New photochemical tools for time-resolved structural studies

The current bottleneck preventing the wider application of time-resolved structural techniques is the ability to quickly and accurately trigger protein function across an ensemble of molecules either in solution or in crystallo. The fastest and most accurate approach to achieve protein function synchronisation is by using protein photo caging approaches. During this project, a new class of protein photo cleavable crosslinking reagents was designed and synthesised. This novel crosslinking scaffold carries two α-bromoacetate groups, which were shown to react with protein surface cysteines efficiently and cleanly. The crosslinker is released from the protein by UV irradiation, by photolysis of ortho-nitrobenzyl groups, cleaving the surface “staple” from the cysteine residues, leaving only a small methyl carboxylate group on the cysteine side chains. The cysteine “anchors” can be easily introduced by site-directed mutagenesis. This new set of reagents were developed as a new approach to protein photocaging which decouples the protein activation step from the protein chemistry being investigated. The photocage does not target residues directly involved in function but rather aims to restrict the conformational space explored by the protein, limiting essential dynamics and preventing function. This approach opens avenues for more widely applicable protein triggering methodology, potentially allowing for time resolved experiments to be performed in wider variety of protein classes than those investigated to date.
To test these reagents, aspartate α-decarboxylase (ADC) was chosen as a model system. This enzyme catalyses the conversion of aspartate to β-alanine, a precursor of coenzyme A. ADC is expressed as an inactive zymogen which cleaves post-translationally, yielding a catalytic pyruvoyl group. The cleavage requires an additional activating partner, PanZ. Protein homogeneity is an essential requirement for a successful time resolved experiment and, therefore, the mechanism by which PanZ activates ADC was investigated. The crystal structure of the ADC-PanZ protein-protein complex was determined at high-resolution, as well as those of several ADC mutants. With the aid of complementary techniques (SAXS, ITC, MS, NMR, in cellulo studies) the molecular mechanism by which this protein-protein interaction promotes ADC activation was elucidated. The protein complex formation causes the formation of a strained, activation-competent conformation of the ADC peptide backbone at the site of cleavage. Furthermore, a novel negative feedback mechanism for pantothenate and CoA biosynthesis regulation in bacteria was discovered and proposed.