Proteins in crystallized form do not necessarily visualize what happens naturally

Scientists from the Laser Based Microscopy research group at VU Amsterdam’s LaserLaB have performed various measurements to characterize the complete photocycle of Photoactive Yellow Protein (PYP), with the protein in crystallized form and in solution.

08/28/2020 | 2:05 PM

The results, which were published today in Nature Communications, show that the results of time-resolved crystallography (TRC) experiments, in which the proteins are in an unnatural, crystallized state, cannot automatically be extrapolated to the way proteins work in the form in which they occur in organisms.
The LaserLaB research group, led by Marloes Groot, Professor of Biophotonics and Medical Imaging, has shown that the kinetics (the rate at which the chemical reaction takes place) of the photocycle and the structural intermediates are different in the crystals. The way proteins work in an artificial crystalline form, as used in TRC experiments, does therefore not always visualize the way they work in an organism. As Groot points out: ‘This illustrates the fact that comparative spectroscopic experiments, in which measurements can take place on proteins in both crystalline and liquid state, are needed to help identify structural intermediates under natural conditions.’
Time-resolved crystallography experiments
Time-resolved crystallography is a technique for studying minor structural changes in proteins during a reaction using X-ray scattering. TRC experiments yield clear, distinct images of proteins in action, but they require an expensive, complex research infrastructure, and the proteins need to be in an organized crystallized state, not dissolved in a watery environment. Groot adds: ‘We were therefore wondering whether TRC actually visualizes the way a protein works in natural conditions, as the unusual conditions that a crystalline protein finds itself in could affect the structural transformations.’

Photoactive Yellow Protein
To ascertain whether TRC visualizes the way a protein works in natural conditions, the researchers studied Photoactive Yellow Protein, as this is often used as a model system in TRC experiments. The research team, working with Klaas Hellingwerf, Professor in Microbiology at the University of Amsterdam, used advanced laser spectroscopy table-top techniques that enable proteins to be studied both in crystallized form and in solution. Electronic and vibrational spectroscopy were used to measure the complete reaction in the protein in both states. The measurements followed the reactions from 100 femtoseconds (a millionth of a millionth of a second) after the start of the reaction to one second, and showed differences both in the reaction kinetics and in the structures of the intermediates formed.

Image caption: The test setup