N-terminal (N<super>α</super>) protein acetylation, one of the most common post-translational modifications in eukaryotes, plays a pivotal role in the stability, activity and targeting of certain proteins (Chapter 1). This protein modification is significantly less frequent in prokaryotes. In <italic>Escherichia coli</italic>, the only N<super>α</super>-acetyltransferases identified are RimI, RimJ, and RimL, which acetylate the ribosomal proteins S18, S5 and L7/L12, respectively. Although most eukaryotic proteins are not acetylated when ectopically expressed in <italic>E. coli</italic>, partial or complete N<super>α</super>-acetylation has been reported for several recombinant proteins. Just recently, it was demonstrated that N<super>α</super>-acetylation of the thymosin α1 fusion proteins is catalyzed by RimJ. For most other proteins, however, the underlying mechanism of N<super>α</super>-acetylation remains unknown.
We recently observed that the Z-domain protein, a small three-helix bundle protein derived from the <italic>Staphylococcal</italic> protein A, is N<super>α</super>-acetylated only under certain conditions. We decided to use the Z-domain as a model protein to study the N<super>α</super>-acetylation in <italic>E. coli</italic>. We revealed that the N<super>α</super>-acetylation of the Z-domain depends on the <italic>E. coli</italic> strains, expression vectors and amino acid residues near the N-terminus, and is enhanced by high cellular levels of RimJ (Chapter 2). In order to systematically study the sequence dependence of the N-terminal methionine cleavage and RimJ-mediated N<super>α</super>-acetylation in <italic>E. coli</italic>, the Z-domain variants differing by the second or third amino acid residue were expressed and analyzed by mass spectrometry (Chapter 3). The initiating methionine residue of the Z-domain was removed only when a small and uncharged amino acid residue was in the second position. Only subsequent to the cleavage of the initiating methionine residue, the RimJ-catalyzed N-terminal acetylation mainly occurred at the N-terminal serine and threonine residues and was significantly enhanced by a hydrophobic or negatively charged residue in the penultimate position.
Although primarily used for analysis of N-terminal acetylation, mass spectrometry often requires careful sample preparation and expensive instrumentation. Therefore, in order to find a simple and sensitive method to analyze the acetylation status of proteins, we developed a fluorogenic derivatization method using 4-chloro-7-nitrobenzofurazan (NBD-Cl) (Chapter 4). The unacetylated protein selectively reacted with NBD-Cl at neutral pH to provide high fluorescence. In contrast, the N<super>α</super>-acetylated protein was essentially non-fluorescent under the same conditions despite the presence of many internal lysine residues. This method should be particularly useful for a large scale high-throughput proteomic analysis of protein N<super>α</super>-acetylation.
| Identifer | oai:union.ndltd.org:TCU/oai:etd.tcu.edu:etd-12062012-095056 |
| Date | 06 December 2012 |
| Creators | Bernal Perez, Lina Fernanda |
| Contributors | Youngha Ryu, Sergei V Dzyuba, Jean-Luc Montchamp, Giridhar R Akkaraju |
| Publisher | Texas Christian University |
| Source Sets | Texas Christian University |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf, application/octet-stream |
| Source | http://etd.tcu.edu/etdfiles/available/etd-12062012-095056/ |
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