Hiroyuki Furusawa

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.

Transient Kinetic Studies of pH-Dependent Hydrolyses by Exo-type Carboxypeptidase P on a 27-MHz Quartz Crystal Microbalance

pH-Dependent kinetic parameters (k(on), k(off), and k(cat)) of protein (myoglobin) hydrolyses catalyzed by exo-enzyme (carboxypeptidase P, CPP) were obtained by using a protein-immobilized quartz crystal microbalance (QCM) in acidic aqueous solutions. The formation of the enzyme-substrate (ES) complex (k(on)), the decay of the ES complex (k(off)), and the formation of the product (k(cat)) could be analyzed by transient kinetics as mass changes on the QCM plate. The Kd (k(off)/k(on)) value was different from the Michaelis constant Km calculated from (k(off) + k(cat))/k(on) due to k(cat) > k(off). The rate-determining step was the binding step (k(on), and the catalytic rate k(cat) was faster than other k(on) and k(off) values. In the range of pH 2.5-5.0, values of k(on) gradually increased with decreasing pH showing a maximum at pH 3.7, values of k(off) were independent of pH, and k(cat) increased gradually with decreasing pH. As a result, the apparent rate constant (k(cat)/Km) showed a maximum at pH 3.7 and gradually increased with decreasing pH. The optimum pH at 3.7 of k(on) is explained by the optimum binding ability of CPP to the COOH terminus of the substrate with hydrogen bonds. The increase of k(cat) at the lower pH correlated with the decrease of alpha-helix contents of the myoglobin substrate on the QCM.

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.

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.

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.

Direct monitoring of allosteric recognition of type IIE restriction endonuclease EcoRII.

EcoRII is a homodimer with two domains consisting of a DNA-binding N terminus and a catalytic C terminus and recognizes two specific sequences on DNA. It shows a relatively complicated cleavage reaction in bulk solution. After binding to either recognition site, EcoRII cleaves the other recognition site of the same DNA (cis-binding) strand and/or the recognition site of the other DNA (trans-binding) strand. Although it is difficult to separate these two reactions in bulk solution, we could simply obtain the binding and cleavage kinetics of only the cis-binding by following the frequency (mass) changes of a DNA-immobilized quartz-crystal microbalance (QCM) responding to the addition of EcoRII in aqueous solution. We obtained the maximum binding amounts (Δmmax), the dissociation constants (Kd), the binding and dissociation rate constants (kon and koff), and the catalytic cleavage reaction rate constants (kcat) for wild-type EcoRII, the N-terminal-truncated form (EcoRII N-domain), and the mutant derivatives in its C-terminal domain (K263A and R330A). It was determined from the kinetic analyses that the N-domain, which covers the catalytic C-domain in the absence of DNA, preferentially binds to the one DNA recognition site while transforming EcoRII into an active form allosterically, and then the secondary C-domain binds to and cleaves the other recognition site of the DNA strand.

In vitro selection and evaluation of RNA aptamers that recognize arginine-rich-motif model peptide on a quartz-crystal microbalance.

To study RNA-peptide interactions, we performed an in vitro selection of RNA on a simple alpha-helical peptide-immobilized quartz-crystal microbalance (QCM) and evaluated the association constants (10(7) M-1) of the selected RNA to the model peptide on the same QCM plate.

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.

70 S ribosomes bind to Shine-Dalgarno sequences without required dissociations.

In all living systems, proteins are synthesized on ribosomes by translating sequences of mRNA. Translation processes are complex and highly regulated by various translation factors together with ribosomes. It is known that translation initiation is the most dynamic step in building an initiation complex composed of a ribosome, mRNA, and an initiator tRNA. 2] In bacteria, it is thought that the 70S ribosome must dissociate into a 30S and 50S subunit triggered by initiation factors to interact with mRNA. It is generally accepted that the free 30S ribosomal subunit binds to a Shine–Dalgarno (SD) sequence, a 3–10 nucleotide purine-rich sequence (for example, AGGAGG), on a mRNA in the first step of the initiation translation, and then the 50S subunit is recruited to the mRNA–30S complex with an initiator tRNA on an AUG start codon downstream of the SD sequence. On the other hand, the preassembled 70S ribosome can translate only a leaderless mRNA lacking the SD sequence with an AUG start codon at the 5’ terminus of the mRNA. 5] Although most free ribosomes exist in the 70S form under physiological conditions, very few studies have investigated the binding of 70S to a mRNA with a SD sequence. 5] The interACHTUNGTRENNUNGaction of the intact 70S ribosome with the SD sequence has either been overlooked or not established in the literature. In this communication, we report that the intact 70S ribosome can bind directly to mRNA with the SD and AUG sequences without a required dissociation. Binding kinetics were studied using a mRNA-immobilized 27 MHz quartz-crystal microACHTUNGTRENNUNGbalance (QCM; Figure 1), which is known to be a very sensitive mass measuring device in aqueous solutions. QCM resonance frequencies decrease linearly upon mass increases on the QCM electrode to the nanogram level. Calibrations of the 27 MHz QCM in aqueous solutions are described in the Supporting Information (S1), and 1 Hz of frequency decrease was calibrated as an increase in 0.18 ng cm 2 of ribosomes in aqueous solution. E. coli 70S ribosomes and 30S and 50S ribosomal subunits were prepared and purified as described previously. Initiator tRNAs (tRNA) were prepared from an overexpressed strain and purified. fMet-tRNA was enzymatically methionylated by methionyl-tRNA synthetase and then formylated by methionyl-tRNA formyltransferase. mRNAs were prepared by in vitro transcription using a T7 RNA polymerase. For biotinylation of the 3’ terminus of the mRNA, the transcript was oxidized with NaIO4 and then modified with biotin hydrazide. We prepared several kinds of biotinylated mRNAs with SD+ AUG or SD +UUG sequences, the mRNA without the SD sequence (non-SD), the mRNA in which the SD sequence is hybridized with the antisense DNA (anti-SD), and the mRNA in which a 5’untranslated region (5’-UTR) is hybridized with the antisense DNA (anti-UTR). The sequence of the transcripts are shown in Table 1, and preparation methods of mRNA and the sequence of antisense DNAs are described in the Supporting Information (S2 and S3). AFFINIX Q4 with four 500 mL cells equipped with a 27 MHz QCM plate at the bottom of each cell was used as a QCM instrument. Biotinylated mRNA was linked by avidin– biotin interactions on a QCM plate covered with NeutrAvidinB as previously reported. After immobilization of mRNA, ribosomes were injected into aqueous buffer (10 mm HEPES-KOH, pH 7.3, 100 mm NH4Cl, 5 mm MgACHTUNGTRENNUNG(OAc)2, 0.5 mm CaCl2, 25 8C) in a QCM cell. Experimental procedures are described in the Supporting Information (S4). Figure 2 A shows typical time courses of frequency decreases (mass increases) corresponding to additions of 30S, 50S, and 70S ribosomes in aqueous solutions. The 30S subunit showed relative binding to mRNA with SD and AUG sequences (SD+ AUG), but the 50S subunit showed little specific binding. The [a] Dr. S. Takahashi, R. Akita, Dr. H. Matsuno, Dr. H. Furusawa, Prof. Y. Okahata Frontier Collaborative Research Centre Department of Biomolecular Engineering, Tokyo Institute of Technology Nagatsuda, Midori-ku, Yokohama 226-8501 (Japan) Fax: (+81)45-924-5781 E-mail : yokahata@bio.titech.ac.jp [b] Dr. Y. Shimizu, Prof. T. Ueda Department of Medical Genome Sciences Graduate School of Frontier Sciences, University of Tokyo FSB401, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562 (Japan) Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author. Figure 1. Experimental setup for measuring the binding kinetics of ribosomes (70S, 50S, 30S, and crosslinked 70S) with/without fMet-tRNA to a biotinylated mRNA-NeutrAvidin-immobilized 27-MHz quartz-crystal microACHTUNGTRENNUNGbalance (QCM, AFFINIX Q4) in aqueous solutions.

Enzyme Reactions on a 27 MHz Quartz Crystal Microbalance

A quartz crystal microbalance (QCM) is known as a useful tool to detect gravimetric molecular interactions. We have developed a 27-MHz QCM (Affinix Q4) to detect various biomolecular interactions such as DNA-DNA hybridization, DNA-protein interactions, glycolipid-protein interactions, and protein-protein interactions. In this chapter, we show that the 27-MHz QCM is also useful to detect the kinetics of enzyme reactions, because all the steps of enzyme reactions, such as the enzyme binding process to substrates, the enzyme catalytic reaction, and the release of enzyme from the product, accompany mass changes. We introduce here kinetic analyses of enzyme reactions on DNA (DNA polymerization, DNA ligation, and DNA cleavage) and enzyme reactions on glycans (glycosylation, phosphorylation, and mutation of enzymes) by using the substrate-immobilized 27-MHz QCM in solution.