Synthetic Fluorescent Supercharged DRNN Scaffold VX Binding Protein

Synthetic Fluorescent Supercharged DRNN Scaffold VX Binding Protein

A September 1, 2022 news article by Kitta Macpherson published on the Physics.org website announced a new application of synthetic protein design that will have dramatic life saving impact in the near future and spawn numerous additional custom made solutions. A team of scientists, a number of whom were from Rutgers University, used a protein core scaffold originating from the CheA phosphotransferase protein domain occurring in Thermotoga maritima (Macpherson, 2022; McCann et al., 2022). From the scaffold, McCann et al. (2022), created an artificial VX nerve agent detection protein that can be used in conjunction with additional supporting equipment to give immediate alerts when minute traces of VX nerve agent are present in the environment. The protein is designed to eliminate problems previous detection systems encountered with false triggering due to pesticides and other chemicals closely resembling VX nerve agent (Macpherson, 2022; McCann et al., 2022). 

Historically, an original team, Vu et al (2011) identified and detailed a conserved histidine phosphotransfer domain four/five-helix bundle motif from the Thermotoga maritima histidine autokinase CheA  (Figure 1). A subsequent group, Murphy et al, (2012) used the four-helix bundle from CheA  to create a flexible protein backbone with designated locations that incorporate non-native amino acids. 

Figure 1. 1tqg.pdb file rendered in Bragi software
(Research Collaboratory for Structural Bioinformatics (RCSB), 2004). 

They identified this structure as DRNN (Figure 2). Their goal in producing this structure was to create an energetically suitable structure not found in nature by modifying a protein found in nature. The task they chose was to mutate the CheA protein bundle while still maintaining its folding character and stability. A significant post-creation finding is that DRNN is thermally stable up to 140 degrees Centigrade. 

Figure 2. 2LCH.pdb file rendered in Bragi software (RCSB, 2011).

Thus, to create the VX biosensor protein, McCann et al. (2022) purchased a modified version of the DRNN from Genscript Biotech Corporation (RCSB, 2012). A key design feature used in the creation of the VX sensor protein was a completely internal binding site, one that could be custom designed to incorporate maximum amino acid-specific attachment points in order to gain the highest affinity and specificity for VX molecules. In addition to this internal binding design, the molecule was additionally computer enhanced to supercharge surface side chains by changing them to glutamate. The electo-repulsion of these side chains causes the protein to unfold in low ionic solutions and refold in higher ionic solutions. In this manner, the authors were able to unfold the protein and insert the custom designed internal binding site. In addition a highly fluorescent molecule of tryptophan was buried in the core of the molecule and fluorescence is reduced when the molecule is folded. Florescence is further reduced when a VX molecule binds to the binding site. In this way the florescence can be used by an inexpensive fluorimeter. An even more intriguing aspect is that the authors claim that a sensor built from this protein should only cost around $5000 (McCann et al., 2022). 

References

Macpherson, K. (2022, September 1). Protein that Could Prevent Chemical Warfare

Attack Created. Physics.org. Retreived on October 6, 2022 from https://phys.org/news/2022-09-protein-chemical-warfare.html

McCann, J. J., Pike, D. H., Brown, M. C., Crouse, D. T., Nanda, V., & Koder, R. L. (2022). Computational Design of a Sensitive, Selective Phase-Changing Sensor Protein for the VX Nerve Agent. Science Advances, 8(27), eabh3421. https://doi.org/10.1126/sciadv.abh3421

Murphy, G. S., Mills, J. L., Miley, M. J., Machius, M., Szyperski, T., & Kuhlman, B. (2012). Increasing Sequence Diversity with Flexible Backbone Protein Design: The Complete Redesign of a Protein Hydrophobic Core. Structure (London, England : 1993), 20(6), 1086–1096. https://doi.org/10.1016/j.str.2012.03.026

Research Collaboratory for Structural Bioinformatics. (2004). CheA Phosphotransferase Domain from Thermotoga maritima. Protein Data Bank. Retreived on October 7, 2022 from https://www.rcsb.org/structure/1TQG - CheA

Research Collaboratory for Structural Bioinformatics. (2011). Solution NMR Structure of a Protein With a Redesigned Hydrophobic Core, Northeast Structural Genomics Consortium Target OR38. Protein Data Bank. Retreived on October 7, 2022 from https://www.rcsb.org/structure/2LCH

Research Collaboratory for Structural Bioinformatics. (2012). Crystal Structure of Computationally Redesigned Four-Helix Bundle. Protein Data Bank. Retreived on October 7, 2022 from https://www.rcsb.org/structure/3U3B

Vu, A., Hamel, D. J., Zhou, H., & Dahlquist, F. W. (2011). The Structure and Dynamic Properties of the Complete Histidine Phosphotransfer Domain of the Chemotaxis Specific Histidine Autokinase CheA from Thermotoga maritima. Journal of Biomolecular NMR, 51(1-2), 49–55. https://doi.org/10.1007/s10858-011-9540-2