We suggested for the introduction of a prolyl residue into a protein that if the N-terminus residue is glycine, an unfavorable interaction in the folded state caused by the introduction of the prolyl residue can be substantially avoided by use of mutant lysozymes in which Gly-Pro and Pro-Gly sequences are introduced to positions 101-102 in the loop region of the lysozymes [Ueda, T., Tamura, T., Maeda, Y., Hashimoto, Y., Miki, T., Yamada, H., and Imoto, T. (1993) Protein Eng. 6, 183-187]. In order to determine whether or not the information obtained is applicable to other regions, we prepared mutant lysozymes with Gly-Pro and Pro-Gly sequences at positions 47, which is located in the β-sheet, positions 70-71, which are located in the loop, positions 117-118, which are located in the β-turn, and positions 121-122, which are located in the 310-helix. The free energy changes of the native and mutant lysozymes for unfolding were determined at pH 5.5 and 35°C. However, a mutant lysozyme with the Gly-Pro sequence was not always stabler than that with the Pro-Gly sequence at the same site. On the other hand, in order to determine whether or not strain caused by these sequences exists in the folded or unfolded state. The structures of these mutant lysozymes were determined by use of energy minimization. On comparison of the differenced in the free energy change between the mutant lysozymes with Gly-Pro and Pro-Gly sequences at the same site with those in their total local conformational energies, it was found there is a good correlation between them. Therefore, it was suggested that the difference in total local conformational energy caused by the introduction of a Gly-Pro or Pro-Gly sequence could be estimated by use of the energy minimized structure. Moreover, the correlation indicated that the differences in the free energy change between Gly-Pro and Pro-Gly lysozymes may be reflected by the differenced in the total local conformational energies in their folded state. It was suggested that the energy levels in the unfolded states of mutant lysozymes with Gly-Pro and Pro-Gly sequences at the same site in a Gdn-HCI solution were almost identical.
|Number of pages||7|
|Journal||Journal of biochemistry|
|Publication status||Published - Dec 1995|
All Science Journal Classification (ASJC) codes
- Molecular Biology