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Synthesis and mass spectrometric structural chracterization of ubiquitin conjugates

Synthesis and mass spectrometric structural chracterization of ubiquitin conjugates

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JUNG, Ji Eun, 2011. Synthesis and mass spectrometric structural chracterization of ubiquitin conjugates [Dissertation]. Konstanz: University of Konstanz

@phdthesis{Jung2011Synth-12841, title={Synthesis and mass spectrometric structural chracterization of ubiquitin conjugates}, year={2011}, author={Jung, Ji Eun}, address={Konstanz}, school={Universität Konstanz} }

Jung, Ji Eun 2011-05-02T10:28:34Z 2011 Synthesis and mass spectrometric structural chracterization of ubiquitin conjugates 2011-05-02T10:28:34Z Jung, Ji Eun Ubiquitin (Ub) is a small-conserved protein consisting of 76 amino acids, and plays an important role in different intracellular processes through covalently conjugation with another proteins mediated by the enzymes E1, E2 and E3. Ubiquitin is found in all eukaryotic cells as a monomer or as a isopeptide-linked polymers called polyubiquitin chains and those are attached to the substrate or to itself by a covalently interaction between the ε-amino group of an internal lysine residue in the bound ubiquitin and a glycine residue in the C-terminal of the consecutive ubiquitin moiety. Ubiquitin contains seven lysine residues (Lys6, 11, 27, 29, 33, 48 and 63) and each of these lysines can be modified for different polyubiquitin chain formation. Importantly, it appears that the specific lysine residue of ubiquitin used for ubiquitin-ubiquitin chain formation determines the biochemical and biological function, thus requiring a detailed chemical structure-function evaluation of the respective ubiquitin conjugates. For example, Lys48-linked polyubiquitin chains serve as a signal targeting the modified proteins for degradation by the 26S proteasome, while Lys63-linked polyubiquitin chains have been linked to non-proteolytic processes. So, an attractive possibility is that the different functions of different polyubiquitin chains are mediated by proteins that selectively interact with the respective chain. However, the present knowledge about the structures, biochemical activities, and biological functions of polyubiquitin chains differentiated by branching sites and isomeric structures is rather limited. So, this is to a major study due to the fact that appropriate tools including the preparation of defined polyubiquitin chains, the structural chracterization of these conjugates, and antibodies that are specific for defined polyubiquitin chains ("linkage-specific antibodies") are hitherto missing or not known and are difficult to develop.<br /><br /><br />In the first study of this thesis, a series of chemically defined polyubiquitin chains that differ by the lysine residue used for Ub-Ub conjugation was synthesized by chemical and biochemical means and characterized by high performance mass spectrometry. Advanced methods of semi-automated solid phase peptide synthesis and chemoselective ligation have been developed and successfully applied to the preparation and molecular characterization of linear ubiquitin and polyubiquitin chains. According to chemoselective ligation-based approach, the ubiquitin conjugation reaction was performed in solution, under slightly alkaline conditions, between the N-chloroacetylated ubiquitin and the ubiquitin containing one cysteine residue with a free thiol group in the sequence by thioether linkage formation. The recombinant "donor" ubiquitin in which the C-terminal amino group is replaced by a cysteine, was prepared by bacteria cell expression and the synthetic "acceptor" ubiquitin in which the N-terminal amino group is chloroacetylated was manually synthesized on TGA resin according to the Fmoc chemistry. This approach was successfully applied for the preparation of linear (mono-)ubiquitin conjugates, which will be used for moiety of branched Lys63-NH<sub>2</sub>ε-linked di-ubiquitin conjugate. In-situ simultaneous halide exchange was employed for the conversion of the N-chloroacetylated to N-iodoacetylated ubiquitin acceptor peptide for increasing reactivity in order to conjugate with recombinant His-tagged ubiquitin-Cys donor component. As an example, a linear- (mono-)ubiquitin conjugatates; ClAc-Ub (54-76) with His-tagged Ub(G53C) 11, and ClAc-Ub (61-76) with His-tagged Ub(N60C) 12, were successfully prepared, and the structure of the 10.70 kDa and 10.63 kDa ubiquitin adducts ascertained by ESI ion trap mass spectrometry. To assess the biochemical activity of the synthetic ubiquitin conjugates containing a cysteine residue; the linear (mono-)ubiquitin conjugates 11, 12 were analyzed in vitro in auto-ubiquitination assays with two different enzymes (E6-AP and HectH9). The synthetic linear ubiquitin conjugation system with efficiency similar to wild-type ubiquitin, indicates the feasibility of the mutated linear ubiquitins by thioether ligation approaches in producing biochemically active conjugates.<br /><br /><br />Based on the performance of linear (mono-)ubiquitin conjugates by chemoselective ligation, the synthesis of oligo-ubiquitin conjugates with defined Lys-ε-amino branching sites was designed and developed. The preparation of specific isomeric Lys-branched conjugates also utilizes a chemoselective ligation approach with chemically stable thioether linkages using (i), an activated N-chloroacylated partial ubiquitin acceptor peptide containing the specific lysine linkage residue, prepared by manual synthesis, and (ii), thioether ligation to a ubiquitin donor peptide that contains a C-terminal Cys residue at the defined conjugation site. According to the strategy, K63-linked di-ubiquitin conjugate 13, was successfully prepared and characterized by HPLC, circular dichroism spectroscopy and ESI ion trap MS which was determined the average molecular weight (21383.97 Da) are consistent with the correct element formula C<sub>932</sub>H<sub>1508</sub>N<sub>282</sub>O<sub>287</sub>S<sub>4</sub> by. The synthetic approach of specific Lys linked ubiquitin conjugates should be feasible as a general route for the preparation of oligo-ubiquitin conjugates that can be used (i), to evaluate the biochemical properties of isomeric poly-ubiquitin conjugates, and (ii), for the preparation of antibodies that selectively recognize different ubiquitin chains depending on the specific lysine residue of ubiquitin used for chain formation.<br /><br /><br />With the advent of recombinant DNA technology and the availability of numerous expression systems, preparation of Lys63- or Lys48- linked di-/tetra- ubiquitin chain components by recombinant Escherichia coli cell expression combination with enzymatic synthesis in vitro were efficiently available. The method involves a series of enzymatic reactions in which proximally and distally blocked monoubiquitins (or chains) are conjugated to produce a particular chain in high yield. Individual chains are then deblocked and joined in another round of reaction. To perform successful bioanalytical applications on molecular structure chracterization of different lysine linked ubiquitin conjugates, efficient and sensitive analytical methods such as a mass spectrometer with MALDI-TOF, Nano-ESI, Ion Trap, CD spectroscopy and molecular modelling were have been required. After separated by 15 % Tris-Tricine SDS-PAGE, the specific lysine linkage ubiquitin modifications were subjected by treatment of passive elution. The purified Lys48- or Lys63-linked di-/tera- ubiquitin chains were characterized by FTICR high resolution mass spectrometry, showing multiple charge distributions with experimental molecular masses of<br />K63 Di-Ub - m<sub>exp</sub> = 17157,25 Da, m<sub>calc</sub> = 17157,39 Da<br /><br />K63 Tetra-Ub - m<sub>exp</sub> = 34330,38 Da, m<sub>calc</sub> = 34326,40 Da<br /><br />K48 Di-Ub - m<sub>exp</sub> = 17201,18 Da, m<sub>calc</sub> = 17201,29 Da<br />.<br /><br />A comparative application of high resolution mass spectrometry together with CD spectroscopy has been successfully applied for the conformational differentiation of defined Lys48- and Lys63-linked di-ubiquitin conjugates, conformation-specific methods are required for structural bases distinguishing of different specific lysine linked di-ubiquitin conjugates. Ion mobility mass spectrometry (IM–MS) has been recently emerging as a highly effective tool for the separation and identification of structural transitions of ubiquitin ions in the gas phase. Investigating gas-phase structures of protein ions can lead to an improved understanding of intramolecular forces that play an important role in protein folding. Hence, the separative capacity of IMS is highly complementary to MS analysis. The structural comparison of Lys48- and Lys63-linked di-ubiquitin conjugates showed that, Lys63 linked chain is considerably more elongated than Lys48 linked chain. Collisional cross-section distributions appear identical for compact structures that exist for the +13 to +17 charge states of both conjugates, while elongated conformers showed distinct differences for the specific lysine-ε-amino linkages. Elongated conformers exist for both types of conjugates in the cross-section distributions of charge states +14 to +19, with collisional cross-sections for these ions ranging from ~3100 Å2 for [K48 di-ubiquitin +14H]<sup>14+</sup> to ~3700 Å<sup>2</sup> for [K63 di-ubiquitin +19H]<sup>19+</sup>. For the elongated charge states of conformers, cross-sections of K63 linked di-ubiquitin are on average 3.5 % larger than those of K48 linked di-ubiquitin, indicating that K48 linked conjugates adopt a more compact structure than K63 linked conjugates. The ion mobility-MS results were in full agreement with a comparative molecular modeling study of the Lys63- and Lys48- linked conjugates, suggesting a more compact Lys48 linked conformation to be required for targeting conjugated substrate proteins for proteolytic, proteasome degradation. These results of the gas phase structures are consistent with previous NMR spectroscopy data in solution phase. Characterizing the structures of proteins in the absence of solvent is suggested to be useful for (і), the comparison of gas-phase results with structural information from solution provides insight about how solvent interactions influence structure; (іі), the characterization of gas phase ion structures may become an important factor in the development of new analytical techniques for studying mixtures of proteins conformers.<br /><br />In the last part of the thesis, the binding of synthesized specific Lys linked ubiquitin conjugates to the monoclonal specific K63 linkage ubiquitin antibody (clone HWA4C4) was studied by affinity-mass spectrometry, immunoblotting methods such as dot blot and western blot. These immuno-analytical techniques were a powerful tool for characterising recognition specificities of different lysine linked ubiquitin conjugates. Affinity-MS data proved the binding of the synthetic ubiquitin peptides containing in their sequences a lysine63 residue to the K63 linkage ubiquitin antibody. The results of the dot blot test confirmed the specifically binding of the K63-Ub antibody to the partial ubiquitin peptides containing Lys6 or Lys63 attached to a Gly-Gly dipeptide. In contrast, for partial ubiquitin sequences contained the Lys11, Lys33 and Lys48 were no response observed. And also Western blot observed that the K63 linkage ubiquitin antibody gave an intense positive response only for the recombinant K63-linked di-/tetra- ubiquitin conjugates but no response for control ubiquitin and K48-linked di-ubiquitin. The comparison of the three methods provided consistent results to reveal differences in binding affinities and specificities by the K63 linkage ubiquitin antibody, showing that antibody binding is influenced by ubiquitin peptides containing in their sequences a lys63 residue and its secondary structure. For mass spectrometric epitope identification, an affinity column was prepared using the purified K63-Ub antibody. The epitope elucidation of Ub(57-69)_K63-GG peptide was accomplished by excision and extraction experiments. The results indicate that the fragments obtained in the elution fraction are underlined by an arrow, showing the overlapping epitope fragments, confirming that the epitope fragment (59-66)-GG containing Lys63 linkage site has intensive affinity for the K63-Ub antibody. Accordingly, the affinity determination of different lysine linkage ubiquitin peptides against to ubiquitin antibody and epitope identification provide a better understanding of ubiquitin antibody related to neurogenerative diseases. Furthermore, the elucidation and analysis of antigenic ubiquitin epitopes are of crucial importance for the understanding of the binding between an antibody and an antigen and will provide a starting point for the design of diagnostic applications or for the development of new vaccines. terms-of-use eng

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