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Probing the transmembrane structure and topology of microsomal cytochrome-p450 by solid-state NMR on temperature-resistant bicelles.

Yamamoto K, Gildenberg M, Ahuja S, Im SC, Pearcy P, Waskell L, Ramamoorthy A - Sci Rep (2013)

Bottom Line: Though the importance of high-resolution structure and dynamics of membrane proteins has been well recognized, optimizing sample conditions to retain the native-like folding and function of membrane proteins for Nuclear Magnetic Resonance (NMR) or X-ray measurements has been a major challenge.While bicelles have been shown to stabilize the function of membrane proteins and are increasingly utilized as model membranes, the loss of their magnetic-alignment at low temperatures makes them unsuitable to study heat-sensitive membrane proteins like cytochrome-P450 and protein-protein complexes.Our results reveal that the N-terminal region of rabbit cytochrome-P4502B4, that is usually cleaved off to obtain crystal structures, is helical and has a transmembrane orientation with ~17° tilt from the lipid bilayer normal.

View Article: PubMed Central - PubMed

Affiliation: Biophysics and Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055, USA.

ABSTRACT
Though the importance of high-resolution structure and dynamics of membrane proteins has been well recognized, optimizing sample conditions to retain the native-like folding and function of membrane proteins for Nuclear Magnetic Resonance (NMR) or X-ray measurements has been a major challenge. While bicelles have been shown to stabilize the function of membrane proteins and are increasingly utilized as model membranes, the loss of their magnetic-alignment at low temperatures makes them unsuitable to study heat-sensitive membrane proteins like cytochrome-P450 and protein-protein complexes. In this study, we report temperature resistant bicelles that can magnetically-align for a broad range of temperatures and demonstrate their advantages in the structural studies of full-length microsomal cytochrome-P450 and cytochrome-b5 by solid-state NMR spectroscopy. Our results reveal that the N-terminal region of rabbit cytochrome-P4502B4, that is usually cleaved off to obtain crystal structures, is helical and has a transmembrane orientation with ~17° tilt from the lipid bilayer normal.

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The use of temperature resistant bicelles at lower temperatures enables the measurement of the transmembrane structure and topology of the functional form of microsomal cytochrome P450 2B4.(A) A UV-Vis spectrum shows that the overall folding of the catalytic site of a heat-sensitive membrane protein, cytochrome P450 2B4, can be stabilized in the functional form using temperature resistant bicelles, composed of DLPC and DHPC. (B) One-dimensional cross-polarization experiments of 100 μl of magnetically-aligned DLPC/DHPC bicelles (q = [DLPC]/[DHPC] = 4.0) containing a 0.61 mM of a uniformly-15N-labeled cytochrome P450 2B4 shows anisotropic 15N chemical shifts at 15°C. (C) Two-dimensional HIMSELF experiment reveal that cytochrome P450 2B4 has a helical structure in the N-terminal transmembrane region. The amino acid sequence (D) and a helical wheel representation (E) of the N-terminal transmembrane region of cytochrome P450 2B454. The full length amino acid sequence is given in the Supporting Information. Hydrophobic and hydrophilic amino acids are in black and blue, respectively. (F) A model depicting the structure and topology of cytochrome P450 in lipid bilayers; the soluble domain structure is adapted from the crystal structure for amino acid residues 28–491444546. The transmembrane structure of residues 1–27, obtained using the structure assembly simulation, I-TASSER55, is shown in the lipid bilayer region; the structure obtained with the highest C-score55 was chosen. The transmembrane domain may not be a straight α-helix due to the presence of Gly residues. This result is consistent with the imperfect wheel-like pattern of resonances in the 2D HIMSELF spectrum shown in Figure 3(C).
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f3: The use of temperature resistant bicelles at lower temperatures enables the measurement of the transmembrane structure and topology of the functional form of microsomal cytochrome P450 2B4.(A) A UV-Vis spectrum shows that the overall folding of the catalytic site of a heat-sensitive membrane protein, cytochrome P450 2B4, can be stabilized in the functional form using temperature resistant bicelles, composed of DLPC and DHPC. (B) One-dimensional cross-polarization experiments of 100 μl of magnetically-aligned DLPC/DHPC bicelles (q = [DLPC]/[DHPC] = 4.0) containing a 0.61 mM of a uniformly-15N-labeled cytochrome P450 2B4 shows anisotropic 15N chemical shifts at 15°C. (C) Two-dimensional HIMSELF experiment reveal that cytochrome P450 2B4 has a helical structure in the N-terminal transmembrane region. The amino acid sequence (D) and a helical wheel representation (E) of the N-terminal transmembrane region of cytochrome P450 2B454. The full length amino acid sequence is given in the Supporting Information. Hydrophobic and hydrophilic amino acids are in black and blue, respectively. (F) A model depicting the structure and topology of cytochrome P450 in lipid bilayers; the soluble domain structure is adapted from the crystal structure for amino acid residues 28–491444546. The transmembrane structure of residues 1–27, obtained using the structure assembly simulation, I-TASSER55, is shown in the lipid bilayer region; the structure obtained with the highest C-score55 was chosen. The transmembrane domain may not be a straight α-helix due to the presence of Gly residues. This result is consistent with the imperfect wheel-like pattern of resonances in the 2D HIMSELF spectrum shown in Figure 3(C).

Mentions: Though many crystal structures of the soluble domain of cyt-P450 have been reported in the literature, high-resolution structure of the full-length protein is unknown. The N-terminal 60-residues segment containing the hydrophobic domain is usually cleaved off to obtain a single crystal for structural studies by X-ray crystallography44454647. In addition, the full-length protein is quite unstable and highly sensitive to heat. Therefore, the thermally stable magnetic-alignment of DLPC/DHPC bicelles is a breakthrough to investigate the structure of a heat-sensitive full-length membrane-bound protein like cytochrome P450 2B4 in fluid lamellar phase lipid bilayers. To optimize the experimental conditions, we first confirmed the functional folding of the catalytic site of cytochrome P450 2B4 by performing carbon monoxide assays and also tested the stability of the protein in these bicelles for various temperatures. As shown in Figure 3(A), these assays indicated that the temperature resistant bicelles retained the stable native structural folding of cytochrome P450 in a biologically active state (P450 instead of P420) under NMR sample conditions. Using this functional form, the transmembrane structure and topology of cytochrome P450 2B4 are then determined using solid-state NMR experiments on magnetically-aligned DLPC/DHPC bicelles containing a uniformly-15N-labeled-P450.


Probing the transmembrane structure and topology of microsomal cytochrome-p450 by solid-state NMR on temperature-resistant bicelles.

Yamamoto K, Gildenberg M, Ahuja S, Im SC, Pearcy P, Waskell L, Ramamoorthy A - Sci Rep (2013)

The use of temperature resistant bicelles at lower temperatures enables the measurement of the transmembrane structure and topology of the functional form of microsomal cytochrome P450 2B4.(A) A UV-Vis spectrum shows that the overall folding of the catalytic site of a heat-sensitive membrane protein, cytochrome P450 2B4, can be stabilized in the functional form using temperature resistant bicelles, composed of DLPC and DHPC. (B) One-dimensional cross-polarization experiments of 100 μl of magnetically-aligned DLPC/DHPC bicelles (q = [DLPC]/[DHPC] = 4.0) containing a 0.61 mM of a uniformly-15N-labeled cytochrome P450 2B4 shows anisotropic 15N chemical shifts at 15°C. (C) Two-dimensional HIMSELF experiment reveal that cytochrome P450 2B4 has a helical structure in the N-terminal transmembrane region. The amino acid sequence (D) and a helical wheel representation (E) of the N-terminal transmembrane region of cytochrome P450 2B454. The full length amino acid sequence is given in the Supporting Information. Hydrophobic and hydrophilic amino acids are in black and blue, respectively. (F) A model depicting the structure and topology of cytochrome P450 in lipid bilayers; the soluble domain structure is adapted from the crystal structure for amino acid residues 28–491444546. The transmembrane structure of residues 1–27, obtained using the structure assembly simulation, I-TASSER55, is shown in the lipid bilayer region; the structure obtained with the highest C-score55 was chosen. The transmembrane domain may not be a straight α-helix due to the presence of Gly residues. This result is consistent with the imperfect wheel-like pattern of resonances in the 2D HIMSELF spectrum shown in Figure 3(C).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3757361&req=5

f3: The use of temperature resistant bicelles at lower temperatures enables the measurement of the transmembrane structure and topology of the functional form of microsomal cytochrome P450 2B4.(A) A UV-Vis spectrum shows that the overall folding of the catalytic site of a heat-sensitive membrane protein, cytochrome P450 2B4, can be stabilized in the functional form using temperature resistant bicelles, composed of DLPC and DHPC. (B) One-dimensional cross-polarization experiments of 100 μl of magnetically-aligned DLPC/DHPC bicelles (q = [DLPC]/[DHPC] = 4.0) containing a 0.61 mM of a uniformly-15N-labeled cytochrome P450 2B4 shows anisotropic 15N chemical shifts at 15°C. (C) Two-dimensional HIMSELF experiment reveal that cytochrome P450 2B4 has a helical structure in the N-terminal transmembrane region. The amino acid sequence (D) and a helical wheel representation (E) of the N-terminal transmembrane region of cytochrome P450 2B454. The full length amino acid sequence is given in the Supporting Information. Hydrophobic and hydrophilic amino acids are in black and blue, respectively. (F) A model depicting the structure and topology of cytochrome P450 in lipid bilayers; the soluble domain structure is adapted from the crystal structure for amino acid residues 28–491444546. The transmembrane structure of residues 1–27, obtained using the structure assembly simulation, I-TASSER55, is shown in the lipid bilayer region; the structure obtained with the highest C-score55 was chosen. The transmembrane domain may not be a straight α-helix due to the presence of Gly residues. This result is consistent with the imperfect wheel-like pattern of resonances in the 2D HIMSELF spectrum shown in Figure 3(C).
Mentions: Though many crystal structures of the soluble domain of cyt-P450 have been reported in the literature, high-resolution structure of the full-length protein is unknown. The N-terminal 60-residues segment containing the hydrophobic domain is usually cleaved off to obtain a single crystal for structural studies by X-ray crystallography44454647. In addition, the full-length protein is quite unstable and highly sensitive to heat. Therefore, the thermally stable magnetic-alignment of DLPC/DHPC bicelles is a breakthrough to investigate the structure of a heat-sensitive full-length membrane-bound protein like cytochrome P450 2B4 in fluid lamellar phase lipid bilayers. To optimize the experimental conditions, we first confirmed the functional folding of the catalytic site of cytochrome P450 2B4 by performing carbon monoxide assays and also tested the stability of the protein in these bicelles for various temperatures. As shown in Figure 3(A), these assays indicated that the temperature resistant bicelles retained the stable native structural folding of cytochrome P450 in a biologically active state (P450 instead of P420) under NMR sample conditions. Using this functional form, the transmembrane structure and topology of cytochrome P450 2B4 are then determined using solid-state NMR experiments on magnetically-aligned DLPC/DHPC bicelles containing a uniformly-15N-labeled-P450.

Bottom Line: Though the importance of high-resolution structure and dynamics of membrane proteins has been well recognized, optimizing sample conditions to retain the native-like folding and function of membrane proteins for Nuclear Magnetic Resonance (NMR) or X-ray measurements has been a major challenge.While bicelles have been shown to stabilize the function of membrane proteins and are increasingly utilized as model membranes, the loss of their magnetic-alignment at low temperatures makes them unsuitable to study heat-sensitive membrane proteins like cytochrome-P450 and protein-protein complexes.Our results reveal that the N-terminal region of rabbit cytochrome-P4502B4, that is usually cleaved off to obtain crystal structures, is helical and has a transmembrane orientation with ~17° tilt from the lipid bilayer normal.

View Article: PubMed Central - PubMed

Affiliation: Biophysics and Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055, USA.

ABSTRACT
Though the importance of high-resolution structure and dynamics of membrane proteins has been well recognized, optimizing sample conditions to retain the native-like folding and function of membrane proteins for Nuclear Magnetic Resonance (NMR) or X-ray measurements has been a major challenge. While bicelles have been shown to stabilize the function of membrane proteins and are increasingly utilized as model membranes, the loss of their magnetic-alignment at low temperatures makes them unsuitable to study heat-sensitive membrane proteins like cytochrome-P450 and protein-protein complexes. In this study, we report temperature resistant bicelles that can magnetically-align for a broad range of temperatures and demonstrate their advantages in the structural studies of full-length microsomal cytochrome-P450 and cytochrome-b5 by solid-state NMR spectroscopy. Our results reveal that the N-terminal region of rabbit cytochrome-P4502B4, that is usually cleaved off to obtain crystal structures, is helical and has a transmembrane orientation with ~17° tilt from the lipid bilayer normal.

Show MeSH
Related in: MedlinePlus