A reporter system that discriminates EF-hand-sensor motifs from signal-modulators at the single-motif level |
| |
Authors: | Joel Osuna Humberto FloresPaul Gaytán |
| |
Institution: | Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de, Mexico |
| |
Abstract: | The T-protein is a single-polypeptide bi-functional enzyme composed of a chorismate mutase domain fused to a prephenate dehydrogenase domain (TyrA). We replaced the chorismate mutase domain with canonical or pseudo-Ca2+-binding motifs (EF-hand). Canonical-EF-hand-motifs differentiate from pseudo-EF-hand-motifs by experimenting a Ca2+-dependent conformational change. The Ca2+-free EF-hand-TyrA fusion-proteins showed TyrA activity at the T-protein level. Canonical-EF-hand-TyrA fusions showed a Ca2+-dependent loss of TyrA activity, but a pseudo-EF-hand-TyrA fusion showed high TyrA activity level in excess-Ca2+ conditions. Because TyrA activity exhibits robust changes in response to Ca2+-dependent-EF-hand conformational alterations, TyrA could be a good Ca2+-reporter enzyme. A chimeric canonical/pseudo-EF-hand strategy is proposed to confer pseudo-EF-hand motifs with a Ca2+-dependent conformational change. |
| |
Keywords: | Monofunctional TyrA Multidomain protein Protein design Site-directed mutagenesis Calcium-sensor system |
本文献已被 ScienceDirect 等数据库收录! |