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Engineered nanostructured β-sheet peptides protect membrane proteins.

Tao H, Lee SC, Moeller A, Roy RS, Siu FY, Zimmermann J, Stevens RC, Potter CS, Carragher B, Zhang Q - Nat. Methods (2013)

Bottom Line: We designed β-strand peptides that stabilize integral membrane proteins (IMPs). β-strand peptides self-assemble in solution as filaments and become restructured upon association with IMPs; resulting IMP-β-strand peptide complexes resisted aggregation when diluted in detergent-free buffer and were visible as stable, single particles with low detergent background in electron micrographs. β-strand peptides enabled clear visualization of flexible conformations in the highly dynamic ATP-binding cassette (ABC) transporter MsbA.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California, USA.

ABSTRACT
We designed β-strand peptides that stabilize integral membrane proteins (IMPs). β-strand peptides self-assemble in solution as filaments and become restructured upon association with IMPs; resulting IMP-β-strand peptide complexes resisted aggregation when diluted in detergent-free buffer and were visible as stable, single particles with low detergent background in electron micrographs. β-strand peptides enabled clear visualization of flexible conformations in the highly dynamic ATP-binding cassette (ABC) transporter MsbA.

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Structures of β-strand peptides designed to stabilize IMPs. (a) Designed BP sequences (BP-1, 2, and 3) feature facial amphiphilicity with alternate hydrophobic (red) and hydrophilic (blue) residues, and differ in the number of N-Me amino acids (green). (b) A cartoon representation of proposed β-barrel architecture assembled from BPs (blue strands) by interstrand H-bonding in which the hydrophobic alkyl chains (space filling spheres) associate with and sequester the IMP surfaces (yellow α-helices). (c) CD spectra of BP-1, 2 and 3 indicate secondary structures with β-sheet character. (d) Electron micrographs of negatively stained BP-1 (BP-2 and BP-3 shown in Supplementary Fig. 2) show self-assembled filamentous structures ~3 nm in diameter (inset). The scale bar represents 30 nm.
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Figure 1: Structures of β-strand peptides designed to stabilize IMPs. (a) Designed BP sequences (BP-1, 2, and 3) feature facial amphiphilicity with alternate hydrophobic (red) and hydrophilic (blue) residues, and differ in the number of N-Me amino acids (green). (b) A cartoon representation of proposed β-barrel architecture assembled from BPs (blue strands) by interstrand H-bonding in which the hydrophobic alkyl chains (space filling spheres) associate with and sequester the IMP surfaces (yellow α-helices). (c) CD spectra of BP-1, 2 and 3 indicate secondary structures with β-sheet character. (d) Electron micrographs of negatively stained BP-1 (BP-2 and BP-3 shown in Supplementary Fig. 2) show self-assembled filamentous structures ~3 nm in diameter (inset). The scale bar represents 30 nm.

Mentions: Toward the goal of stabilization and structural studies of IMPs, we designed three BPs, with the general sequence Ac-(Oct)Gly-Ser-Leu-Ser-Leu-Asp-(Oct)Gly-Asp-NH2 (Fig. 1a), to include the following notable features: (1) alternate hydrophobic and hydrophilic residues to confer facial amphiphilicity in an extended, β-strand conformation9; (2) a short, eight amino acid sequence of appropriate length to span the central nonpolar region of membrane bilayers (~3 nm)8; (3) elongated alkyl side chains at each end of the peptide to increase hydrophobicity, a modification important for IMP stabilization as implicated in lipopeptide detergent design3; and (4) incorporation of N-methyl (N-Me) amino acids that diminish H-bond donors to regulate interstrand association and avoid the formation of insoluble macroscopic fibril structures10, 11. It is notable that the same sequence lacking N-Me amino acids was barely soluble in aqueous solution and most organic solvents, making it difficult to purify as a result of its tendency to aggregate due to strong H-bond interactions, whereas the three analogous peptides (BP-1, BP-2 and BP-3, with one, two and three N-Me substituents, respectively) showed moderate water solubility (~3 mM for BP-1 and BP-2, and ~1 mM for BP-3). Overall, these BPs, distinct from a majority of amphiphiles, were designed to sequester IMP hydrophobic surfaces by forming an ordered, stabilizing β-barrel-like structure, and to decrease dynamic dissociation from IMP surfaces by introducing intermolecular H-bond interactions (Fig. 1b).


Engineered nanostructured β-sheet peptides protect membrane proteins.

Tao H, Lee SC, Moeller A, Roy RS, Siu FY, Zimmermann J, Stevens RC, Potter CS, Carragher B, Zhang Q - Nat. Methods (2013)

Structures of β-strand peptides designed to stabilize IMPs. (a) Designed BP sequences (BP-1, 2, and 3) feature facial amphiphilicity with alternate hydrophobic (red) and hydrophilic (blue) residues, and differ in the number of N-Me amino acids (green). (b) A cartoon representation of proposed β-barrel architecture assembled from BPs (blue strands) by interstrand H-bonding in which the hydrophobic alkyl chains (space filling spheres) associate with and sequester the IMP surfaces (yellow α-helices). (c) CD spectra of BP-1, 2 and 3 indicate secondary structures with β-sheet character. (d) Electron micrographs of negatively stained BP-1 (BP-2 and BP-3 shown in Supplementary Fig. 2) show self-assembled filamentous structures ~3 nm in diameter (inset). The scale bar represents 30 nm.
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Related In: Results  -  Collection

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Figure 1: Structures of β-strand peptides designed to stabilize IMPs. (a) Designed BP sequences (BP-1, 2, and 3) feature facial amphiphilicity with alternate hydrophobic (red) and hydrophilic (blue) residues, and differ in the number of N-Me amino acids (green). (b) A cartoon representation of proposed β-barrel architecture assembled from BPs (blue strands) by interstrand H-bonding in which the hydrophobic alkyl chains (space filling spheres) associate with and sequester the IMP surfaces (yellow α-helices). (c) CD spectra of BP-1, 2 and 3 indicate secondary structures with β-sheet character. (d) Electron micrographs of negatively stained BP-1 (BP-2 and BP-3 shown in Supplementary Fig. 2) show self-assembled filamentous structures ~3 nm in diameter (inset). The scale bar represents 30 nm.
Mentions: Toward the goal of stabilization and structural studies of IMPs, we designed three BPs, with the general sequence Ac-(Oct)Gly-Ser-Leu-Ser-Leu-Asp-(Oct)Gly-Asp-NH2 (Fig. 1a), to include the following notable features: (1) alternate hydrophobic and hydrophilic residues to confer facial amphiphilicity in an extended, β-strand conformation9; (2) a short, eight amino acid sequence of appropriate length to span the central nonpolar region of membrane bilayers (~3 nm)8; (3) elongated alkyl side chains at each end of the peptide to increase hydrophobicity, a modification important for IMP stabilization as implicated in lipopeptide detergent design3; and (4) incorporation of N-methyl (N-Me) amino acids that diminish H-bond donors to regulate interstrand association and avoid the formation of insoluble macroscopic fibril structures10, 11. It is notable that the same sequence lacking N-Me amino acids was barely soluble in aqueous solution and most organic solvents, making it difficult to purify as a result of its tendency to aggregate due to strong H-bond interactions, whereas the three analogous peptides (BP-1, BP-2 and BP-3, with one, two and three N-Me substituents, respectively) showed moderate water solubility (~3 mM for BP-1 and BP-2, and ~1 mM for BP-3). Overall, these BPs, distinct from a majority of amphiphiles, were designed to sequester IMP hydrophobic surfaces by forming an ordered, stabilizing β-barrel-like structure, and to decrease dynamic dissociation from IMP surfaces by introducing intermolecular H-bond interactions (Fig. 1b).

Bottom Line: We designed β-strand peptides that stabilize integral membrane proteins (IMPs). β-strand peptides self-assemble in solution as filaments and become restructured upon association with IMPs; resulting IMP-β-strand peptide complexes resisted aggregation when diluted in detergent-free buffer and were visible as stable, single particles with low detergent background in electron micrographs. β-strand peptides enabled clear visualization of flexible conformations in the highly dynamic ATP-binding cassette (ABC) transporter MsbA.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, California, USA.

ABSTRACT
We designed β-strand peptides that stabilize integral membrane proteins (IMPs). β-strand peptides self-assemble in solution as filaments and become restructured upon association with IMPs; resulting IMP-β-strand peptide complexes resisted aggregation when diluted in detergent-free buffer and were visible as stable, single particles with low detergent background in electron micrographs. β-strand peptides enabled clear visualization of flexible conformations in the highly dynamic ATP-binding cassette (ABC) transporter MsbA.

Show MeSH