Solid-state NMR-based structure determination of membrane proteins and large protein complexes faces the challenge of limited spectral resolution when the proteins are uniformly 13C-labeled. A strategy to meet this challenge is chemical ligation combined with site-specific or segmental labeling. While chemical ligation has been adopted in NMR studies of water-soluble proteins, it has not been demonstrated for membrane proteins. Here we show chemical ligation of the influenza M2 protein, which contains a transmembrane (TM) domain and two extra-membrane domains. The cytoplasmic domain, which contains an amphipathic helix (AH) and a cytoplasmic tail, is important for regulating virus assembly, virus budding, and the proton channel activity. A recent study of uniformly 13C-labeled full-length M2 by spectral simulation suggested that the cytoplasmic tail is unstructured. To further test this hypothesis, we conducted native chemical ligation of the TM segment and part of the cytoplasmic domain. Solid-phase peptide synthesis of the two segments allowed several residues to be labeled in each segment. The post-AH cytoplasmic residues exhibit random-coil chemical shifts, low bond order parameters, and a surface-bound location, thus indicating that this domain is a dynamic random coil on the membrane surface. Interestingly, the protein spectra are similar between a model membrane and a virus-mimetic membrane, indicating that the structure and dynamics of the post-AH segment is insensitive to the lipid composition. This chemical ligation approach is generally applicable to medium-sized membrane proteins to provide site-specific structural constraints, which complement the information obtained from uniformly 13C, 15N-labeled proteins.
[NMR paper] Chemical Ligation of the Influenza M2 Protein for Solid-State NMR Characterization of the Cytoplasmic Domain.
Chemical Ligation of the Influenza M2 Protein for Solid-State NMR Characterization of the Cytoplasmic Domain.
Related Articles Chemical Ligation of the Influenza M2 Protein for Solid-State NMR Characterization of the Cytoplasmic Domain.
Protein Sci. 2015 May 13;
Authors: Kwon B, Tietze D, White PB, Liao SY, Hong M
Abstract
Solid-state NMR-based structure determination of membrane proteins and large protein complexes faces the challenge of limited spectral resolution when the proteins are uniformly (13) C-labeled. A strategy to...
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05-15-2015 08:02 PM
Chemical Ligation of the Influenza M2 Protein for Solid-State NMR Characterization of the Cytoplasmic Domain
Chemical Ligation of the Influenza M2 Protein for Solid-State NMR Characterization of the Cytoplasmic Domain
Abstract
Solid-state NMR-based structure determination of membrane proteins and large protein complexes faces the challenge of limited spectral resolution when the proteins are uniformly 13C-labeled. A strategy to meet this challenge is chemical ligation combined with site-specific or segmental labeling. While chemical ligation has been adopted in NMR studies of water-soluble proteins, it has not been demonstrated for membrane proteins. Here we show chemical ligation of the...
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05-13-2015 02:01 PM
Conformational analysis of the full-length M2 protein of the influenza A virus using solid-state NMR
Conformational analysis of the full-length M2 protein of the influenza A virus using solid-state NMR
Abstract
The influenza A M2 protein forms a proton channel for virus infection and mediates virus assembly and budding. While extensive structural information is known about the transmembrane helix and an adjacent amphipathic helix, the conformation of the N-terminal ectodomain and the C-terminal cytoplasmic tail remains largely unknown. Using two-dimensional (2D) magic-angle-spinning solid-state NMR, we have investigated the secondary structure and dynamics of full-length M2 (M2FL) and...
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10-07-2013 08:31 AM
[NMR paper] Conformational analysis of the full-length M2 protein of the influenza a virus using solid-state NMR.
Conformational analysis of the full-length M2 protein of the influenza a virus using solid-state NMR.
http://www.bionmr.com//www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--media.wiley.com-assets-2250-98-WileyOnlineLibrary-Button_120x27px_FullText.gif Related Articles Conformational analysis of the full-length M2 protein of the influenza a virus using solid-state NMR.
Protein Sci. 2013 Sep 10;
Authors: Liao SY, Fritzsching KJ, Hong M
Abstract
The influenza A M2 protein forms a proton channel for virus infection and mediates virus...
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09-12-2013 11:02 PM
Conformational analysis of the full-length M2 protein of the influenza a virus using solid-state NMR
Conformational analysis of the full-length M2 protein of the influenza a virus using solid-state NMR
Abstract
The influenza A M2 protein forms a proton channel for virus infection and mediates virus assembly and budding. While extensive structural information is known about the transmembrane (TM) helix and an adjacent amphipathic helix (AH), the conformation of the N-terminal ectodomain and the C-terminal cytoplasmic tail remains largely unknown. Using 2D magic-angle-spinning (MAS) solid-state NMR, we have investigated the secondary structure and dynamics of full-length M2 (M2FL) and...
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09-10-2013 08:44 PM
Segmental isotopic labeling of a 140 kDa dimeric multi-domain protein CheA from Escherichia coli by expressed protein ligation and protein trans-splicing
Segmental isotopic labeling of a 140 kDa dimeric multi-domain protein CheA from Escherichia coli by expressed protein ligation and protein trans-splicing
Abstract Segmental isotopic labeling is a powerful labeling tool to facilitate NMR studies of larger proteins by not only alleviating the signal overlap problem but also retaining features of uniform isotopic labeling. Although two approaches, expressed protein ligation (EPL) and protein trans-splicing (PTS), have been mainly used for segmental isotopic labeling, there has been no single example in which both approaches have been...
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07-02-2012 06:18 AM
[NMR paper] Magic-angle spinning solid-state NMR spectroscopy of the beta1 immunoglobulin binding domain of protein G (GB1): 15N and 13C chemical shift assignments and conformational analysis.
Magic-angle spinning solid-state NMR spectroscopy of the beta1 immunoglobulin binding domain of protein G (GB1): 15N and 13C chemical shift assignments and conformational analysis.
Related Articles Magic-angle spinning solid-state NMR spectroscopy of the beta1 immunoglobulin binding domain of protein G (GB1): 15N and 13C chemical shift assignments and conformational analysis.
J Am Chem Soc. 2005 Sep 7;127(35):12291-305
Authors: Franks WT, Zhou DH, Wylie BJ, Money BG, Graesser DT, Frericks HL, Sahota G, Rienstra CM
Magic-angle spinning...
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12-01-2010 06:56 PM
[NMR paper] Transmembrane domain of M2 protein from influenza A virus studied by solid-state (15)
Transmembrane domain of M2 protein from influenza A virus studied by solid-state (15)N polarization inversion spin exchange at magic angle NMR.
Related Articles Transmembrane domain of M2 protein from influenza A virus studied by solid-state (15)N polarization inversion spin exchange at magic angle NMR.
Biophys J. 2000 Aug;79(2):767-75
Authors: Song Z, Kovacs FA, Wang J, Denny JK, Shekar SC, Quine JR, Cross TA
The M2 protein from the influenza A virus forms a proton channel in the virion that is essential for infection. This tetrameric protein...
Chemical ligation of the influenza M2 protein for solid-state NMR characterization of the cytoplasmic domain
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