Introducing Cysteines into Nanobodies for Site-Specific Labeling.

We have developed a generally applicable methodology for cysteine mutagenesis of nanobody (Nb) framework region serine residues. This strategy allows for subsequent labeling with thiol-reactive compounds without disrupting Nb antigen binding. We provide a protocol for production, labeling, and affinity determination of cysteine-engineered Nbs (cys-Nbs) with Alexa Fluor 488-maleimide and the mercury compound para-chloromercuribenzoic acid (PCMB). Alexa Fluor 488- and PCMB-labeled cys-Nbs can be used for immunofluorescence microscopy and experimental phasing in crystallography, respectively.

Introducing site-specific cysteines into nanobodies for mercury labelling allows de novo phasing of their crystal structures.

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.