Time-resolved crystallography and protein design: signalling photoreceptors and optogenetics

Abstract

Time-resolved X-ray crystallography and solution scattering have been successfully conducted on proteins on time-scales down to around 100 ps, set by the duration of the hard X-ray pulses emitted by synchrotron sources. The advent of hard X-ray free-electron lasers (FELs), which emit extremely intense, very brief, coherent X-ray pulses, opens the exciting possibility of time-resolved experiments with femtosecond time resolution on macromolecular structure, in both single crystals and solution. The X-ray pulses emitted by an FEL differ greatly in many properties from those emitted by a synchrotron, in ways that at first glance make time-resolved measurements of X-ray scattering with the required accuracy extremely challenging. This opens up several questions which I consider in this brief overview. Are there likely to be chemically and biologically interesting structural changes to be revealed on the femtosecond time-scale? How shall time-resolved experiments best be designed and conducted to exploit the properties of FELs and overcome challenges that they pose? To date, fast time-resolved reactions have been initiated by a brief laser pulse, which obviously requires that the system under study be light-sensitive. Although this is true for proteins of the visual system and for signalling photoreceptors, it is not naturally the case for most interesting biological systems. To generate more biological targets for time-resolved study, can this limitation be overcome by optogenetic, chemical or other means?

The birth of a new field

“…Broadly, three types of experiments were first attempted-those in which hydrated protein nanocrystals were sprayed across the pulsed beam (serial femtosecond nanocrystallography, SFX), those in which the hard X-ray beam of micrometre dimensions traverses many biomolecules in a liquid jet (fast solution scattering, FSS-see contributions by Haldrup [4], Mendez et al [5] and Pande et al [6]), and single particle (SP) imaging, in which a beam of submicrometre dimensions scatters from an SP such as a virus [7][8][9]. Before long many other experimental arrangements had also been tried during this exciting first 4 years, including fixed samples scanned across the beam for the study of two-dimensional membrane protein crystals [10], time-resolved SFX [11] (see also Moffat [12]), and new types of sample delivery devices, such as those based on the lipid cubic phase [13,14] and on electrospraying [15].…”

Microfluidics: From crystallization to serial time-resolved crystallography

“…Prior to the development of serial methods for crystallography, these types of experiments were only feasible on very large, stable crystals where the structural change or enzymatic reaction was reversible and would naturally reset to the initial state, allowing for a large number of repetitions of this pump-probe cycle. 19,[196][197][198][199][200][201][202][203] The advent of serial crystallography has opened up the potential for timeresolved experiments to smaller crystals and targets that are highly sensitive to radiation damage, as well as reaction pathways that are irreversible within the limitations of the crystal lattice. 177,201,[204][205][206] Furthermore, the development of next-generation X-ray sources has expanded the range of available timescales from femtoseconds to seconds or longer.…”

“…Calculations by Schmidt, based on the diffusivity of glucose and assuming no significant barriers to diffusion within the crystal, suggest that the size of microcrystals is critical for the success of chemically triggered time-resolved experiments (Table III). 201,218 Furthermore, the use of high concentrations of triggering molecules is important to facilitate diffusion and achieve effective reaction initiation. 219 With these experimental requirements in mind, many of the injection strategies for serial crystallography are uniquely posed to usher in a new era of dynamic protein structural studies.…”

Blue protein with red fluorescence

“…It is important to note that these proteins have a completely different fold, and hence the mode of binding is very different. Bacteriophytochromes are well-studied photoswitches; their mechanism of photoconversion and structures of biliverdin in red (Pr) to far-red (Pfr) absorption (29)(30)(31) have been determined by time-resolved (30,38) and pump-probe methods (39). Despite the difference in topology of the binding site, it would be interesting to study whether Sandercyanin has photoswitching properties similar to bacteriophytochromes.…”