CUI: Advanced Imaging of Matter
Cluster of ExcellenceCUI: Advanced
Imaging of Matter
Imaging of Matter
Watching protein cross-linkers break – and reform remarkably fast
13 November 2024
Photo: Jessica Harich
An inside view of the experimental chamber used to conduct the measurements at the X-ray free-electron laser PAL-XFEL. The chamber houses the liquid jet in which the disulfide molecules were dissolved. The UV and X-ray laser flashes are focused to a small spot on the jet in the middle of the Helium-filled chamber.
An international team of scientists has used extremely short X-ray flashes to understand the first steps in the light-induced breaking of disulfide bridges – the natural cross-linkers that hold most protein structures in place. The study, published in the journal “Nature Communications”, followed the change in X-ray absorption of sulfur atoms and delivered unexpected insights which may help design new materials that better resist damage from ultraviolet radiation.
Sulfur plays an essential role in life, in particular in the form of disulfide bridges in proteins. These bridges serve as natural cross-linkers to provide structural stability and help regulate biological processes in the cell. Nevertheless, they are quite susceptible to damage and can easily be broken using, for instance, ultraviolet (UV) light, which can result in a loss of protein function and in some cases diseases. However, breaking disulfide bonds has been useful in chemical catalysis, sensors, pharmaceuticals, polymers and nanomaterials.
For a long time, scientist have debated whether disulfides always break symmetrically to form two identical sulfur-centered radicals. One influencing factor is the energy of the exciting light. UV light of wavelengths shorter than 200 nanometers can break sulfur-sulfur as well as neighboring carbon-sulfur bonds. For UV light of longer wavelengths (i.e. lower energy) the case has been confusing. In the gas phase, only the sulfur-sulfur bond of disulfides is broken, but in solution, several accounts suggest a different behavior.
An international research team led by the University of Hamburg in collaboration with the Korea Advanced Institute of Science and Technology (KAIST) and the Pohang Accelerator Laboratory (PAL) has now succeeded in resolving this puzzling discrepancy with time-resolved X-ray absorption spectroscopy.
The extreme speed is one of the challenges
One of the difficulties in understanding disulfide bond cleavage is that it occurs ultrafast. Therefore, scientists excited the disulfide molecules with a UV laser flash of 100 femtoseconds (that’s 0.0000000000001 seconds) followed by an even shorter X-ray pulse. This kind of measurement can only be done with an X-ray free-electron laser (XFEL) because only XFELs can deliver the ultrashort X-ray pulses required to capture atoms while they are in motion.
By comparing the X-ray absorption spectra of the sulfur atoms with quantum chemical calculations, the scientists were able to identify the earliest photoproducts of L-cystine in aqueous solution and follow their evolution, from one hundred femtoseconds to just under a nanosecond: Within 140 femtoseconds the disulfide molecule splits symmetrically in half between the two sulfur atoms. “This means that disulfides in gas-phase and liquid phase behave initially the same. But we saw the majority of the initial photoproducts vanish surprisingly fast while the parent molecule L-cystine reformed. This reverse reaction is impossible in the gas phase, but not uncommon in solution, where the surrounding medium keeps the reaction partners close for a while,” explains Jessica Harich, researcher at University of Hamburg who co-led the study.
The reformation is followed by a second reaction product
“We also observed a second reaction product that emerged when the first product started to recombine to its parent L-cystine. This recombination happens at relatively high energy, that is, a part of the UV light’s energy is still stored in the reformed L-cystine molecules, enough to split the carbon-sulfur bond – the bridgehead so to speak – and produce secondary products,” adds Nils Huse, who is a professor of physics at Universität Hamburg and a researcher in the Cluster of Excellence “CUI: Advanced Imaging of Matter.”
These results now provide an answer to the long-standing question of how disulfides behave in solution. The new results clearly show the same primary reaction step as observed in isolated molecules, but they also reveal the delayed emergence of other molecular sulfur species in solution which were not directly generated by the UV flash of light. The ultrafast and dominant reformation of the initial disulfide structure suggests that nature may use disulfide bond cleavage as a way to dynamically dissipate the destructive energy of UV light by sacrificing disulfide bonds for a short stretch of time, a mechanism that may constitute a blueprint for designing materials that are more resistant to damage from ultraviolet radiation.
Original publication:
M. Ochmann, J. Harich, R. Ma et al.
UV photochemistry of the L-cystine disulfide bridge in aqueous solution investigated by femtosecond X-ray absorption spectroscopy
Nat Commun 15, 8838 (2024)
