Dynamic Interaction Between Membrane-Bound Full-Length Cytochrome P450 and Cytochrome b5 Observed by Solid-State NMR Spectroscopy

Yamamoto, Kazutoshi; Dürr, U.; Xu, Jiadi; Im, Sang Choul; Waskell, Lucy; Ramamoorthy, Ayyalusamy

doi:10.1038/srep02538

Cited by 56 publications

(88 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…39 Lowering the liquid-crystalline-to-gel phase transition temperature of planar bilayers generally enhances the stabilization of the biceller phase, as reported previously. 26 (Figure 4). 36 On the basis of the optimization of DDPC based bicelles, we found that DHepPC is an excellent detergent to improve the stability of bicelles that can render an increased range of temperatures for which DDPC:DHexPC bicelles magnetically align and therefore are well suited for solid-state NMR structural studies of membrane-associated proteins.…”

Section: ■ Results and Discussionmentioning

confidence: 93%

Temperature-Resistant Bicelles for Structural Studies by Solid-State NMR Spectroscopy

et al. 2015

Self Cite

View full text Add to dashboard Cite

Three-dimensional structure determination of membrane proteins is important to fully understand their biological functions. However, obtaining a high-resolution structure has been a major challenge mainly due to the difficulties in retaining the native folding and function of membrane proteins outside of the cellular membrane environment. These challenges are acute if the protein contains a large soluble domain, as it needs bulk water unlike the transmembrane domains of an integral membrane protein. For structural studies on such proteins either by nuclear magnetic resonance (NMR) spectroscopy or X-ray crystallography, bicelles have been demonstrated to be superior to conventional micelles, yet their temperature restrictions attributed to their thermal instabilities are a major disadvantage. Here, we report an approach to overcome this drawback through searching for an optimum combination of bicellar compositions. We demonstrate that bicelles composed of 1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC) and 1,2-diheptanoyl-sn-glycero-3-phosphocholin (DHepPC), without utilizing additional stabilizing chemicals, are quite stable and are resistant to temperature variations. These temperature-resistant bicelles have a robust bicellar phase and magnetic alignment over a broad range of temperatures, between -15 and 80 °C, retain the native structure of a membrane protein, and increase the sensitivity of solid-state NMR experiments performed at low temperatures. Advantages of two-dimensional separated-local field (SLF) solid-state NMR experiments at a low temperature are demonstrated on magnetically aligned bicelles containing an electron carrier membrane protein, cytochrome b5. Morphological information on different DDPC-based bicellar compositions, varying q ratio/size, and hydration levels obtained from (31)P NMR experiments in this study is also beneficial for a variety of biophysical and spectroscopic techniques, including solution NMR and magic-angle-spinning (MAS) NMR for a wide range of temperatures.

show abstract

Section: ■ Results and Discussionmentioning

confidence: 93%

Temperature-Resistant Bicelles for Structural Studies by Solid-State NMR Spectroscopy

et al. 2015

Self Cite

View full text Add to dashboard Cite

show abstract

“…6,11 Moreover, CYP activity is dependent on the presence of certain redox partners, which attach to the membrane via transmembrane helices. 12,13 Thus, it is important to gain a deeper understanding of the role of membranes on the abovementioned processes.…”

Section: Introductionmentioning

confidence: 99%

Effect of Cholesterol on the Structure of Membrane-Attached Cytochrome P450 3A4

Navrátilová

Paloncýová

Kajšová

et al. 2015

J. Chem. Inf. Model.

View full text Add to dashboard Cite

Cholesterol is a widely researched component of biological membranes that significantly influences membrane properties. Human cytochrome P450 3A4 (CYP3A4) is an important drug-metabolizing enzyme, wherein the catalytic domain is attached to a membrane by an N-terminal α-helical transmembrane anchor. We analyzed the behavior of CYP3A4 immersed in a 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) membrane with various amounts of cholesterol. The presence of cholesterol caused ordering and thickening of the membrane and led to greater immersion and inclination of CYP3A4 toward the membrane. Cholesterol also lowered the flexibility of and tended to concentrate around membrane-immersed parts of CYP3A4. Further, the pattern of the CYP3A4 active-site access channels was altered in the presence of cholesterol. In summary, cholesterol in the membrane affected the positioning and structural features of CYP3A4, which in turn may have implications for the activity of this enzyme in various membranes and membrane parts with different cholesterol content.

show abstract

“…For example, most membrane proteins are unstable in solution but they fold natively in a lipid bilayer environment to form stable structures. This is why studies on a membrane protein should be carried out in a near-native membrane environment to obtain physiologically meaningful information about the protein [18-20]. This is now well accepted by the structural biology community, even though some of the challenges still remain to be overcome for high-resolution studies in a membrane environment.…”

Section: Introductionmentioning

confidence: 99%

Cellular solid-state NMR investigation of a membrane protein using dynamic nuclear polarization

Yamamoto

Caporini

et al. 2015

Biochimica et Biophysica Acta (BBA) - Biomembranes

Self Cite

View full text Add to dashboard Cite

While an increasing number of structural biology studies successfully demonstrate the power of high-resolution structures and dynamics of membrane proteins in fully understanding their function, there is considerable interest in developing NMR approaches to obtain such information in a cellular setting. As long as the proteins inside the living cell tumble rapidly in the NMR timescale, recently developed in-cell solution NMR approaches can be applied towards the determination of 3D structural information. However, there are numerous challenges that need to be overcome to study membrane proteins inside a cell. Research in our laboratory is focused on developing a combination of solid-state NMR and biological approaches to overcome these challenges with a specific emphasis on obtaining high-resolution structural insights into electron transfer biological processes mediated by membrane-bound proteins like mammalian cytochrome b5, cytochrome P450 and cytochrome P450 reductase. In this study, we demonstrate the feasibility of using the signal-enhancement rendered by dynamic nuclear polarization (DNP) magic angle spinning (MAS) NMR spectroscopy for in-cell studies on a membrane-anchored protein. Our experimental results obtained from 13C-labeled membrane-anchored cytochrome b5 in native Escherichia coli cells show a ~16-fold DNP signal enhancement (ε). Further, results obtained from a 2D 13C/13C chemical shift correlation MAS experiment demonstrates that it is highly possible to suppress the background signals from other cellular contents for high-resolution structural studies on membrane proteins. We believe that this study would pave new avenues for high-resolution 3D structural studies on a variety of membrane-associated proteins and their complexes in the cellular context to fully understand their functional roles in physiological processes.

show abstract

Dynamic Interaction Between Membrane-Bound Full-Length Cytochrome P450 and Cytochrome b5 Observed by Solid-State NMR Spectroscopy

Cited by 56 publications

References 30 publications

Temperature-Resistant Bicelles for Structural Studies by Solid-State NMR Spectroscopy

Temperature-Resistant Bicelles for Structural Studies by Solid-State NMR Spectroscopy

Effect of Cholesterol on the Structure of Membrane-Attached Cytochrome P450 3A4

Cellular solid-state NMR investigation of a membrane protein using dynamic nuclear polarization

Contact Info

Product

Resources

About