An NMR method for the determination of protein binding interfaces using TEMPOL-induced chemical shift perturbations

作者：Moriya Jun; Sakakura Masayoshi; Tokunaga Yuji; Prosser R Scott; Shimada Ichio^*

来源：BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS, 2009, 1790(10): 1368-1376.

DOI：10.1016/j.bbagen.2009.06.001

• C-13 NMR
• Paramagnetic shift
• Protein-protein interaction
• Surface analysis
• TEMPOL

Background: The determination of protein-protein interfaces is of crucial importance to understand protein function and to guide the design of compounds. To identify protein-protein interface by NMR spectroscopy, C-13 NMR paramagnetic shifts induced by freely diffusing 4-hydroxy-2, 2, 6, 6-tetramethyl-piperidine-1-oxyl (TEMPOL) are promising, because TEMPOL affects distinct C-13 NMR chemical shifts of the solvent accessible nuclei belonging to proteins of interest, while C-13 nuclei within the interior of the proteins may be distinguished by a lack of such shifts.
Method: We measured the C-13 NMR paramagnetic shifts induced by TEMPOL by recording C-13-C-13 TOCSY spectra for ubiquitin in the free state and the complex state with yeast ubiquitin hydrolase1 (YUH1).
Results: Upon complexation of ubiquitin with YUH1, C-13 NMR paramagnetic shifts associated with the protein binding interface were reduced by 0.05 ppm or more. The identified interfacial atoms agreed with the prior X-ray crystallographic data.
Conclusions: The TEMPOL-induced C-13 chemical shift perturbation is useful to determine precise protein-protein interfaces. General significance: The present method is a useful method to determine protein-protein interface by NMR, because it has advantages in easy sample preparations, simple data analyses, and wide applicabilities.

• 出版日期2009-10