Cytochrome-P450鈥揅ytochrome-b5 Interaction in a Membrane Environment Changes 15N Chemical Shift Anisotropy Tensors

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• 作者：Manoj Kumar Pandey ; Subramanian Vivekanandan ; Shivani Ahuja ; Rui Huang ; Sang-Choul Im ; Lucy Waskell ; Ayyalusamy Ramamoorthy
• 刊名：Journal of Physical Chemistry B
• 出版年：2013
• 出版时间：November 7, 2013
• 年：2013
• 卷：117
• 期：44
• 页码：13851-13860
• 全文大小：460K
• 年卷期：v.117,no.44(November 7, 2013)
• ISSN：1520-5207

文摘

It has been well realized that the dependence of chemical shift anisotropy (CSA) tensors on the amino acid sequence, secondary structure, dynamics, and electrostatic interactions can be utilized in the structural and dynamic studies of proteins by NMR spectroscopy. In addition, CSA tensors could also be utilized to measure the structural interactions between proteins in a protein鈥損rotein complex. To this end, we report the experimentally measured backbone amide-¹⁵N CSA tensors for a membrane-bound 16.7 kDa full-length rabbit cytochrome-b₅ (cytb₅), in complexation with a 55.8 kDa microsomal rabbit cytochrome P450 2B4 (cytP4502B4). The ¹⁵N-CSAs, determined using the ¹⁵N CSA/¹⁵N鈥?sup>1H dipolar coupling transverse cross-correlated rates, for free cytb₅ are compared with those for the cytb₅ bound to cytP4502B4. An overall increase in backbone amide-¹⁵N transverse cross-correlated rates for the cytb₅ residues in the cytb₅鈥揷ytP450 complex is observed as compared to the free cytb₅ residues. Due to fast spin鈥搒pin relaxation (T₂) and subsequent broadening of the signals in the complex, we could measure amide-¹⁵N CSAs only for 48 residues of cytb₅ as compared to 84 residues of free cytb₅. We observed a change in ¹⁵N CSA for most residues of cytb₅ in the complex, as compared to free cytb₅, suggesting a dynamic interaction between the oppositely charged surfaces of anionic cytb₅ and cationic cytP450. The mean values of ¹⁵N CSA determined for residues in helical, sheet, and turn regions of cytb₅ in the complex are 鈭?84.5, 鈭?46.8, and 鈭?46.2 ppm, respectively, with an overall average value of 鈭?65.5 ppm (excluding the values from residues in more flexible termini). The measured CSA value for residues in helical conformation is slightly larger as compared to previously reported values. This may be attributed to the paramagnetic effect from Fe(III) of the heme in cytb₅, which is similar to our previously reported values for the free cytb₅.