Reversible Switching of Fluorophore Property Based on Intrinsic Conformational Transition of Adenylate Kinase during Its Catalytic Cycle

Abstract

Adenylate kinase shows a conformational transition (OPEN and CLOSED forms) during substrate binding and product release to mediate the phosphoryl transfer between ADP and ATP/AMP. The protein motional characteristics will be useful to construct switching systems of fluorophore properties caused by the catalytic cycle of the enzyme. This paper demonstrates in situ reversible switching of a fluorophore property driven by the conformational transition of the enzyme. The pyrene-conjugated mutant adenylate kinase is able to switch the monomer/excimer emission property of pyrene on addition of ADP or P(1)P(5)-di(adenosine-5')pentaphosphate (Ap5A, a transition state analog). The observation under the dilute condition (~0.1 μM) indicates that the emission spectral change was caused by the motion of a protein molecule and not led by protein-protein interactions through π-π stacking of pyrene rings. The switching can be reversibly conducted by using hexokinase-coupling reaction. The fashion of the changes in emission intensities at various ligand concentrations is different between ADP, Mg(2+)-bound ADP, and Mg(2+)-bound Ap5A. The emission property switching is repeatable by a sequential addition of a substrate in a one-pot process. It is proposed that the property of a synthetic molecule on the enzyme surface is switchable in response to the catalytic cycle of adenylate kinase.

Excimer Emission Properties on Pyrene-Labeled Protein Surface: Correlation between Emission Spectra, Ring Stacking Modes, and Flexibilities of Pyrene Probes

“…The CD spectra of pyrene-labeled Adk tm s in the 1 L a absorption band region were collected for the characterization of pyrene-pyrene association in terms of its exciton coupling ( Figure S5 in Supporting Information). According to our previous report, 26 the CLOSED C1-Adk tm shows a negative Cotton effect at the 1 L a absorption band, whereas the OPEN C1-Adk tm shows no typical exciton coupling-type signal ( Figure S5b). In contrast, the CLOSED C2-Adk tm showed no distinct CD signal in the 1 L a absorption band region ( Figure S5c).…”

“…Protein expression and purification was carried out in the same manner as the method descried in a previous report. 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 32 Adk tm was confirmed by MALDI-TOF-MS analyses ( Figure S12 in Supporting Information). Measurements of UV-vis, steady-state fluorescence and CD spectra.…”

“…This trend is also the same as that previously observed in C1-Adk tm . 26 The fluorescence quantum yields of the pyrene-labeled Adk tm s determined under N 2 atmosphere or air were similar, indicating that the emissions were all from the singlet excited states. C3-Adk tm was found to have the smallest Φ F among the three modified Adk tm s regardless of the presence or the absence of Ap 5 A. UV-vis and excitation spectra of pyrene-labeled Adk tm s. All the pyrene-labeled Adk tm s showed absorption bands in the range from 300 nm to 370 nm.…”

“…The synthesis of pyrenyl iodoacetamide C1 was described in a previous report. 26 The syntheses of pyrenyl iodoacetamides C2 and C3 are described above (see the detailed 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 30 synthetic procedures in Supporting Information). The plasmid pEAK91 harboring Adk tm (A55C/C77S/V169C) was prepared from the plasmid coding the double mutant of E. coli.…”

“…C1-Adk tm is the same modified enzyme as reported previously. 26 In these proteins, a common scaffold biomolecule (Adk tm ) with fixed sites for conjugation (Cys55 and Cys169) allows us to selectively extract the effect of structural flexibility of the linker moiety on the ring stacking modes of pyrenes at the protein surface. As a result, it was found that the excimer emission intensity in the CLOSED forms of Adk tm s is highly dependent on the linker length in spite of similar distance between the two pyrene-labeled cysteine residues.…”