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The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications
Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
Vise andre og tillknytning
Rekke forfattare: 112019 (engelsk)Inngår i: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 10, artikkel-id 4403Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Specialized epitope tags are widely used for detecting, manipulating or purifying proteins, but often their versatility is limited. Here, we introduce the ALFA-tag, a rationally designed epitope tag that serves a remarkably broad spectrum of applications in life sciences while outperforming established tags like the HA-, FLAG (R)- or myc-tag. The ALFA-tag forms a small and stable a-helix that is functional irrespective of its position on the target protein in prokaryotic and eukaryotic hosts. We characterize a nanobody (NbALFA) binding ALFA-tagged proteins from native or fixed specimen with low picomolar affinity. It is ideally suited for super-resolution microscopy, immunoprecipitations and Western blotting, and also allows in vivo detection of proteins. We show the crystal structure of the complex that enabled us to design a nanobody mutant (NbALFA(PE)) that permits efficient one-step purifications of native ALFA-tagged proteins, complexes and even entire living cells using peptide elution under physiological conditions.

sted, utgiver, år, opplag, sider
2019. Vol. 10, artikkel-id 4403
HSV kategori
Forskningsprogram
biokemi
Identifikatorer
URN: urn:nbn:se:su:diva-175701DOI: 10.1038/s41467-019-12301-7ISI: 000488232600011PubMedID: 31562305OAI: oai:DiVA.org:su-175701DiVA, id: diva2:1369975
Tilgjengelig fra: 2019-11-13 Laget: 2019-11-13 Sist oppdatert: 2020-01-29bibliografisk kontrollert
Inngår i avhandling
1. Structural Insights into Botulinum Neurotoxins and the ALFA-tag System: Structural and Functional Studies of Proteins Related to the Botulinum Neurotoxins and Design of a Novel Epitope Tag
Åpne denne publikasjonen i ny fane eller vindu >>Structural Insights into Botulinum Neurotoxins and the ALFA-tag System: Structural and Functional Studies of Proteins Related to the Botulinum Neurotoxins and Design of a Novel Epitope Tag
2020 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

This thesis is divided into two sections; the first part describes our work in the field of botulinum neurotoxins (presented in papers I, II, III, and manuscript IV) and the second part summarizes our work involving the design of a new biochemical tool (presented in paper V).

Botulinum neurotoxins (BoNTs) produced by the anaerobic bacterium Clostridium botulinum are the most poisonous substances known to date. They have a conserved structure that consists of three domains (receptor-binding, translocation, and catalytic domain), each of which has a distinct function. The receptor-binding domain binds to neuronal receptors, and after endocytosis the translocation domain shuttles the catalytic domain into the cytosol, where it cleaves neuronal proteins of the SNARE family, which are part of the vesicle-membrane fusion machinery.

In paper I, we studied proteins of unknown function (OrfX1, OrfX2, OrfX3, and P47), which are co-expressed with certain BoNTs. We solved the crystal structures of OrfX2 and P47, and their structural resemblance to tubular lipid binding proteins (TULIP) together with lipid binding studies, led us to conclude that OrfX1 and P47 are able to bind phosphatidyl inositol phosphates (PIPs) in vitro.

In paper II, we studied the binding of BoNT/B, /DC and /G to their protein receptor synaptotagmin (Syt). We determined their affinities to synaptotagmins from different species, and concluded that residue F50 in bovine Syt-II is responsible for its increased affinity towards BoNT/DC. In addition, we studied the interaction between BoNT/G and Syt-II via STD-NMR. Our results showed the binding to be similar to BoNT/B and Syt-II, and that the N-terminal region of the Syt peptide is important for the binding of BoNTs to synaptotagmin, even though it is not part of the binding interface.

In paper III and manuscript IV, we present the identification of a novel BoNT serotype named BoNT/X. We showed that BoNT/X cleaves the non-canonical substrates VAMP4, VAMP5 and Ykt6, as well as the canonical substrate VAMP1-3 at a new cleavage site, distinct from other BoNTs. In addition, we present the cryo-EM structure of BoNT/X in complex with its non-toxic interaction partner NTNH. Our pH stability experiments revealed that BoNT/X-NTNH remain bound at neutral to moderately high pH, in contrast with what is observed for BoNT/A-NTNH.

In paper V we present the design of a novel epitope tag named the ALFA system. The ALFA tag is a short α-helical protein tag that is highly stable and electroneutral. The ALFA nanobody has a very high affinity for the tag and is small enough to allow for high performance in high-resolution microscopy. The crystal structure of the ALFA nanobody in complex with the tag led to a modified version of the ALFA nanobody that can release the tag via competitive elution with free ALFA peptide. Our results showed that this system outperforms several commercially available systems in protein purification and high-resolution microscopy.

sted, utgiver, år, opplag, sider
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2020. s. 60
Emneord
botulinum neurotoxin
HSV kategori
Forskningsprogram
biokemi
Identifikatorer
urn:nbn:se:su:diva-178474 (URN)978-91-7911-002-4 (ISBN)978-91-7911-003-1 (ISBN)
Disputas
2020-03-13, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (engelsk)
Opponent
Veileder
Merknad

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

Tilgjengelig fra: 2020-02-19 Laget: 2020-01-29 Sist oppdatert: 2020-02-07bibliografisk kontrollert

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