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Introducing site-specific cysteines into nanobodies for mercury labelling allows de novo phasing of their crystal structures
Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avis › Tidsskriftartikel › Forskning › peer review
Dokumenter
- qh5047
Forlagets udgivne version, 1,75 MB, PDF-dokument
DOI
- https://doi.org/10.1107/S2059798317013171
Forlagets udgivne version
- Simon Boje Hansen
- Nick Stub Laursen ,
- Gregers Rom Andersen
- Kasper R Andersen
The generation of high-quality protein crystals and the loss of phase information during an X-ray crystallography diffraction experiment represent the major bottlenecks in the determination of novel protein structures. A generic method for introducing Hg atoms into any crystal independent of the presence of free cysteines in the target protein could considerably facilitate the process of obtaining unbiased experimental phases. Nanobodies (single-domain antibodies) have recently been shown to promote the crystallization and structure determination of flexible proteins and complexes. To extend the usability of nanobodies for crystallographic work, variants of the Nb36 nanobody with a single free cysteine at one of four framework-residue positions were developed. These cysteines could be labelled with fluorophores or Hg. For one cysteine variant (Nb36-C85) two nanobody structures were experimentally phased using single-wavelength anomalous dispersion (SAD) and single isomorphous replacement with anomalous signal (SIRAS), taking advantage of radiation-induced changes in Cys-Hg bonding. Importantly, Hg labelling influenced neither the interaction of Nb36 with its antigen complement C5 nor its structure. The results suggest that Cys-Hg-labelled nanobodies may become efficient tools for obtaining de novo phase information during the structure determination of nanobody-protein complexes.
| Originalsprog | Engelsk |
|---|---|
| Tidsskrift | Acta crystallographica Section D: Structural biology |
| Vol/bind | 73 |
| Nummer | Pt 10 |
| Sider (fra-til) | 804-813 |
| Antal sider | 10 |
| ISSN | 2059-7983 |
| DOI | |
| Status | Udgivet - 2017 |
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