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

Kubala, MH, Kovtun, O, Alexandrov, K and Collins, BM (2010) Structural and thermodynamic analysis of the GFP:GFP-nanobody complex. Protein Science, 19 12: 2389-2401. doi:10.1002/pro.519


Author Kubala, MH
Kovtun, O
Alexandrov, K
Collins, BM
Title Structural and thermodynamic analysis of the GFP:GFP-nanobody complex
Journal name Protein Science   Check publisher's open access policy
ISSN 0961-8368
1469-896X
1359-5040
Publication date 2010-12-01
Year available 2010
Sub-type Article (original research)
DOI 10.1002/pro.519
Open Access Status DOI
Volume 19
Issue 12
Start page 2389
End page 2401
Total pages 13
Place of publication Hoboken, NJ, United States
Publisher Wiley-Blackwell Publishing
Language eng
Abstract The green fluorescent protein (GFP)-nanobody is a single-chain V(H)H 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
Formatted abstract
The green fluorescent protein (GFP)-nanobody is a single-chain V HH 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. Published by Wiley-Blackwell. © 2010 The Protein Society.
Keyword GFP
Nanobody
ITC
Thermodynamics
Heat capacity
Q-Index Code C1
Q-Index Status Confirmed Code
Grant ID DP0986496
401626
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2011 Collection
Institute for Molecular Bioscience - Publications
 
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Created: Sun, 19 Dec 2010, 10:02:45 EST