartz-crystal microbalance-admittance (QCM-A) technique for in situ monitoring of conformational changes in pre-30S translation initiation complex in response to the binding of fMet-tRNA(fMet) in aqueous solution. The addition of fMet-tRNA(fMet) caused changes in the physical property (increased dehydration and elasticity) in 30S ribosomal subunit in the presence of mRNA and IF2/guanosine 5'-triphosphate (GTP) on the QCM plate. Furthermore, two sequential changes triggered by the addition of fMet-tRNA(fMet) were observed in 30S ribosomal subunit bound to mRNA in the presence of IF2/GTP and IF3. These observations suggest that the structural changes in 30S ribosomal subunit caused by the binding of fMet-tRNA(fMet) with IF2/GTP in the presence of IF3 could act as a switch to regulate the orderly processing in the construction of translation initiation complex, because the structural distinction can be a guidepost in the process for the relevant biomolecules.

Arginine arrangement of bacteriophage λ N-peptide plays a role as a core motif in GNRA tetraloop RNA binding.

A simple α-helical N-model-peptide was designed to investigate the role of the arginine-rich motif of bacteriophage λ N-peptide in selective binding with boxB RNA. The five-arginine arrangement of native N-peptide was retained; all other residues were replaced with alanine. In vitro selection of RNA (30 random-nucleotide region) was carried out with N-model-peptide immobilized on a 27 MHz quartz-crystal microbalance (QCM). Selected RNAs were evaluated on the same QCM plate to obtain binding constants (Ka =10(7) -10(8) M(-1) ). Many selected RNAs contained GNR(N)A-type loops (similar to the boxB RNA motif recognized by the native N-peptide). Fragments and minimal RNAs containing the GNRA-type loop also bound to N-model-peptide (Ka =10(6) -10(7) M(-1) ). The RNA recognition specificity of the peptide was studied by changing the "closing" U-A base pair and one base in the tetraloop of the RNA aptamers, and by peptide mutations (18th residue of N-model-peptide). It was concluded that the five-arginine arrangement of the peptide performs selective recognition of the GNRA tetraloop and GNR(N)A pentaloop RNA structures, and that substitution of another functional amino acid residue at the 18th position in N-peptide adds the recognition ability for a loop-RNA sequence.

Translation enhancer improves the ribosome liberation from translation initiation.

For translation initiation in bacteria, the Shine-Dalgarno (SD) and anti-SD sequence of the 30S subunit play key roles for specific interactions between ribosomes and mRNAs to determine the exact position of the translation initiation region. However, ribosomes also must dissociate from the translation initiation region to slide toward the downstream sequence during mRNA translation. Translation enhancers upstream of the SD sequences of mRNAs, which likely contribute to a direct interaction with ribosome protein S1, enhance the yields of protein biosynthesis. Nevertheless, the mechanism of the effect of translation enhancers to initiate the translation is still unknown. In this paper, we investigated the effects of the SD and enhancer sequences on the binding kinetics of the 30S ribosomal subunits to mRNAs and their translation efficiencies. mRNAs with both the SD and translation enhancers promoted the amount of protein synthesis but destabilized the interaction between the 30S subunit and mRNA by increasing the dissociation rate constant (koff) of the 30S subunit. Based on a model for kinetic parameters, a 16-fold translation efficiency could be achieved by introducing a tandem repeat of adenine sequences (A20) between the SD and translation enhancer sequences. Considering the results of this study, translation enhancers with an SD sequence regulate ribosomal liberation from translation initiation to determine the translation efficiency of the downstream coding region.

Direct monitoring of initiation factor dynamics through formation of 30S and 70S translation-initiation complexes on a quartz crystal microbalance.

Translation initiation is a dynamic and complicated process requiring the building a 70S initiation complex (70S-IC) composed of a ribosome, mRNA, and an initiator tRNA. During the formation of the 70S-IC, initiation factors (IFs: IF1, IF2, and IF3) interact with a ribosome to form a 30S initiation complex (30S-IC) and a 70S-IC. Although some spectroscopic analyses have been performed, the mechanism of binding and dissociation of IFs remains unclear. Here, we employed a 27 MHz quartz crystal microbalance (QCM) to evaluate the process of bacterial IC formation in translation initiation by following frequency changes (mass changes). IFs (IF1, IF2, and IF3), N-terminally fused to biotin carboxyl carrier protein (bio-BCCP), were immobilized on a Neutravidin-covered QCM plate. By using bio-BCCP-IF2 immobilized to the QCM, three steps of the formation of ribosomal initiation complex could be sequentially observed as simple mass changes in real time: binding of a 30S complex to the immobilized IF2, a recruitment of 50S to the 30S-IC, and formation of the 70S-IC. The kinetic parameters implied that the release of IF2 from the 70S-IC could be the rate-limiting step in translation initiation. The IF3-immobilized QCM revealed that the affinity of IF3 for the 30S complex decreased upon the addition of mRNA and fMet-tRNA(fMet) but did not lead to complete dissociation from the 30S-IC. These results suggest that IF3 binds and stays bound to ICs, and its interaction mode is altered during the formation of 30S-IC and 70S-IC and is finally induced to dissociate from ICs by 50S binding. This methodology demonstrated here is applicable to investigate the role of IFs in translation initiation driven by other pathways.

Traveling Time of a translating ribosome along messenger RNA monitored directly on a quartz crystal microbalance.

During translation, the biosynthesis of polypeptides is dynamically regulated. The translation rate along messenger RNA (mRNA), which is dependent on the codon, structure, and sequence, is not always constant. However, methods for measuring the duration required for polypeptide elongation on an mRNA of interest have not been developed. In this work, we used a quartz crystal microbalance (QCM) technique to monitor mRNA translation in an Escherichia coli cell-free translation system in real time. This method permitted us to evaluate the translation of proteins of interest fused upstream of a streptavidin-binding peptide (SBP) fusion protein. The translation of mRNA encoding the SBP fusion protein alone was observed as a mass increase on a streptavidin-modified QCM plate. Addition of the protein of interest resulted in a delay in the mass change corresponding to the traveling time of the ribosome along the coding region of the protein of interest. With this technique, the lengths of coding sequences, codon usages, influences of unique sequences, and various protein-coding sequences were evaluated. The results showed that the traveling time of the translating ribosome depends on the length of the coding region translated but is also affected by the sequence itself. Differences in the time lags for various proteins imply that mRNA coding sequences may regulate gene expression.

Single-molecular enzymatic elongation of hyaluronan polymers visualized by high-speed atomic force microscopy.

Using high-speed scanning atomic force microscopy, we directly observed single-molecular enzymatic elongation of hyaluronan polymer chains at intervals of 10 s on a mica or lipid bilayer surface, on which Pasteurella multocida hyaluronic acid synthase (pmHAS) was immobilized. The reaction was started by the addition of both UDP-glucuronic acid and UDP-N-acetylglucosamine monomers. The average catalytic elongation rate constant (k(cat)) was found to be 1.8 mer s(-1) from one active enzyme physically adsorbed on a mica surface. When pmHAS was immobilized by inserting its hydrophobic tail part into lipid bilayers, most of the enzymes retained their activity, and the k(cat) values were found to be in the range 1-10 mer s(-1) for 29 enzymes (average was k(cat) = 2-4 mer s(-1)). These k(cat) values were lowest level of k(cat) = 1-100 s(-1) obtained in bulk solution by radioisotope methods.

Kinetics of iterative carbohydrate transfer to polysaccharide catalyzed by chondroitin polymerase on a highly sensitive flow-type 27†MHz quartz-crystal microbalance.

Using a highly sensitive flow-type 27 MHz quartz crystal microbalance, we could detect a small mass change during stepwise and alternating one-sugar transfer of glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc) to an acceptor, catalyzed by chondroitin polymerase from Escherichia coli strain K4 (K4CP), and analyze the elongation mechanism of K4CP. K4CP was found to bind strongly to a chondroitin acceptor (K(d)=0.97 µM). Although the binding affinity and the catalytic rate constant for each monomer were considerably different, the apparent catalytic efficiency (k(cat)/K(m)) was similar (6.3×10(4) M(-1) s(-1) for GlcA transfer and 3.4×10(4) M(-1) s(-1) for the GalNAc transfer). This is reasonable for the smooth alternating elongation of GlcA and GalNAc on the acceptor. This is the first study to report the determination of kinetic parameters for enzymatic, alternated, sugar elongation.

Real-time monitoring of a stepwise transcription reaction on a quartz-crystal microbalance.

We monitored real-time DNA transcription by T7 RNAP using a 27-MHz DNA-immobilized quartz-crystal microbalance (QCM) in buffer solution to investigate the stepwise reaction of transcription. We designed a template double-stranded DNA that consisted of a T7 promoter, a stall position (15 bp downstream from the promoter), and a 73-bp transcription region. Based on the frequency (mass) changes of the template-immobilized QCM in response to the addition of T7 RNAP and monomers of NTP, we obtained the kinetic parameters of each step of the T7 RNAP reactions: the enzyme-binding rate (k(on)) to and the dissociation rate (k(off)) from the promoter, the proceeding rate (k(for)) from the promoter to the forward stall position, the polymerization rate (k(cat)) of RNA along DNA, and the release rate (k(r)) from the end of the template DNA. We found that k(cat) (120 s⁻¹) was extremely large compared with k(off) (0.014 s⁻¹), k(for) (0.062 s⁻¹), and k(r) (0.014 s⁻¹), revealing that the rate-limiting steps of T7 RNAP involve the binding to the promoter, the movement to the stall position, and the release from DNA. These kinetic parameters were compared with values for other DNA-binding enzymes.

In situ monitoring of a trace intermediate during DNA phosphorylation by T4 polynucleotide kinase for transient kinetic studies.

Formation and decomposition of the enzyme-substrate (ES) complex during phosphorylation by T4 polynucleotide kinase (T4 PNK) of dsDNAs were monitored using a highly sensitive quartz crystal microbalance (QCM) to determine kinetic parameters, which were characterised in comparison with those of other enzymes such as DNA polymerase and exo- and endo-nucleases.

Single-molecule force spectroscopy for studying kinetics of enzymatic dextran elongations.

Catalytic elongation of dextran by a single molecule of dextransucrase (DSase) was directly monitored by observing the movements of the positions of a rupture peak, which represented the adhesive force between an isomaltoheptaose (dextran 7-mer)-immobilized probe and a DSase-immobilized mica surface. This was initiated with the addition of sucrose monomers. From the histograms of the rupture peaks after elongation reactions on each individual enzyme and the continuous peak shift of certain single enzymes, the catalytic elongation rate constant (k(cat)) was ascertained to be 1.2-2.7 s(-1).

Small mass-change detectable quartz crystal microbalance and its application to enzymatic one-base elongation on DNA.

A highly sensitive 27 MHz quartz crystal microbalance instrument with an automatic flow injection system was developed to obtain realistic minimal frequency noise (±0.05 Hz) and to obtain a stable signal baseline (±1 Hz/h) by controlling the temperature of each part in the quartz crystal microbalance (QCM) system using three Peltier devices with a resolution of ±0.001 °C and by optimizing the flow system to prevent fluctuation of the internal pressure of the QCM. The improved QCM with an automatic flow injection system enabled detection of small mass changes such as binding of biotin to a streptavidin-immobilized QCM with a high signal-to-noise ratio. We also applied this device to enzyme reactions of one-base elongation by DNA polymerase (Klenow fragment, KF). We immobilized dsDNAs including the protruding end of dA, dG, dT, or dC on the QCM electrode and ran complementary dNTP monomers with KF into the QCM flow cell. We could directly detect the enzymatic one-base elongation of DNA as a small mass increase, and we found the difference in the reaction rate for each monomer.

Kinetic studies of dextransucrase enzyme reactions on a substrate- or enzyme-immobilized 27 MHz quartz crystal microbalance.

Catalytic elongation by dextransucrase (DSase) was monitored directly on a dextran-acceptor- or DSase-immobilized 27 MHz quartz crystal microbalance (QCM). Kinetic parameters for the binding of the enzyme to the dextran acceptor (k(on), k(off), and K(d)) and enzymatic elongation in the presence of a sucrose monomer (K(m) for sucrose and k(cat)) were determined. The kinetic parameters obtained by both methods were consistent.

Affinity purification of multifunctional polymer nanoparticles.

We report that multifunctional polymer nanoparticles approximately the size of a large protein can be "purified", on the basis of peptide affinity just as antibodies, using an affinity chromatography strategy. The selection process takes advantage of the thermoresponsiveness of the nanoparticles allowing "catch and release" of the target peptide by adjusting the temperature. Purified particles show much stronger affinity (K(dapp) ≈ nM) and a narrower affinity distribution than the average of particles before purification (K(dapp) > µM) at room temperature but can release the peptide just by changing the temperature. We anticipate this affinity selection will be general and become an integral step for the preparation of "plastic antibodies" with near-homogeneous and tailored affinity for target biomacromolecules.

Kinetic analyses of bindings of Shiga-like toxin to clustered and dispersed Gb3 glyco-arrays on a quartz-crystal microbalance.

One-, two-, four-, and eight-branched globotriaosyl saccharides (Gb(3): Gal-alpha1,4-Gal-beta1,4-Glc), whose reducing ends were biotinylated, were prepared (1Gb(3)-bio, 2Gb(3)-bio, 4Gb(3)-bio, and 8Gb(3)-bio, respectively). They are dispersively immobilized as a glyco-array in the matrix of biotinylated maltotriose (Glc(3)-bio) on a streptavidin-covered 27 MHz quartz-crystal microbalance (QCM). The binding kinetics of the verotoxin B subunit (VTB) to various branched Gb(3)-bio ligands in the Glc(3)-bio matrix could be obtained from frequency decreases (mass increases) of the QCM. VTB can recognize the Gb(3) unit but not the Glc(3) unit, where VTB is a pentamer having five binding sites for one Gb(3) unit per each B subunit (having a total of 15 binding sites for Gb(3)). By changing the Gb(3) multivalency, the Gb(3) packing density, and the Gb(3) cluster size in the Glc(3) matrix, association constants (K(a)), maximum amounts bound (Delta m(max)), and binding and dissociation rate constants (k(on) and k(off)) were obtained. When 15 sites of VTB were recognized by 16 Gb(3) units, K(a) was 100 times larger than that when 15 sites of VTB were recognized by only 2 Gb(3) units, with a 6-fold-larger k(on) and a 25-fold-smaller k(off). When the Gb(3) multivalency was changed by covering with two 1Gb(3)-bio, 2Gb(3)-bio, 4Gb(3)-bio, or 8Gb(3)-bio ligands on two pockets of one streptavidin, the K(a) values increased with increasing branch number from one to eight. When the Gb(3) cluster size was changed from eight 1Gb(3)-bio units to one 8Gb(3)-bio unit in the matrix, the K(a) values increased but the Delta m(max) values decreased with increasing cluster size from eight 1Gb(3)-bio units to one 8Gb(3)-bio unit. This is the first example of systematically obtaining all kinetic parameters of sugar-binding proteins to sugars on a glyco-array by changing the sugar multivalency, the sugar packing density, and the sugar cluster size in the matrix.

Vertically aligned multilayer films of monodispersed helical polypeptides with micrometer thickness via simple cast.

Utilizing the zwitterionic alpha-helix peptide bearing a cationic and anionic group at the N- and C-terminus, respectively, we first demonstrated that the vertically aligned multilayer film can be prepared by a simple cast and slow evaporation. The tilt angle of the peptide remained unchanged with ca. 30 degrees in the range between submicrometer and several micrometers in thickness. The key designs allowing simple vertical alignment of the helical peptide multilayer films were (i) monodispersity of the peptide, (ii) electrostatic interaction between anionic substrate and the cationic group bearing at the N-terminus of the peptide, and (iii) interlayer electrostatic interaction among terminal groups of the peptide.

Real-time monitoring of cell-free translation on a quartz-crystal microbalance.

The efficiency of protein synthesis is often regulated post-transcriptionally by sequences within the mRNA. To investigate the reactions of protein translation, we established a system that allowed real-time monitoring of protein synthesis using a cell-free translation mixture and a 27 MHz quartz-crystal microbalance (QCM). Using an mRNA that encoded a fusion polypeptide comprising the streptavidin-binding peptide (SBP) tag, a portion of Protein D as a spacer, and the SecM arrest sequence, we could follow the binding of the SBP tag, while it was displayed on the 70S ribosome, to a streptavidin-modified QCM over time. Thus, we could follow a single turnover of protein synthesis as a change in mass. This approach allowed us to evaluate the effects of different antibiotics and mRNA sequences on the different steps of translation. From the results of this study, we have determined that both the formation of the initiation complex from the 70S ribosome, mRNA, and fMet-tRNA(fMet) and the accommodation of the second aminoacyl-tRNA to the initiation complex are rate-limiting steps in protein synthesis.

Added mass effect on immobilizations of proteins on a 27 MHz quartz crystal microbalance in aqueous solution.

During the immobilization process of proteins onto an Au-surface of a 27 MHz quartz crystal microbalance (QCM) in aqueous solutions, apparent large frequency changes (DeltaF(water)) were observed compared with those in the air phase (DeltaF(air)) due to the interaction with surrounding water of proteins. On the basis of an energy-transfer model for the QCM, the apparent added mass in the aqueous solution [(-DeltaF(water))/(-DeltaF(air)) - 1] could be explained by frictional forces at the interface of proteins with aqueous solutions. When [(-DeltaF(water))/(-DeltaF(air)) - 1] values for various proteins were plotted against values relating to the friction (antimobility), such as values of the molecular weight divided by the sedimentation coefficient (Mw/s), the inverse of the diffusion coefficient (1/D), and the volume divided by the surface area (volume/surface area = apparent radius) of proteins, there were good linear correlations. Thus, observations of the larger DeltaF(water) than DeltaF(air) for protein immobilizations on the QCM can be simply explained by the friction effect at the interface between proteins and the aqueous solution. Thus, QCM would be a mass sensor based on mechanical oscillation motion even in aqueous solutions.

In situ monitoring of conformational changes of and peptide bindings to calmodulin on a 27 MHz quartz-crystal microbalance.

Conformational changes of calmodulin (CaM) by additions of Ca(2+) ions and bindings of CaM-binding peptides to Ca(2+)/CaM followed by conformational changes were monitored by a CaM-immobilized 27 MHz quartz-crystal microbalance (QCM) with an admittance analysis. Both the binding and the conformational change events could be detected from the time-dependence of frequency decreases (mass increases) and energy dissipation decreases (elasticity increases), respectively. When Ca(2+) ions were injected to a QCM cell on which biotinylated CaM was immobilized with avidin-biotin interactions, a frequency increase (a mass decrease) and an energy dissipation decrease (an elasticity increase) were observed because of the dehydration and the elasticity increase caused by conformational changes from the flexible CaM to the rigid Ca(2+)/CaM exposing the hydrophobic surface. In the case of the addition of CaMKII-peptide in the Ca(2+)/CaM-immobilized QCM, the immediate frequency decrease (the mass increase) due to the binding of the peptide to Ca(2+)/CaM and the following energy dissipation decrease (the elasticity increase) with a time lag were observed. This suggests that the interaction of the CaMKII-peptide to Ca(2+)/CaM follows an allosteric binding mode. Binding kinetics of the peptide to Ca(2+)/CaM (k(1) and k(-1)) and kinetics of the following conformational change of Ca(2+)/CaM (k(2) and k(-2)) could be obtained. This technique is useful to investigate biomolecular interactions involving the conformational and/or viscoelastic property changes that are biologically important.