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Esben Meldgaard Høgh Quistgaard

Nanobody mediated crystallization of an archeal mechanosensitive channel

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaperJournal articleResearchpeer-review

Standard

Nanobody mediated crystallization of an archeal mechanosensitive channel. / Löw, Christian; Yau, Yin Hoe; Pardon, Els; Jegerschöld, Caroline; Wåhlin, Lisa; Quistgaard, Esben M; Moberg, Per; Geifman-Shochat, Susana; Steyaert, Jan; Nordlund, Pär.

In: P L o S One, Vol. 8, No. 10, 10.2013, p. e77984.

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaperJournal articleResearchpeer-review

Harvard

Löw, C, Yau, YH, Pardon, E, Jegerschöld, C, Wåhlin, L, Quistgaard, EM, Moberg, P, Geifman-Shochat, S, Steyaert, J & Nordlund, P 2013, 'Nanobody mediated crystallization of an archeal mechanosensitive channel', P L o S One, vol. 8, no. 10, pp. e77984. https://doi.org/10.1371/journal.pone.0077984

APA

Löw, C., Yau, Y. H., Pardon, E., Jegerschöld, C., Wåhlin, L., Quistgaard, E. M., ... Nordlund, P. (2013). Nanobody mediated crystallization of an archeal mechanosensitive channel. P L o S One, 8(10), e77984. https://doi.org/10.1371/journal.pone.0077984

CBE

Löw C, Yau YH, Pardon E, Jegerschöld C, Wåhlin L, Quistgaard EM, Moberg P, Geifman-Shochat S, Steyaert J, Nordlund P. 2013. Nanobody mediated crystallization of an archeal mechanosensitive channel. P L o S One. 8(10):e77984. https://doi.org/10.1371/journal.pone.0077984

MLA

Vancouver

Löw C, Yau YH, Pardon E, Jegerschöld C, Wåhlin L, Quistgaard EM et al. Nanobody mediated crystallization of an archeal mechanosensitive channel. P L o S One. 2013 Oct;8(10):e77984. https://doi.org/10.1371/journal.pone.0077984

Author

Löw, Christian ; Yau, Yin Hoe ; Pardon, Els ; Jegerschöld, Caroline ; Wåhlin, Lisa ; Quistgaard, Esben M ; Moberg, Per ; Geifman-Shochat, Susana ; Steyaert, Jan ; Nordlund, Pär. / Nanobody mediated crystallization of an archeal mechanosensitive channel. In: P L o S One. 2013 ; Vol. 8, No. 10. pp. e77984.

Bibtex

@article{77b125cd66924acb93176db37c47c69e,
title = "Nanobody mediated crystallization of an archeal mechanosensitive channel",
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.",
keywords = "Animals, Archaeal Proteins, Camelids, New World, Crystallography, X-Ray, Mechanotransduction, Cellular, Single-Domain Antibodies, Thermoplasma, Journal Article, Research Support, Non-U.S. Gov't",
author = "Christian L{\"o}w and Yau, {Yin Hoe} and Els Pardon and Caroline Jegersch{\"o}ld and Lisa W{\aa}hlin and Quistgaard, {Esben M} and Per Moberg and Susana Geifman-Shochat and Jan Steyaert and P{\"a}r Nordlund",
year = "2013",
month = "10",
doi = "10.1371/journal.pone.0077984",
language = "English",
volume = "8",
pages = "e77984",
journal = "P L o S One",
issn = "1932-6203",
publisher = "public library of science",
number = "10",

}

RIS

TY - JOUR

T1 - Nanobody mediated crystallization of an archeal mechanosensitive channel

AU - Löw, Christian

AU - Yau, Yin Hoe

AU - Pardon, Els

AU - Jegerschöld, Caroline

AU - Wåhlin, Lisa

AU - Quistgaard, Esben M

AU - Moberg, Per

AU - Geifman-Shochat, Susana

AU - Steyaert, Jan

AU - Nordlund, Pär

PY - 2013/10

Y1 - 2013/10

N2 - 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.

AB - 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.

KW - Animals

KW - Archaeal Proteins

KW - Camelids, New World

KW - Crystallography, X-Ray

KW - Mechanotransduction, Cellular

KW - Single-Domain Antibodies

KW - Thermoplasma

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1371/journal.pone.0077984

DO - 10.1371/journal.pone.0077984

M3 - Journal article

C2 - 24205053

VL - 8

SP - e77984

JO - P L o S One

JF - P L o S One

SN - 1932-6203

IS - 10

ER -