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

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, ¹³C NMR paramagnetic shifts induced by freely diffusing 4-hydroxy-2, 2, 6, 6-tetramethyl-piperidine-1-oxyl (TEMPOL) are promising, because TEMPOL affects distinct ¹³C NMR chemical shifts of the solvent accessible nuclei belonging to proteins of interest, while ¹³C nuclei within the interior of the proteins may be distinguished by a lack of such shifts.

Method

We measured the ¹³C NMR paramagnetic shifts induced by TEMPOL by recording ¹³C–¹³C TOCSY spectra for ubiquitin in the free state and the complex state with yeast ubiquitin hydrolase1 (YUH1).

Results

Upon complexation of ubiquitin with YUH1, ¹³C 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 ¹³C 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.