Breaking the cage – Implementing photocages to address spatiotemporal challenges in chemical biology

Abstract

Visualizing atoms and molecules in motion remains a formidable challenge within structural chemical biology. Significant advances in the generation of ultrashort and bright X-ray pulses have provided the technical framework to facilitate the study of complex biomolecules with atomic resolution. For the first time, it was possible to convince nature’s machinery to reveal their temporal structural dynamics by initiating native reactions in photoresponsive systems and observe the molecular dance of life by ultrafast time-resolved serial femtosecond crystallography. However, given the exceedingly small fraction of naturally photoactive proteins within nature’s diverse repertoire of proteins, the vast majority remain elusive to detailed, time-resolved structural studies.

A major objective in the forefront of structural biology is to extend the scope of time-resolved X-ray diffraction beyond light responsive proteins to include substrate dependent systems. Photocages constitute a generic method of introducing a biologically relevant substrate to its associated protein with spatiotemporal control. Numerous challenges remain, some of which are addressed in this thesis.

In this work, the native reaction between cytochrome c oxidase and oxygen released from an oxygen photocage is studied by time-resolved serial femtosecond crystallography. These are difficult experiments, both from a theoretical and technical perspective. Nevertheless, we can provide structural evidence for enzymatic turnover following the release of oxygen from the photocage. In this thesis, novel oxygen photocages and singlet oxygen responsive materials have been developed towards addressing contemporary challenges in various scientific fields. An unexpectedly successful study of structural rearrangements in a pH-responsive ion channel following acidification by a photoacid via time-resolved X-ray solution scattering further cements the scientific versatility provided by photocages in the study of temporal dynamics of proteins.