Protein-nucleic acid interactions play essential roles in all living cells in various cellular functions. The study of these interactions can reveal important structural and functional information. UV cross-linking of nucleic acids to proteins in combination with mass spectrometry is a powerful technique to identify proteins, peptides and the amino acids involved in intermolecular interactions within nucleic acid-protein complexes. However, the mass spectrometric identification of cross-linked nucleic acid-protein heteroconjugates in complex mixtures and MS/MS characterisation of the specific sites of cross-linking is a challenging task. In this investigation, novel tools and methods have been developed for the investigation of DNA-protein interactions using UV cross-linking and mass spectrometry. These tools were developed towards their application for the characterisation of the complex between the eukaryotic DNA repair protein Tyrosyl-DNA phosphodiesterase 1 (Tdp1) and its DNA substrates. DNA-Tdp1 UV cross-linking was optimised using purified recombinant human Tdp1 and radioactively labelled DNA oligonucleotides containing UV photoactivatable 4- thio-thymidine or 5-iodouracil. Tdp1-DNA heteroconjugates were detected by SDS PAGE and Phosphorimaging. In order to analyse the DNA-Tdp1 heteroconjugates by mass spectrometry, they must first be enriched and hydrolysed by a protease and a nuclease. Here, a novel sample preparation protocol was developed for the enrichment of Tdp1 oligonucleotide-peptide heteroconjugates. Detection and analysis of oligonucleotide-peptide heteroconjugates using mass spectrometry is a challenging task. As a tool to optimise the various parameters involved, a synthetic DNA oligonucleotide-peptide heteroconjugate was constructed using click chemistry. RP-HPLC/ESI-FT-ICR-MS on a Bruker 12T SolariX in conjunction with CID fragmentation was used to unambiguously identify the site of the cross-link. Lastly, a novel 18O labeling approach was introduced to facilitate the identification of DNA-protein cross-links. This approach was shown to be suitable for the labeling of heteroconjugate species by testing it with the click heteroconjugate.

The concept of 3D proteomics is a technique that couples chemical cross-linking with mass spectrometry and has emerged as a tool to study protein conformations and protein-protein interactions. In this thesis I present my work on improving the analytical workflow and developing applications for 3D proteomics in the structural analysis of proteins and protein complexes through four major tasks. I. As part of the technical development of an analytical workflow for 3D proteomics, a cross-linked peptide library was created by cross-linking a mixture of synthetic peptides. Analysis of this library generated a large dataset of cross-linked peptides. Characterizing the general features of cross-linked peptides using this dataset allowed me to optimize the settings for mass spectrometric analysis and to establish a charge based enrichment strategy for cross-linked peptides. In addition to this, 1185 manually validated high resolution fragmentation spectra gave an insight into general fragmentation behaviours of cross-linked peptides and facilitated the development of a cross-linked peptide search algorithm. II. The advanced 3D proteomics workflow was applied to study the architecture of the 670 kDa 15-subunit Pol II-TFIIF complex. This work established 3D proteomics as a structure analysis tool for large multi-protein complexes. The methodology was validated by comparing 3D proteomics analysis results and the X-ray crystallographic data on the 12-subunit Pol II core complex. Cross-links observed from the Pol II–TFIIF complex revealed interactions between the Pol II and TFIIF at the peptide level, which also reflected the dynamic nature of Pol II-TFIIF structure and implied possible Pol II conformational changes induced by TFIIF binding. III. Conformational changes of flexible protein molecules are often associated with specific functions of proteins or protein complexes. To quantitatively measure the differences between protein conformations, I developed a quantitative 3D proteomics strategy which combines isotope labelling and cross-linking with mass spectrometry and database searching. I applied this approach to detect in solution the conformational differences between complement component C3 and its active form C3b in solution. The quantitative cross-link data confirmed the previous observation made by X-ray crystallography. Moreover, this analysis detected the spontaneous hydrolysis of C3 in both C3 and C3b samples. The architecture of hydrolyzed C3-C3(H2O) was proposed based on the quantified cross-links and crystal structure of C3 and C3b, which revealed that C3(H2O) adopted the functional domain arrangement of C3b. This work demonstrated that quantitative 3D proteomics is a valuable tool for conformational analysis of proteins and protein complexes. IV. Encouraged by the achievements in the above applications with relatively large amounts of highly purified material, I explored the application of 3D proteomics on affinity purified tagged endogenous protein complexes. Using an on-beads process which connected cross-linking and an affinity purification step directly, provided increased sensitivity through minimized sample handling. A charge-based enrichment step was carried out to improve the detection of cross-linked peptides. The occurrence of cross-links between complexes was monitored by a SILAC based control. Cross-links observed from low micro-gram amounts of single-step purified endogenous protein complexes provided insights into the structural organization of the S. cerevisiae Mad1-Mad2 complex and revealed a conserved coiled-coil interruption in the S. cerevisiae Ndc80 complex. With this endeavour I have demonstrated that 3D proteomics has become a valuable tool for studying structure of proteins and protein complexes.

3D proteomics combines chemical cross-linking with mass spectrometry to study the structure of protein assemblies and protein-protein interactions both in vitro and in vivo by providing distance constraints that indicate which residues are in close spatial proximity. I addressed the main bottleneck of this technology: the reliable identification of cross-linked peptides. Reporter ion signatures for cross-linked peptides were developed, by fragmenting model compounds containing two lysine residues joined by a cross-linker backbone or a lysine residue modified with a hydrolysed cross-linker. The reporter ion signatures showed 97% specificity at 90% sensitivity and segregated cross-linked from modified and linear peptides. They decreased the false discovery rate of the identification of cross-linked peptides from 5% to 1% in a large dataset. The signatures permit data sorting during and after mass spectrometry acquisition. The advanced 3D proteomics workflow was applied to study the protein-protein interactions in Mycoplasma pneumoniae cells. In lysates of the bacterium we identified 128 protein-protein interactions (of which 24 are novel) and obtained in vivo topological data on 208 proteins, even for cases where high-resolution structures are not yet available. We showed that our data are in excellent agreement with crystal structures of proteins and complexes where available. We defined a network of ribosomal and RNA polymerase proteins that reveals an intricate link between transcription and translation in bacteria. We demonstrated that the method is suitable for identification of homomultimeric protein complexes by exploiting peptide pairs of identical amino acid sequence. The technology has the potential to provide a complete protein interaction network map after the selective enrichment of cross-lined peptides is achieved. The method was next applied to investigate the structure of condensin and cohesin complexes, which play a crucial role in stabilization of chromosome structure during mitosis. The complexes were purified, cross-linked and their linkage map created. The condensin coiled coil cross-linked on the entire length was modeled. The information was used to direct the analysis of in situ cross-linked condensin in intact chromosomes. I found two high confidence linkages between SMC2 and SMC4 coiled coils and identified H2A as a potential condensin receptor on chromosomes.

A recent shift towards proteomics has seen many structural genomics initiatives set up for high-throughput structure determination using traditional methods of x-ray crystallography and NMR. The next step in the proteomic revolution focuses on the interplay of multi-protein complexes and transient protein-protein interactions, which are involved in many cellular functions. Greater understanding of protein-protein interactions will inevitably lead to better comprehension of the regulation of cellular process, which has implications in biomedical sciences and biotechnology. Even though many high-resolution initiatives focus on proteins and protein complexes, their structure-determination success rates are still low. An emerging approach uses chemical cross-linking and mass spectrometry to derive a set of sparse distance constrains, which can be used for building models of proteins and to map out residues in protein interaction interface based on partial structural information. This technique allows low-resolution identification of protein structures and their interactions in cases where traditional structure determination techniques did not produce results. Chemical cross-linkers have been successfully used for many years in identifying interacting proteins. However, recent advances in mass spectrometry have allowed the identification of exact insertion points of low-abundance cross-links and hence has opened up a new perspective on the use of cross-linkers in combination with computational structure prediction. For protein interaction studies, the approach uses chemical cross-linking information with molecular docking, so that the cross-links are treated as explicit constraints in the calculations. This study focuses on a low-cost and rapid approach to structure prediction, where partial structural information and distance constraints can be used to obtain the relative orientation of interacting proteins and domains, specifically as a rescue strategy where traditional high resolution structure determination methods were unsuccessful. This hybrid biochemical/bioinformatics approach was applied in the determination of structure of the latexin:carboxypeptidase A complex, and succeeded in achieving 4 Å rmsd compared to the crystal structure determined subsequently (Mouradov et al., 2006). Application of the bioinformatics/biochemical approach to multi-domain proteins was carried out on murine acyl-CoA thioesterase 7 (Acot7). X-ray crystallography provided structures of the two separate domains of Acot7, however the full length protein did not crystalise. Combining chemical cross-linking, mass spectrometry, molecular docking and homology modeling we were able to reconstruct how the two domains are arranged in the full length protein (Forwood et al., 2007). Limitations of this technique caused by the enormous complexity of the cross-linking reaction mixtures were identified and emphasized by analysing a large (four protein) complex of DNA polymerase III, where only one inter-protein cross-link was identified. A rapid and cost-effective method for identification of cross-linked peptides using a commercially available cross-linker was developed as part of the overall aim of streamlining the hybrid biochemical/bioinformatics in order for it to become a generally applicable technique for rapid protein structure characterisation (King et al., 2008). Finally an in-house software package was developed for assignment of cross-linked peptides based on m/z values.

A recent shift towards proteomics has seen many structural genomics initiatives set up for high-throughput structure determination using traditional methods of x-ray crystallography and NMR. The next step in the proteomic revolution focuses on the interplay of multi-protein complexes and transient protein-protein interactions, which are involved in many cellular functions. Greater understanding of protein-protein interactions will inevitably lead to better comprehension of the regulation of cellular process, which has implications in biomedical sciences and biotechnology. Even though many high-resolution initiatives focus on proteins and protein complexes, their structure-determination success rates are still low. An emerging approach uses chemical cross-linking and mass spectrometry to derive a set of sparse distance constrains, which can be used for building models of proteins and to map out residues in protein interaction interface based on partial structural information. This technique allows low-resolution identification of protein structures and their interactions in cases where traditional structure determination techniques did not produce results. Chemical cross-linkers have been successfully used for many years in identifying interacting proteins. However, recent advances in mass spectrometry have allowed the identification of exact insertion points of low-abundance cross-links and hence has opened up a new perspective on the use of cross-linkers in combination with computational structure prediction. For protein interaction studies, the approach uses chemical cross-linking information with molecular docking, so that the cross-links are treated as explicit constraints in the calculations. This study focuses on a low-cost and rapid approach to structure prediction, where partial structural information and distance constraints can be used to obtain the relative orientation of interacting proteins and domains, specifically as a rescue strategy where traditional high resolution structure determination methods were unsuccessful. This hybrid biochemical/bioinformatics approach was applied in the determination of structure of the latexin:carboxypeptidase A complex, and succeeded in achieving 4 Å rmsd compared to the crystal structure determined subsequently (Mouradov et al., 2006). Application of the bioinformatics/biochemical approach to multi-domain proteins was carried out on murine acyl-CoA thioesterase 7 (Acot7). X-ray crystallography provided structures of the two separate domains of Acot7, however the full length protein did not crystalise. Combining chemical cross-linking, mass spectrometry, molecular docking and homology modeling we were able to reconstruct how the two domains are arranged in the full length protein (Forwood et al., 2007). Limitations of this technique caused by the enormous complexity of the cross-linking reaction mixtures were identified and emphasized by analysing a large (four protein) complex of DNA polymerase III, where only one inter-protein cross-link was identified. A rapid and cost-effective method for identification of cross-linked peptides using a commercially available cross-linker was developed as part of the overall aim of streamlining the hybrid biochemical/bioinformatics in order for it to become a generally applicable technique for rapid protein structure characterisation (King et al., 2008). Finally an in-house software package was developed for assignment of cross-linked peptides based on m/z values.

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La maladie d’Alzheimer (MA) est un désordre neuro-dégénératif chronique fatal et la forme la plus répandue des démences chez l’adulte qui touchent plus de 28 millions de personnes dans le monde. En absence de traitement pour les démences neurodégénérative dont la maladie d’Alzheimer, le coût de celles-ci est estimé à 1 trillion d’USD en 2018 ce qui représente des enjeux économiques et sociétaux majeurs au niveau national et mondial. La MA est une forme d’amylose qui est caractérisée par l’agrégation du peptide amyloïde beta (Aβ) dans le cerveau des patients. Le facteur de risque génétique principal de la forme tardive (après 65 ans) de cette maladie est l’isoforme E4 de l’apolipoprotéine E (apoE) qui intervient dans le transport et le métabolisme des lipides et interagit avec le peptide Aβ. La modulation de la structure des isoformes d’apoE et de leur interaction avec l’Aβ apparaît comme une cible prometteuse dans la conception rationnelle de thérapies de la maladie. Celle-ci nécessite néanmoins une compréhension approfondie des propriétés structurales et dynamiques des deux partenaires moléculaires. Dans le cadre de cette thèse, nous avons étudié la structure de trois isoformes (E2, E3 et E4) de l’apoE par différentes techniques de biologie structurale et principalement par la réticulation chimique couplée à la spectrométrie de masse (CXMS) quantitative et par la bioinformatique structurale. Ces données complémentées par la spectroscopie infrarouge ont permis de construire des modèles structuraux de l’apoE2, E3 et E4. Nous avons mis en évidence l’interaction des domaines N- et C-terminal et la présence de multiples conformations de l’apoE chez les trois isoformes. Nos données suggèrent un équilibre entre deux principales conformations de l’apoE dont la population relative diffère entre les trois isoformes. Nous proposons les interfaces à cibler dans le cadre de thérapie visant à moduler les propriétés structurales des isoformes de l’apoE.Nous avons également mis en évidence que chaque isoforme d’apoE (E2, E3 et E4) interfère avec l’agrégation d’Aβ. Le peptide interagit avec les deux domaines N- et C- terminal de l’apoE. L’étude quantitative de l’interaction par CXMS a révélé des différences entre les cross-links formés en présence des isoformes. La modélisation du complexe apoE-Aβ a permis de mettre en évidence les interfaces impliquées dans l’interaction. Celle-ci possède une composante hydrophobe et électrostatique qui diffère chez les isoformes d’apoE. Nous proposons un mécanisme de l’interaction apoE-Aβ qui est initié par les propriétés hydrophobes des deux partenaires et qui est stabilisé par la suite via des contacts électrostatiques. Par ailleurs, une étude permettant d’explorer le potentiel de la nouvelle chimie de réticulation des résidues acides de protéine dans des applications en protéomique structurale a été effectuée. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

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