Applications of ultrafast physics to structural biology reveal the complex dance of molecular "coherence" with unprecedented clarity. Understanding how molecules respond to stimuli such as light, for example during photosynthesis, is fundamental to biology. Scientists have been working to uncover how these changes operate in multiple areas, and by bringing two of these areas together, researchers are paving the way for a new era of understanding the molecular reactions of proteins crucial to life.
A large international research team led by Professor Jasper van Thor from the Department of Life Sciences at Imperial College London recently reported their findings in the journal Nature Chemistry.
Crystallography is a powerful technique in structural biology that takes "snapshots" of how molecules are arranged. After several large-scale experiments and years of theoretical research, the team behind the new study combined this technique with another technique for mapping the vibrations of a molecule's electronic and nuclear configurations, known as spectroscopy.
Demonstrating the new technique at powerful X-ray laser facilities around the world, the team showed that when the molecules in the proteins they studied were optically excited, their initial movements were the result of "coherence." This suggests that this is a vibrational effect rather than subsequent movement of functional parts of the biological response.
This important difference, shown experimentally for the first time, highlights how spectral physics can shed new light on the classic crystallographic methods of structural biology.
Professor Van Tol said: "Every process that sustains life is carried out by proteins, but to understand how these complex molecules do their job it is necessary to understand the arrangement of their atoms and how this structure changes during reactions. Using spectroscopy methods we can now see directly in the form of images by solving their crystal structures "We now have the tools to understand and even control molecular dynamics at extremely fast timescales approaching atomic resolution. We hope that by sharing the methodological details of this new technique, we will encourage researchers in the field of time-resolved structural biology as well as ultrafast laser spectroscopy to explore the crystal structure of coherent processes."
Technology combination
Combining these technologies requires the use of X-ray free electron laser (XFEL) facilities, including the Linac Coherent Light Source (LCLS) in the United States, the SPring-8 Angstrom Compact Free Electron Laser (SACLA) in Japan, PAL-XFEL in South Korea and, most recently, the European XFEL in Hamburg.
Members of the team have been working at XFEL since 2009, harnessing and understanding the motion of reactive proteins on the femtosecond (billionth of a second) time scale, known as femtosecond chemistry. After excitation with a laser pulse, X-rays are used to take a "snapshot" of the structure.
In 2016, the technology achieved initial success, describing in detail the changes that occur in biological proteins induced by light. However, the researchers still need to solve a key question: Where does the tiny molecular "movement" on the femtosecond time scale originate directly after the first laser light pulse? Previous research has assumed that all movements correspond to biological responses, that is, their functional movements. But using the new method, the team found in experiments that this was not the case.
coherent control
To come to this conclusion, they created "coherence control" - shaping laser light to control the movement of proteins in a predictable way. After initial success at Stanford's LCLS in 2018, to check and validate the approach, they conducted a total of six experiments at XFEL facilities around the world, each time forming large teams and forming international collaborations. They then combined these experimental data with theoretical methods modified from droplet chemistry so that they could be applied to X-ray crystallographic data rather than spectroscopic data.
The conclusion is that ultrafast motions measured precisely on picometer and femtosecond time scales do not belong to biological reactions but to the vibrational coherence of the remaining ground state. This means that the molecules "left over" after the femtosecond laser pulse dominate the subsequently measured motion, but only within the so-called vibrational coherence time.
Professor Van Thor said: "We conclude that in our experiments, even without including coherence control, conventional time-resolved measurements are actually dominated by motions from the dark 'reactant' ground state that are not related to light-induced biological reactions. Instead, these motions are opposed to those measured by conventional vibrational spectroscopy "