Spectra collected from individual crystalline amino acids suspended in DMSO showed XEOL to arise predominantly from aromatic amino acids (). The λmax
of the aromatic amino acids varied between 380 and 537 nm. No XEOL was observed between 300 and 900 nm from any other amino acid tested apart from cystine. Cystine yielded a weak XEOL signal with a λmax
of 808 nm that is likely to arise from the disulfide bond. The spectral shapes observed are broadly consistent with previously recorded XEOL data for tyrosine, tryptophan and phenylalanine powders and for tryptophan dissolved in 1:1 water:ethylene glycol (Carter et al., 1965
; Steen, 1967
; Nelson et al., 1967
; Nummedal & Steen, 1969
). The XEOL spectra bleached after continued exposure to X-rays, as previously observed in other inorganic systems (Rogalev & Goulon, 2002
). The spectra were not observed to change as a function of crystal orientation. There were some differences between the λmax
values observed here and those in the early literature. These are likely to be a consequence of a combination of differences in solvent polarity, the flux density of incident X-rays and the temperature and state of the samples (powder, solution or crystalline).
Glycerol, MPD and DMSO did not exhibit XEOL, consistent with their chemical structures. Paraffin oil demonstrated a broad and strong XEOL signal across the same wavelength range as proteins and is therefore an unsuitable cryoprotectant for use in XEOL studies.
XEOL spectra were recorded from HEWL, apoferritin and holoferritin, NAL and thermolysin. These complex spectra showed distinct features for each protein (); the spectra did not show any change as a function of crystal orientation. Comparison of the protein XEOL with the XEOL of individual amino acids and consideration of the amino-acid composition of each protein indicated that the protein XEOL is not the simple sum of the luminescence of its constituent amino acids. The λmax of the protein XEOL spectra are also red-shifted compared with those of the amino acids in DMSO. These observations indicate that XEOL, like fluorescence, is sensitive to local environment (i.e. polarity and nonradiative transfer) and that the luminescence of specific amino acids is quenched or altered when incorporated into a protein. This is particularly evident when the XEOL spectra of apoferritin and holoferritin are compared, where there is both an overall red shift and a change in spectral shape. This possibly arises from a combination of the increased protein dielectric constant and both radiative and nonradiative transfer in the presence of the amorphous iron core of holoferritin.
XEOL spectra were observed to rapidly decay during exposure to X-rays. Longer accumulation times resulted in smoother spectra, but in order to achieve a higher time resolution shorter exposure times were used in conjunction with FFT smoothing. This decay is shown for an apoferritin crystal in . In order to establish whether this decay is correlated to a decay in diffracting power, diffraction data and XEOL spectra were collected simultaneously from apoferritin and holoferritin, HEWL and NAL crystals. The decay of both the total luminescence yield and the peak counts from an apoferritin crystal are compared with the decay in diffracting power in . In this case XEOL follows a single exponential decay. Despite the comparable decay rates observed in this example, further investigation using several proteins showed that XEOL decay correlated poorly with decay in diffracting power. A wide range of XEOL decay rates were observed, even between crystals of the same type. In addition, XEOL was observed to not always follow a single exponential decay, with some crystals better described by a double exponential decay. During these experiments XEOL and diffraction data were collected with the simplest possible experiment design: each diffraction image was collected over the same repeated angular range. The lack of correlation and consistency observed with this simplified experimental setup suggests that XEOL is not (with currently available instrumentation) a reliable metric for following radiation damage.
Figure 3 (a) Decay of apoferritin XEOL spectra as a function of absorbed dose and (b) the concomitant decay of diffracting power and luminescence (both overall and at the peak XEOL wavelength) of the same apoferritin crystal as a function of absorbed dose. XEOL (more ...)
Bleaching of fluorescence is usually associated with the irreversible destruction of the fluorophore (Adam et al., 2009
). In order to determine whether a similar irreversible process occurs during the bleaching of the XEOL signal during X-ray exposure, we collected a series of thermolysin XEOL spectra using a sequence of X-ray pulses spaced such that the XEOL signal could completely decay before the next X-ray pulse occurred (). These data showed several interesting features. Firstly, the XEOL signal does not recover even after extended delays (50 s) between each X-ray pulse. Instead, a progressive bleaching is observed consistent with destruction of the luminescent moieties. Secondly, spectral features with differing λmax
showed a clear difference both in XEOL lifetime after the X-ray shutter closed and in bleaching rate. Previously, almost complete recovery of fluorescence spectra has been observed for crystals subjected to low doses (<1 MGy; Adam et al., 2009
). The lack of XEOL recovery in this study can be attributed to the large absorbed dose per pulse (~3.6 MGy), meaning that even if intermediates only infrequently convert to a permanently damaged state the damaged state becomes highly populated. The non-uniform decay of XEOL spectra suggests that XEOL reflects differential damage rates at distinct sites within the protein. It has been shown that different amino acids are damaged at different rates during X-ray irradiation depending on the type of side chain and its local environment (Garman, 2010
). This is consistent with the wavelength-dependent luminescence decay observed here. This non-uniform decay suggests that further characterization and understanding of macromolecular XEOL will help to reveal how individual amino acids respond to X-ray irradiation and allow us to develop a more complete model of the progression of radiation damage in macromolecules.
Figure 4 Decay in luminescence of a thermolysin crystal as a function of time. XEOL spectra show differential rates of decay as a function of wavelength and a lack of recovery between X-ray pulses. The cumulative dose absorbed by the crystal per pulse is 3.6 MGy. (more ...)