Structural and thermodynamic analysis of the GFP:GFP‐nanobody complex

Marta H. Kubala; Oleksiy Kovtun; Kirill Alexandrov; Brett M. Collins

doi:10.1002/pro.519

journal article Nov 18, 2010

Structural and thermodynamic analysis of the GFP:GFP‐nanobody complex

Marta H. Kubala Oleksiy Kovtun

Kirill Alexandrov

Brett M. Collins

Protein Science Vol. 19 No. 12 pp. 2389-2401 · Wiley

View at Publisher Save 10.1002/pro.519

Abstract

AbstractThe green fluorescent protein (GFP)‐nanobody is a single‐chain VHH antibody domain developed with specific binding activity against GFP and is emerging as a powerful tool for isolation and cellular engineering of fluorescent protein fusions in many different fields of biological research. Using X‐ray crystallography and isothermal titration calorimetry, we determine the molecular details of GFP:GFP‐nanobody complex formation and explain the basis of high affinity and at the same time high specificity of protein binding. Although the GFP‐nanobody can also bind YFP, it cannot bind the closely related CFP or other fluorescent proteins from the mFruit series. CFP differs from GFP only within the central chromophore and at one surface amino acid position, which lies in the binding interface. Using this information, we have engineered a CFP variant (I146N) that is also able to bind the GFP‐nanobody with high affinity, thus extending the toolbox of genetically encoded fluorescent probes that can be isolated using the GFP‐nanobody.

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References

Details

Published: Nov 18, 2010
Vol/Issue: 19(12)
Pages: 2389-2401
License: View

Authors

MRC LMB Cambridge Biomedical Campus, Cambridge CB2 0QH, UK.

K

Kirill Alexandrov

Institute for Molecular Bioscience; The University of Queensland; Queensland, Australia

B

Brett M. Collins

Cite This Article

Marta H. Kubala, Oleksiy Kovtun, Kirill Alexandrov, et al. (2010). Structural and thermodynamic analysis of the GFP:GFP‐nanobody complex. Protein Science, 19(12), 2389-2401. https://doi.org/10.1002/pro.519

Structural and thermodynamic analysis of the GFP:GFP‐nanobody complex

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