Design and synthesis of protein chemical crosslinkers: a modular approach

The study of protein structure and interactions is pivotal in understanding the function and malfunction of complex biological systems. The structures of some proteins are unable to be determined using traditional high resolution biophysical techniques, requiring the development of amenable low resolution alternatives. Chemical Crosslinking Mass Spectrometry (CXMS) is one technique which can be used to probe protein structure through the formation of covalent linkages between protein residues. The formation of these links is facilitated by chemical crosslinking reagents. Widespread use of the CXMS technique has been hampered primarily by analytical challenges pertaining to the detection and identification of crosslinked species using Mass Spectrometry (MS). Attempts to mitigate the challenges have been made by modifying the structure of chemical crosslinkers through the addition of functional groups such as affinity tags, isotope labels and cleavable bonds. Crosslinkers combining more than one type of functional group (combination crosslinkers) present the most promising targets for CXMS applications, combining the benefits of each functional group. However, combination crosslinkers are not commercially available, thus necessitating in-house synthesis. Incorporating more than one functionality also results in more complex molecular structures and synthetic processes, making the crosslinkers difficult to adapt to suit a particular experiment. Consequently, the use of combination crosslinkers has been limited to date to a small number of studies. The research presented in this thesis describes the development of a modular chemical crosslinker design and corresponding synthetic protocol for the synthesis of combination crosslinkers. The modular crosslinker structure can be readily modified to include a range of functional groups using a small number of different reactions, including amide coupling and O-alkylation, and commercially available starting materials such as Boc-serine, from a minimum of five synthetic steps. The utility of the synthetic process was validated through the synthesis of a crosslinker containing an alkyne functional group, which can be used to attach a biotin affinity tag through alkyne-azide Huisgen Cyclisation. Synthesis of two custom designed combination crosslinkers utilising alkyne tags and cleavable bonds is also described. The function of the cleavable bonds was established using collision induced dissociation processes within the mass spectrometer. Ensuring that a crosslinker is effective in probing quaternary structure and protein- protein interactions is essential as the investigation of these structures is a major goal of CXMS. Therefore, a crosslinking assay using Staphylococcus aureus biotin protein ligase, which forms homodimers when substrate bound, was also developed using the commercially available crosslinkers Disuccinimidyl Suberate (DSS) and Dithiobis(succinimidyl) Propionate (DSP), to enable the efficacy of crosslinkers synthesised using the modular synthetic protocol to be determined.Thesis (M.Phil.) -- University of Adelaide, School of Physical Sciences, 2018