Investigating The Correlation Between Protein Dynamics And Biological Activity Of Interleukin-21

The cytokine Interleukin-21(IL-21) which is produced by activated CD4+T cells, T-follicular helper cells, and natural killer T (NKT) cells, has been demonstrated to exert pleiotropic effects on the innate and adaptive immunity. The biological effects of IL-21are mediated via activation of the IL-21receptor complex. Recently, research showed that part of the structurally unstable region of hIL-21around CD loop had been replaced with the structurally more stable region of IL-4. As a result, it demonstrated to exhibit a10-fold increase in biological activity. Protein dynamics studies can disclose internal motions of a protein on different time scales, providing a better link between dynamic structures and biological functions. Nuclear Magnetic Resonance (NMR) is a powerful technique that provides information about internal dynamics associated with configurational energetics in proteins. So in this paper we optimized refolding method firstly, then the backbone resonances of Chim-hIL-21/4were assigned, at last the CPMG constant-time relaxation dispersion experiments had been successfully applied. The main contents of this paper are as follows:1. High-level expression of recombinant proteins in E.coli often resulted in forming inclusion bodies. So refolding of inclusion body proteins into bioactive forms was very important. We optimized the refolding method which helped to achieve high yield and cut down running time. One dimensional1H spectra were acquired for mIL-21samples in phosphate buffer showed that it folded into native strcture.2. The method of protein refolding was established and optimized. For NMR analysis, the protein was expressed in E. coli growing in a minimal medium with15NH4Cl and13C-glucose. Sequential backbone assignments were done by using15N-HSQC, CBCANH, CBCA(CO)NH spectra.3. Then the CPMG relaxation dispersion experiment was done at variable radio-frequency (RF) field in order to get R2values. Datas were fitted into three different models. We found out change of internal motions of Chim-hIL-21/4can lead to conformational exchange, which affected its biological activities.