Cryophotolysis of caged compounds: a technique for trapping intermediate states in protein crystals

Abstract

Caged compounds in combination with protein crystallography represent a valuable tool in studies of enzyme reaction intermediates. To date, photochemical triggering of reactions has been performed close to room temperature. Synchronous reaction initiation has only been achieved with enzymes of relatively slow turnover (<0.1 s À1 ) and caged compounds of high quantum yield. Here X-ray crystallography and microspectrophotometry were used to provide evidence that (nitrophenyl)ethyl (NPE) ester bonds can be photolyzed by UV light at cryotemperatures. NPE-caged ATP in¯ash-cooled crystals of Mycobacterium tuberculosis thymidylate kinase was photolyzed successfully at 100±150 K as assessed by the structural observation of ATP-dependent enzymatic conversion of TMP to TDP after temporarily warming the crystals to room temperature. A new method is proposed in which cryophotolysis combined with temperature-controlled protein crystallography can be used to trap reaction intermediates even in some of the fastest enzymes and/or when only compounds of low quantum yield are available. Raising the temperature after cryophotolysis may allow a transition barrier to be passed and an intermediate to accumulate in the crystal. A comparable method has only been used so far with proteins displaying endogenous photosensitivity. The approach described here opens the way to studying the reaction mechanisms of a much larger number of crystalline enzymes. Furthermore, it is shown that X-ray-induced radiolysis of caged compounds occurs if high-intensity synchrotron beamlines are used. This caveat should be taken into account when deriving data-collection protocols. It could also be used potentially as a way to trigger reactions.

Advances in spectroscopic methods for biological crystals. 1. Fluorescence lifetime measurements

“…However, since in our case the¯uorescence intensity is also dependent on sample volume and geometry, pH changes can only be measured reliably in this way if they are generated in situ within a unique sample. This would be the case if for example a pH jump was generated by liberating caged protons with UV light (Khan et al, 1993), possibly at cryo-temperature (Ursby et al, 2002). Proton release and diffusion throughout the sample could be monitored by recording the decay of uorescence intensity.…”

Serial femtosecond crystallography: A revolution in structural biology

“…While the large solvent channels in crystalline macromolecules often allow for diffusion to take place via soaking with a diffusing substrate 120-122 (assuming the active site is unobstructed and the substrate is sufficiently small relative to the solvent channels), the size of the crystal limits diffusion times, thereby constraining the time regime of reaction intermediates accessible in an experiment. There have been chemically activated time-resolved experiments performed successfully by incorporating photo-activated caged substrates into the crystal 123-125 , thus reducing diffusion times to that of the photo-penetration of the pump laser. However, the incorporation of caged substrates requires extensive knowledge about a given system and is not generally compatible 126 .…”

Cryophotolysis of ortho-Nitrobenzyl Derivatives of Enzyme Ligands for the Potential Kinetic Crystallography of Macromolecules

“…For instance, the cryophotolysis at 100 K of NPEcaged ATP has been demonstrated to occur within crystals of the enzyme thymidylate kinase after x-ray analysis. [19] This method opens up new possibilities for studying reaction intermediates of functional proteins, provided that the intermediates build up in the crystal when a suitable temperature profile is subsequently applied.…”