SurA is a molecular chaperone in the periplasm of E.coli and has been implicated in the maturation of outer membrane proteins (OMPs). SurA consists of four domains, but only two of them, namely the N and C-terminal domains, are necessary for chaperone function. Very little is known about which residues drive the interaction between the N and C-termini that facilitates normal activity. We mutated several conserved residues on the C-terminus and generated additive truncations to observe the effects of each on the fitness of the cell. We found one mutation E(408):A was sufficient to reduce SurA activity by 3-fold, but structural characterization of the mutated protein revealed little variation from wild-‐type SurA. Most notable, we found that when at least 10 residues are removed from the C-‐terminus, the protein is completely non-‐functional. We introduced a random peptide library to substitute these 10 residues and found that ~1.5% of all possible sequences in the library can restore SurA function to at least 50% activity. Moreover, we observed no pattern in the sequences of 26 different variants that were chosen and characterized. Here we show for the first time that SurA can tolerate many mutations at the C-terminus and still be active.
| Identifer | oai:union.ndltd.org:uky.edu/oai:uknowledge.uky.edu:chemistry_etds-1007 |
| Date | 01 January 2012 |
| Creators | Ferrell, Brent |
| Publisher | UKnowledge |
| Source Sets | University of Kentucky |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations--Chemistry |
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