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Title: Nanobody mediated crystallization of an archeal mechanosensitive channel
Authors: Moberg, Per
Quistgaard, Esben M.
Löw, Christian
Yau, Yin Hoe
Pardon, Els
Jegerschöld, Caroline
Wåhlin, Lisa
Geifman-Shochat, Susana
Steyaert, Jan
Nordlund, Pär
Keywords: DRNTU::Science::Biological sciences
Issue Date: 2013
Source: Löw, C., Yau, Y. H., Pardon, E., Jegerschöld, C., Wåhlin, L., Quistgaard, E. M., et al. (2013). Nanobody mediated crystallization of an archeal mechanosensitive channel. PLoS ONE, 8(10), e77984-.
Series/Report no.: PLoS ONE
Abstract: 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.
ISSN: 1932-6203
DOI: 10.1371/journal.pone.0077984
Rights: © 2013 The Authors. This paper was published in PLoS ONE and is made available as an electronic reprint (preprint) with permission of the authors. The paper can be found at the following official DOI: []. One print or electronic copy may be made for personal use only. Systematic or multiple reproduction, distribution to multiple locations via electronic or other means, duplication of any material in this paper for a fee or for commercial purposes, or modification of the content of the paper is prohibited and is subject to penalties under law.
Fulltext Permission: open
Fulltext Availability: With Fulltext
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