Concluding Remarks and Outlook

The function and activity of proteins is often modulated by other proteins they interact with. To understand cellular behaviour at the system level, a complete description of interactions among different proteins and the dynamic nature of these interactions is required.

Identification and quantification of proteins based on measured peptide intensities represents the highest standard for an unbiased and definite determination of protein interaction partners.

Inspired by the display of effectiveness of pBPA in vivo crosslinking in elucidating the mechanistic details of chromatin condensation in S. cerevisiae (Wilkins et al., 2014), the research objective of this PhD thesis was the development of a method for the quantitative identification of histone interaction partners for further decoding of chromatin structure dynamics.

First, the effectiveness of pBPA in vivo crosslinking for capturing histone-protein interactions in living yeast cells was successfully confirmed. Secondly, an immunoprecipitation protocol for pulldown of HA-tagged histone pBPA mutants and their UV-induced crosslink products was established and adapted to SILAC conditions. The targeted enrichment of crosslink products presented an obstacle during this project which could not satisfactorily be solved. Thirdly, initial SILAC analyses were performed and optimized until reproducible results of increased quality were obtained. Optimization led to the identification of numerous chromatin-related proteins, including a confirmed interaction partner. However, all candidate proteins suffered from poor ratios of light and heavy peptides, preventing distinction of their specificities.

During this project it became apparent that the concentration of crosslink products might represent the main bottleneck of this method, preventing sufficient quantification. Low protein counts per crosslink product result in low quantities of respective peptides, which increases the risk of them not being selected for MS/MS analysis.

Therefore, it is of considerable importance to increase the concentration of crosslink products per sample for an effective improvement of ion statistics.

First level of possible optimization could be the in vivo crosslinking approach. For in vivo interaction studies, pBPA is the most frequently used UV-inducible crosslinker amino acid. Although it inserts into C-H bonds within a 3.1 Å reactive radius (De Ruijter et al., 2003), it was reported that pBPA preferentially reacts with methionine’s thioether sidechain even over distances beyond 3.1 Å (Wittelsberger et al., 2006). Consequently, the crosslinking efficiency of pBPA can be altered severely when being introduced in close proximity to a methionine, potentially allowing capturing of abnormal crosslinks. Also, pBPA was shown to produce substantially smaller amounts of mutant protein in comparison to other unnatural amino acids, assumably because of reduced intracellular availability due to low solubility (Chen et al., 2007).

In order to remove these potential interference factors, other crosslinker amino acids (

Figure 1.8) with higher solubility and crosslink efficiency should be tested for increased overall yields of mutant protein and crosslink products. The aryl azide p-azido-L-phenylalanine (pAzF) has been reported to have a high incorporation efficiency, assumably by being less hydrophobic than pBPA, and produces significantly less steric hindrance due to smaller size (Chen et al., 2007; Chou et al., 2011). p-Trifluoromethyl-diazirinyl-L-phenylalanine (tmdF) possesses a higher degree of incorporation efficiency, too, as well as a higher crosslinking efficiency than pBPA at 365 nm (Hino et al., 2011). Both 3’-azibutyl-N-carbamoyl-lysine (ABK) and the pyrrolysine-derived 3-(3-methyl-3H-diazirine-3-yl)-propaminocarbonyl-Nε-L-lysine (DiZPK) have displayed higher incorporation efficiency, flexibility and crosslinking efficiency than pBPA (Ai et al., 2011; Chou et al., 2011; Hino et al., 2011; Zhang et al., 2011).

A major advantage of pBPA is its return to ground state after excitement with 365 nm light and lack of a reaction partner, as well as its capability of re-excitement. On the contrary, the photo-activation of ABK, pAzF

Second level of optimization could be the purification process. As described as in chapter 5.2, an alternative lysis procedure could help to increase the overall yield of protein. More attention should be paid to selective purification strategies such as hydrazide or hydroxylamine chemistry for the enrichment of crosslink products to effectively improve IP pulldown efficiency. Also, scaling up of cell culture quantities or higher amounts of beads for immunoprecipitation should be tested.

A third level of optimization could target the MS/MS analysis. The MS/MS setup is biased to high abundant peptides and tends to neglect their low abundant counterparts. As described as in chapter 5.3, optimization should be performed on behalf of improved peptide detection. Here, prolonged scan intervals in the MS1 scan and an optimized acquisition threshold for MS2 spectra could yield improved peptide detection and better ion statistics for quantification.

Once the SILAC ratios are unambiguous, this method will open enormous possibilities for the investigation of protein-protein interactions in vivo. Combining the demonstrated power of in vivo crosslinking for large-scale capturing of interaction partners (4.1.1) with SILAC-based MS will allow the acquisition of vast amounts of interactome data with single amino acid resolution to effectively help to elucidate biological processes.

In regard to chromatin structure, highly detailed interactomes of histone surfaces could be generated and investigated for changes upon mutation or drug administration. Binding patterns of specific proteins could be mapped and aligned with other candidates to investigate signaling cascades. Also, the addition of directed cell phase arrest would give this approach a spatio-temporal resolution and would allow the investigation of binding characteristics over the course of the cell cycle.

Histone modifications have been described to have cell phase-dependent gradient-like features, e.g., the phosphorylation of H3 S10 is first observable in the pericentromeric heterochromatin during late G2 phase. It subsequently spreads over the chromosomal arms, is completed by prophase, and remains visible during metaphase (Hendzel et al., 1997). Combining cell phase arrest at distinct phases and the absence or presence of a non-phosphorylatable H3 S10A mutation could yield the identification of numerous H3 S10 phosphorylation-dependent interaction partners and allow the development of a conclusive regulation model.

This approach would also be suited to probe other non-histone proteins (e.g., HST2), but also proteins out of chromatin context (e.g., importins, exportins).

6 Acknowledgements

First, I would like to thank Prof. Dr. Heinz Neumann for giving me the opportunity to accomplish this thesis in his workgroup and for his continuous support and supervision during my PhD studies.

I am grateful to Prof. Dr. Henning Urlaub and Prof. Dr. Matthias Dobbelstein for support and agreeing to be my co-examiners. Furthermore, I want to thank Prof. Dr. Blanche Schwappach, Prof. Dr. Steven Johnsen and Prof. Dr. Andre Fischer for participating in my extended thesis committee.

A special thanks goes to Dr. Bryan Wilkins for countless brainstorming sessions, support in writing this thesis and being an awesome person. The contribution from Corinna Krüger is acknowledged, whose help was crucial for the verification of STH1.

I would like to acknowledge all my colleagues of the Applied Synthetic Biology workgroup for the wonderful time both in and out of the lab. Your company resulted in a truly unique and pleasurable working experience.

I am grateful to the staff of the Molecular Structural Biology department of Prof. Dr. Ralf Ficner for providing a well-functioning work environment. A special thanks goes to Marita Kalck for her administrative support.

I would like to acknowledge Dr. Kuan-Ting Pan, Dr. Samir Karaca and Annika Kühn of the Bioanalytical Mass Spectrometry department of Prof. Dr. Henning Urlaub for their help with processing and analyzing my SILAC samples.

Further acknowledgements go to all my friends in Göttingen and all over the world for support and lending an ear.

I would like to thank my girlfriend Freya Gehrke for her support and invaluable help while writing this thesis.

Finally, I would like to thank my parents, Prof. Dr. Klaus Rall and Irene Rall, for their endless faith in me and their support beyond all description. Without you, I wouldn’t be the person I am today.