pH dependent conformational changes of the catalytic domain of deubiquitinase OTU1 from Saccharomyces cerevisiae
| dc.contributor.author | Cadet, Herby | |
| dc.contributor.examiningcommittee | Schreckenbach, Georg (Chemistry) | en_US |
| dc.contributor.examiningcommittee | Bay, Denice (Medical Microbiology) | en_US |
| dc.contributor.supervisor | Khajehpour, Mazdak (Chemistry) O'Neil, Joe (Chemistry) | en_US |
| dc.date.accessioned | 2020-09-09T16:10:01Z | |
| dc.date.available | 2020-09-09T16:10:01Z | |
| dc.date.copyright | 2020-08-25 | |
| dc.date.issued | 2020-08-25 | en_US |
| dc.date.submitted | 2020-08-26T04:21:44Z | en_US |
| dc.degree.discipline | Chemistry | en_US |
| dc.degree.level | Master of Science (M.Sc.) | en_US |
| dc.description.abstract | Proteins often consist of linked domains which are regions of a protein sequence that can fold independently of each other. Domain dynamics are known to play an important role in enabling the formation of large proteins but there has not been much research done into how domains may affect protein folding and enzymatic activity. To gain insight into domain dynamics we will study the catalytic domain ScOTU1 of a model multi-domain deubiquitinase from yeast called OTU1 which catalyzes the breakdown of ubiquitin iso-peptide linkages. ScOTU1 was successfully overexpressed in BL21-DE3 cells and purified using a nickel column where it then underwent a rapid buffer exchange using a gel filtration column. ScOTU1 was studied utilizing two experiments a Ubiquitin-Rhodamine catalytic cleavage assay and a Bis-ANS hydrophobicity assay. Through cleavage of Ubiquitin-Rhodamine it was possible to measure the kcat/Km specificity constant which showed significant activity at pH ranges from eight to seven. The Bis-ANS experiments showed that the ScOTU1 catalytic domain existed in two conformations that were shifted in a pH dependent manner. At high pH the ScOTU1 catalytic domain was present in a primarily open conformation and at low pH the ScOTU1 catalytic domain was present in a primarily closed conformation. The Ubiquitin-Rhodamine and Bis-ANS experiments provided evidence that the current model for catalytic cleavage of the iso-peptide linkages of a polyubiquitin molecule by ScOTU1 follows a conformation selection mechanism for enzyme catalysis which is a two-state mechanism where ScOTU1 is in an equilibrium between a closed and open conformation where at physiological pH the enzyme is primarily in a closed conformation. Through the binding of its polyubiquitin substrate the equilibrium can shift to enable catalysis to occur at physiological pH. | en_US |
| dc.description.note | October 2020 | en_US |
| dc.identifier.uri | http://hdl.handle.net/1993/35027 | |
| dc.language.iso | eng | en_US |
| dc.rights | open access | en_US |
| dc.subject | deubiquitinase | en_US |
| dc.subject | ubiquitin | en_US |
| dc.subject | conformational selection | en_US |
| dc.title | pH dependent conformational changes of the catalytic domain of deubiquitinase OTU1 from Saccharomyces cerevisiae | en_US |
| dc.type | master thesis | en_US |
| local.subject.manitoba | yes | en_US |