Collaborative Research: Enzyme dynamics on biologically relevant time-scales revealed by single-particle CryoEM and X-ray crystallography

University Of Wisconsin-Milwaukee, Milwaukee WI

Investigators

Abstract

Some organisms have proteins known as phytochromes that give them the ability to sense light. Phytochromes are red-light receptors that were first discovered in plants, but are also found in fungi and photosynthetic and non-photosynthetic bacteria. Phytochromes regulate essential processes in plants such as seedling germination and shade avoidance. The soil dwelling myxobacteria, known for the production of secondary metabolites, are unique for their ability to form multicellular fruiting bodies that require light as an environmental signal. The goal of this project is to understand, at the molecular level, how phytochromes in myxobacteria are involved in a signaling cascade that induces developmental changes, such as fruiting body formation when exposed to light. Through a collaboration between the University of Wisconsin-Milwaukee and Northeastern Illinois University, graduate and undergraduate researchers at their respective institutions will work together to determine the molecular mechanism how light is perceived and how the light-signal is converted to a chemical signal by the myxobacterial phytochromes. This will be accomplished using cutting edge methods capable of recording molecular movies of the phytochromes in action. Workshops on data analysis will be organized and will be open to interested researchers. These two-day workshops will introduce modern biochemical imaging methods with near-atomic resolution to graduate and undergraduate researchers alike. Canonical bacteriophytochromes change their structures upon absorption of red-light from a red-light absorbing Pr state to a far-red light absorbing Pfr state. They contain an enzymatic domain, usually a histidine kinase (HK), whose activity is regulated by light. After red-light absorption the HK is less active and far-red light activates it. The light is absorbed by a central pigment, or chromophore, which is a heme-derived biliverdin (BV) in bacteriophytochromes. Upon binding to another protein called a response regulator, bacteriophytochromes trigger an essential two-component signaling pathway, which in turn regulates more downstream processes such as motility and gene expression. There is evidence that light absorption modulates both the HK activity and the response regulator binding. However, little is known about the molecular mechanism and how this is achieved. Evidence of a HK domain rotation has already been obtained by single particle cryo-electron-microscopy (cryoEM). With time-resolved methods that include time-resolved X-ray crystallography (TRX) and time-resolved single particle cryo-electron microscopy (TR-cryoEM), this project aims to unravel the exact sequence of structural changes after light absorption on both ultrafast and physiological time scales with near-atomic spatial resolution. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.