Nanobody mediated crystallization of an archeal mechanosensitive channel

PLoS One. 2013 Oct 21;8(10):e77984. doi: 10.1371/journal.pone.0077984. eCollection 2013.


Mechanosensitive channels (MS) are integral membrane proteins and allow bacteria to survive sudden changes in external osmolarity due to transient opening of their pores. The efflux of cytoplasmic osmolytes reduces the membrane tension and prevents membrane rupture. Therefore these channels serve as emergency valves when experiencing significant environmental stress. The preparation of high quality crystals of integral membrane proteins is a major bottleneck for structure determination by X-ray crystallography. Crystallization chaperones based on various protein scaffolds have emerged as promising tool to increase the crystallization probability of a selected target protein. So far archeal mechanosensitive channels of small conductance have resisted crystallization in our hands. To structurally analyse these channels, we selected nanobodies against an archeal MS channel after immunization of a llama with recombinant expressed, detergent solubilized and purified protein. Here we present the characterization of 23 different binders regarding their interaction with the channel protein using analytical gel filtration, western blotting and surface plasmon resonance. Selected nanobodies bound the target with affinities in the pico- to nanomolar range and some binders had a profound effect on the crystallization of the MS channel. Together with previous data we show that nanobodies are a versatile and valuable tool in structural biology by widening the crystallization space for highly challenging proteins, protein complexes and integral membrane proteins.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Archaeal Proteins / chemistry*
  • Archaeal Proteins / immunology
  • Archaeal Proteins / metabolism
  • Camelids, New World
  • Crystallography, X-Ray
  • Mechanotransduction, Cellular
  • Single-Domain Antibodies / chemistry*
  • Single-Domain Antibodies / immunology
  • Single-Domain Antibodies / metabolism
  • Thermoplasma / chemistry


  • Archaeal Proteins
  • Single-Domain Antibodies

Grants and funding

E.M.Q. was supported by The Danish Council for Independent Research (Medical Sciences; grant 271-09-0187). C.L. was supported by a European Molecular Biology Organization (EMBO) postdoctoral fellowship. E.P. and J.S. were supported by the Fonds Wetenschappelijk Onderzoek-Vlaanderen through research grants G011110N and G049512N, Innoviris Brussels through Impulse Life Science program BRGEOZ132, the Belgian Federal Science Policy Office through IAP7-40 and by SBO program IWT120026 from the Flemish Agency for Innovation by Science and Technology. This research was further supported by grants from the Swedish Research Council, Swedish Cancer Society and the integrated EU project EDICT (European drug initiative on channels and transporters), as well as a Singapore NRF-CRP (National Research Foundation - Competitive Research Programme) grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.