Development of photoaffinity probes to identify protein-protein interactions and map binding regions

Abstract

Proteins are involved in virtually every cellular process. Instead of acting alone, in most cases, they interact with each other and form multiprotein complexes to accomplish their biological activities. The protein-protein interactions (PPIs) within or between these protein complexes are exquisitely regulated to maintain the normal physiological state in all living cells. Therefore, characterization of the complex PPIs network is critical for the protein function annotation. Most of the PPIs rely on noncovalent interactions, which leads to the variation of binding affinity for different PPI pairs. Among these PPIs, the study of weak and transient ones is still tricky and requires great efforts. As a result, attention has been drawn to develop tools to interrogate transient PPIs in the past decades. A representative category of the transient PPIs is the PPIs mediated by protein post-translational modifications (PTMs), a process that significantly contributes to the expansion of proteome and involves in various cellular processes by altering property changes of modified proteins. In particular, histone PTM is an important class of PTMs that play essential roles in the regulation of many critical chromatin-templated processes, such as gene transcription, DNA damage repair, and chromatin assembly. In recent years, photoaffinity probes have been intensively used to identify weak transient binding proteins of histone PTMs. However, there are two limitations that have not yet been overcome: i) in theory, the photoaffinity labeling can provide valuable information regarding the binding regions between the probe and the binding proteins, but successful cases are seldom reported; ii) PTMs involving higher-order chromatin structures cannot be recapitulated by traditional peptide photoaffinity probes. In this thesis, tools to overcome these limitations have been developed. In Chapter 2, the photo-crosslinker and the alkyne handle in the traditional peptide photoaffinity probe, together with an additional cleavable moiety were integrated into a single amino acid, termed ADssC. Besides the identification of protein-protein interactions, it was demonstrated that the ADssC incorporated trifunctional (i.e., photo-crosslinkable, enrichable, and cleavable) probes can efficiently fetch the information regarding the binding regions. In Chapter 3, with the aim to overcome some envisioned limitations of the peptide-based photoaffinity probes, nucleosome-based probes for different purposes were designed. To investigate the crosstalk between DNA methylation and histone methylation, the DNA and histone H3 modified nucleosome probes were reconstituted. UHRF1 is a multidomain protein that bivalently recognizes methylated DNA and methylated lysine 9 on H3. Using the reconstituted nucleosome probes, binding preference of UHRF1 towards nucleosomes in different modification states was investigated. In Chapter 4, peptide photoaffinity probes were designed and used to study PPIs in a system other than histone PTMs. The direct binding partners of a short, essential N-terminal fragment of the human microcephaly protein CDK5RAP2 were identified by the traditional photoaffinity labeling strategy. In the future, ADssC-based trifunctional probes will be synthesized and used to obtain more information regarding the binding regions between this fragment and its binding proteins.