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Dynamic interaction between membrane-bound full-length cytochrome P450 and cytochrome b5 observed by solid-state NMR spectroscopy.

Yamamoto K, Dürr UH, Xu J, Im SC, Waskell L, Ramamoorthy A - Sci Rep (2013)

Bottom Line: Full-length cytochrome-b5 has been shown to be important for the catalytic activity of cytochrome-P450.Functional and solid-state NMR (Nuclear Magnetic Resonance) data reveal interactions between the proteins in fluid lamellar phase bilayers.In addition, our data infer that the backbone structure and geometry of the transmembrane domain of cytochrome-b5 is not significantly altered due to its interaction with cytochrome-P450, whereas the mobility of cytochrome-b5 is considerably reduced.

View Article: PubMed Central - PubMed

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

ABSTRACT
Microsomal monoxygenase enzymes of the cytochrome-P450 family are found in all biological kingdoms, and play a central role in the breakdown of metabolic as well as xenobiotic, toxic and 70% of the drugs in clinical use. Full-length cytochrome-b5 has been shown to be important for the catalytic activity of cytochrome-P450. Despite the significance in understanding the interactions between these two membrane-associated proteins, only limited high-resolution structural information on the full-length cytochrome-P450 and the cytochromes-b5-P450 complex is available. Here, we report a structural study on a functional ~72-kDa cytochromes-b5-P450 complex embedded in magnetically-aligned bicelles without having to freeze the sample. Functional and solid-state NMR (Nuclear Magnetic Resonance) data reveal interactions between the proteins in fluid lamellar phase bilayers. In addition, our data infer that the backbone structure and geometry of the transmembrane domain of cytochrome-b5 is not significantly altered due to its interaction with cytochrome-P450, whereas the mobility of cytochrome-b5 is considerably reduced.

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Related in: MedlinePlus

Two-dimensional15N-HIMSELF spectrum of U-15N-cytb5 (black) and the complex of U-15N-cyt b5 with cyt P450 (red).Spectra were obtained using a Low-E probe30 at 900 MHz. The use of Low-E probe and higher magnetic field is advantageous for this study because the intact cyt P450 is sensitive to heat induced by the radiofrequency pulses. Other experimental parameters include 0.5 ms cross polarization contact time, 64 t1 experiments, 1024 scans, 10.24 ms acquisition time, 25 kHz SPINAL16 decoupling, and 2 s recycle delay. Total measurement time was 18.2 hours at 30°C.
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f3: Two-dimensional15N-HIMSELF spectrum of U-15N-cytb5 (black) and the complex of U-15N-cyt b5 with cyt P450 (red).Spectra were obtained using a Low-E probe30 at 900 MHz. The use of Low-E probe and higher magnetic field is advantageous for this study because the intact cyt P450 is sensitive to heat induced by the radiofrequency pulses. Other experimental parameters include 0.5 ms cross polarization contact time, 64 t1 experiments, 1024 scans, 10.24 ms acquisition time, 25 kHz SPINAL16 decoupling, and 2 s recycle delay. Total measurement time was 18.2 hours at 30°C.

Mentions: To better understand the interaction between the proteins, 2D HIMSELF242526 (Heteronuclear Isotropic Mixing leading to Spin Exchange via the Local Field) or HERSELF (HEteronuclear Rotating-frame Spin Exchange via the Local Field) spectra, which correlate15N chemical shift with 1H-15N dipolar coupling, were obtained from magnetically-aligned bicelles containing the b5-P450 complex (Figure 3). The distinct geometry of the transmembrane α-helix of cyt b5 gives rise to a circular pattern of resonances seen in the upper right region of the 2D spectrum27282930. Figure 3 also shows the resolved signals exhibiting residual1H-15N dipolar couplings associated with the soluble domain resonances in the 100–135 ppm range, which could be due to the weak alignment of this domain. These residual dipolar couplings were found to be larger in the complex than in free cyt-b5. This further confirms the interaction between the soluble domains of cyt b5 and cyt P450.


Dynamic interaction between membrane-bound full-length cytochrome P450 and cytochrome b5 observed by solid-state NMR spectroscopy.

Yamamoto K, Dürr UH, Xu J, Im SC, Waskell L, Ramamoorthy A - Sci Rep (2013)

Two-dimensional15N-HIMSELF spectrum of U-15N-cytb5 (black) and the complex of U-15N-cyt b5 with cyt P450 (red).Spectra were obtained using a Low-E probe30 at 900 MHz. The use of Low-E probe and higher magnetic field is advantageous for this study because the intact cyt P450 is sensitive to heat induced by the radiofrequency pulses. Other experimental parameters include 0.5 ms cross polarization contact time, 64 t1 experiments, 1024 scans, 10.24 ms acquisition time, 25 kHz SPINAL16 decoupling, and 2 s recycle delay. Total measurement time was 18.2 hours at 30°C.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3756332&req=5

f3: Two-dimensional15N-HIMSELF spectrum of U-15N-cytb5 (black) and the complex of U-15N-cyt b5 with cyt P450 (red).Spectra were obtained using a Low-E probe30 at 900 MHz. The use of Low-E probe and higher magnetic field is advantageous for this study because the intact cyt P450 is sensitive to heat induced by the radiofrequency pulses. Other experimental parameters include 0.5 ms cross polarization contact time, 64 t1 experiments, 1024 scans, 10.24 ms acquisition time, 25 kHz SPINAL16 decoupling, and 2 s recycle delay. Total measurement time was 18.2 hours at 30°C.
Mentions: To better understand the interaction between the proteins, 2D HIMSELF242526 (Heteronuclear Isotropic Mixing leading to Spin Exchange via the Local Field) or HERSELF (HEteronuclear Rotating-frame Spin Exchange via the Local Field) spectra, which correlate15N chemical shift with 1H-15N dipolar coupling, were obtained from magnetically-aligned bicelles containing the b5-P450 complex (Figure 3). The distinct geometry of the transmembrane α-helix of cyt b5 gives rise to a circular pattern of resonances seen in the upper right region of the 2D spectrum27282930. Figure 3 also shows the resolved signals exhibiting residual1H-15N dipolar couplings associated with the soluble domain resonances in the 100–135 ppm range, which could be due to the weak alignment of this domain. These residual dipolar couplings were found to be larger in the complex than in free cyt-b5. This further confirms the interaction between the soluble domains of cyt b5 and cyt P450.

Bottom Line: Full-length cytochrome-b5 has been shown to be important for the catalytic activity of cytochrome-P450.Functional and solid-state NMR (Nuclear Magnetic Resonance) data reveal interactions between the proteins in fluid lamellar phase bilayers.In addition, our data infer that the backbone structure and geometry of the transmembrane domain of cytochrome-b5 is not significantly altered due to its interaction with cytochrome-P450, whereas the mobility of cytochrome-b5 is considerably reduced.

View Article: PubMed Central - PubMed

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

ABSTRACT
Microsomal monoxygenase enzymes of the cytochrome-P450 family are found in all biological kingdoms, and play a central role in the breakdown of metabolic as well as xenobiotic, toxic and 70% of the drugs in clinical use. Full-length cytochrome-b5 has been shown to be important for the catalytic activity of cytochrome-P450. Despite the significance in understanding the interactions between these two membrane-associated proteins, only limited high-resolution structural information on the full-length cytochrome-P450 and the cytochromes-b5-P450 complex is available. Here, we report a structural study on a functional ~72-kDa cytochromes-b5-P450 complex embedded in magnetically-aligned bicelles without having to freeze the sample. Functional and solid-state NMR (Nuclear Magnetic Resonance) data reveal interactions between the proteins in fluid lamellar phase bilayers. In addition, our data infer that the backbone structure and geometry of the transmembrane domain of cytochrome-b5 is not significantly altered due to its interaction with cytochrome-P450, whereas the mobility of cytochrome-b5 is considerably reduced.

Show MeSH
Related in: MedlinePlus