Azurin–DNA Conjugate with the Binding Motif of a Transcriptional Regulator, CooA: CO-dependent Modulation of the Electron-transfer Reaction

Abstract

An azurin DNA conjugate (Azu DNA) where the DNA part contains the binding sequence of a carbon monoxide (CO)-dependent transcriptional regulator, CooA, has been prepared. CooA in the CO-bound form (CO CooA) showed specific binding to Azu DNA. The association of CO CooA with Azu DNA affects the electron transfer (ET) between Azu DNA and its redox partner protein, cytochrome c 552 . Consequently, the CO-dependent modulation of ET was achieved. This could be a potential platform for a novel biobased sensor, which exploits the transcriptional regulator as a detection module and whose readout is immediately transduced to a readily processable electronic signal.A transcriptional regulator is a member of signal transducer proteins that specifically sense an environmental stress to treat it by regulating the activity of the appropriate proteins at the transcriptional level. In biological systems, various transcriptional regulators have evolved to respond to all kinds of stresses, which include not only chemical substances but also physical stimuli such as light, 1,2 heat, 3 and osmotic pressure. 4,5 In general, stress detection by transcriptional regulators is highly sensitive and specific, which are essential in eliminating stresses and retaining homeostasis in living cells. Meanwhile, these properties appear attractive in light of constructing a novel biobased sensor device. A whole cell bacterial sensor is a well-known example that has succeeded in correlating the sensibility of the transcriptional regulator with the activity of a reporter protein. 6 9 In the whole cell sensor, the gene of the reporter protein is fused with the promoter system of the transcriptional regulator so as to reflect the stress detection on the expression level of the reporter protein. Fluorescence and colorimetric reaction by the expressed reporter protein are major choices for the final quantification of the stress level. The bacterial biosensor appears simple and versatile because of the in situ use of the transcriptional regulators in living cells. The progress in this field is, however, sluggish till date due to the difficulty in controlling unexpected responses of the biosensor cell to a target stress under incomplete biological information regarding inherent repression mechanisms of stress. 9 Construction of a similar sensing system in vitro is a potential solution to avoid the problem associated with bacterial biosensors. This, however, requires replacement of the reporter protein system exploiting the physiological function with a facile molecular-base signal transducer in vitro.We have recently developed a signal transduction mechanism consisting of a pair of electron-transfer proteins (azurin 10 and cytochrome c 552 11 from Alcaligenes xylosoxidans and Thermus thermophilus, respectively) and a heat-responsive polymer, poly(N-isopropylacrylamide) (PNIPAM), 12 which is introduced on the hydrophobic surface of azurin to modulate protein protein interaction, prerequisite of the electron-transfer (ET) step. 13 Consequently, the appar...

Probing the Active Site of an Azurin Mutant Hot-Wired to Gold Electrodes

“…Because of its robust self-assembly on gold surfaces, azurin is suitable for biosensing applications and has been extensively used in the design of a variety of hybrid bionanostructures. − In this report, we have used conductive atomic force microscopy (C-AFM) to probe the electron transfer properties of surface-assembled azurin from Pseudomonas aeruginosa and its active site mutant (azurin H117G). We have wired the protein variant to the gold surface through reconstitution with external ligands.…”