Photocontrolled reversible self-assembly of dodecamer nitrilase

Abstract

Background

Naturally photoswitchable proteins act as a powerful tool for the spatial and temporal control of biological processes by inducing the formation of a photodimerizer. In this study, a method for the precise and reversible inducible self-assembly of dodecamer nitrilase in vivo (in Escherichia coli) and in vitro (in a cell-free solution) was developed by means of the photoswitch-improved light-inducible dimer (iLID) system which could induce protein–protein dimerization.

Results

Nitrilase was fused with the photoswitch protein AsLOV2-SsrA to achieve the photocontrolled self-assembly of dodecamer nitrilase. The fusion protein self-assembled into a supramolecular assembly when illuminated at 470 nm. Scanning electron microscopy showed that the assembly formed a circular sheet structure. Self-assembly was also induced by light in E. coli. Dynamic light scattering and turbidity assay experiments showed that the assemblies formed within a few seconds under 470-nm light and completely disassembled within 5 min in the dark. Assembly and disassembly could be maintained for at least five cycles. Both in vitro and in vivo, the assemblies retained 90% of the initial activity of nitrilase and could be reused at least four times in vitro with 90% activity.

Conclusions

An efficient method was developed for the photocontrolled assembly and disassembly of dodecamer nitrilase and for scaffold-free reversible self-assembly of multiple oligomeric enzymes in vivo and in vitro, providing new ideas and methods for immobilization of enzyme without carrier.

Keywords

Disassembly / iLID / Nitrilase / Photoswitch / Self-assembly

References

[1] Brady D, Jordaan J. Advances in enzyme immobilisation. Biotechnol Lett, 2009, 31(11): 1639.

[2] Fairman R, Åkerfeldt KS. Peptides as novel smart materials. Curr Opin Struct Biol, 2005, 15(4): 453-463.

[3] Gao X, Zhao CC, Yu T, Yang S, Ren YH, Wei DZ. Construction of a reusable multi-enzyme supramolecular device via disulfide bond locking. Chem Commun, 2015, 51(50): 10131-10133.

[4] Guntas G, Hallett RA, Zimmerman SP, Williams T, Yumerefendi H, Bear JE, Kuhlman B. Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins. Proc Natl Acad Sci USA, 2014, 112(1): 112-117.

[5] Halavaty AS, Moffat K. N- and C-terminal flanking regions modulate light-induced signal transduction in the LOV2 domain of the blue light sensor phototropin 1 from Avena sativa. Biochemistry, 2007, 46(49): 14001-14009.

[6] Harper SM, Christie JM, Gardner KH. Disruption of the LOV-J alpha helix interaction activates phototropin kinase activity. Biochemistry, 2004, 43(51): 16184-16192.

[7] Holmes TC. Novel peptide-based biomaterial scaffolds for tissue engineering. Trends Biotechnol, 2002, 20(1): 16-21.

[8] Jaenicke R. Stability and stabilization of globular proteins in solution. J Biotechnol, 2000, 79(3): 193-203.

[9] Kanekura K, Yagi T, Cammack AJ, Mahadevan J, Kuroda M, Harms MB, Miller TM, Urano F. Poly-dipeptides encoded by the C9ORF72 repeats block global protein translation. Hum Mol Genet, 2016, 25(9): 1803-1813.

[10] Kennedy MJ, Hughes RM, Peteya LA, Schwartz JW, Ehlers MD, Tucker CL. Rapid blue-light-mediated induction of protein interactions in living cells. Nat Methods, 2010, 7(12): 973-975.

[11] King NP, Lai YT. Practical approaches to designing novel protein assemblies. Curr Opin Struct Biol, 2013, 23(4): 632-638.

[12] Levskaya A, Weiner OD, Lim WA, Voigt CA. Spatiotemporal control of cell signalling using a light-switchable protein interaction. Nature, 2009, 461(7266): 997-1001.

[13] Matsumoto T, Isogawa Y, Minamihata K, Tanaka T, Kondo A. Twigged streptavidin polymer as a scaffold for protein assembly. J Biotechnol, 2016, 225: 61-66.

[14] Ni KF, Wang HL, Zhao L, Zhang MJ, Zhang SY, Ren YH. Efficient production of (R)-(−)-mandelic acid in biphasic system by immobilized recombinant E. coli. J Biotechnol, 2013, 167(4): 433-440.

[15] Pieters RJ. Maximising multivalency effects in protein–carbohydrate interactions. Org Biomol Chem, 2009, 7(10): 2013-2025.

[16] Schoffelen S, van Hest JCM. Multi-enzyme systems: bringing enzymes together in vitro. Soft Matter, 2012, 8(6): 1736-1746.

[17] Sengupta P, Jovanovic-Talisman T, Skoko D, Renz M, Veatch SL, Lippincott-Schwartz J. Probing protein heterogeneity in the plasma membrane using PALM and pair correlation analysis. Nat Methods, 2011, 8(11): 969-975.

[18] Smith KH, Tejeda-Montes E, Poch M, Mata A. Integrating top-down and self-assembly in the fabrication of peptide and protein-based biomedical materials. Chem Soc Rev, 2011, 40(9): 4563-4577.

[19] Strickland D, Yao X, Gawlak G, Rosen MK, Gardner KH, Sosnick TR. Rationally improving LOV domain-based photoswitches. Nat Methods, 2010, 7(8): 623-626.

[20] Wang HL, Sun HH, Wei DZ. Discovery and characterization of a highly efficient enantioselective mandelonitrile hydrolase from Burkholderia cenocepacia J2315 by phylogeny-based enzymatic substrate specificity prediction. BMC Biotechnol, 2013, 13(1): 14-24.

[21] Yin TF, Wu YI. Guiding lights: recent developments in optogenetic control of biochemical signals. Pfluegers Arch, 2013, 465(3): 397-408.

[22] Yu T, Gao X, Ren YH, Wei DZ. Assembly of cellulases with synthetic protein scaffolds in vitro. Bioresour Bioprocess, 2015, 2(1): 16-22.

[23] Zimmerman SP, Hallett RA, Bourke AM, Bear JE, Kennedy MJ, Kuhlman B. Tuning the binding affinities and reversion kinetics of a light inducible dimer allows control of transmembrane protein localization. Biochemistry, 2016, 55(37): 5264-5271.

Funding

2060204