Development of a microsecond X-ray protein footprinting facility at the Advanced Light Source

Abstract

X-ray footprinting (XF) is an important structural biology tool used to determine macromolecular conformations and dynamics of both nucleic acids and proteins in solution on a wide range of timescales. With the impending shut-down of the National Synchrotron Light Source, it is ever more important that this tool continues to be developed at other synchrotron facilities to accommodate XF users. Toward this end, a collaborative XF program has been initiated at the Advanced Light Source using the white-light bending-magnet beamlines 5.3.1 and 3.2.1. Accessibility of the microsecond time regime for protein footprinting is demonstrated at beamline 5.3.1 using the high flux density provided by a focusing mirror in combination with a micro-capillary flow cell. It is further reported that, by saturating samples with nitrous oxide, the radiolytic labeling efficiency is increased and the imprints of bound versus bulk water can be distinguished. These results both demonstrate the suitability of the Advanced Light Source as a second home for the XF experiment, and pave the way for obtaining high-quality structural data on complex protein samples and dynamics information on the microsecond timescale.

Keywords: mass spectrometry; microsecond irradiation; protein structure; radiolytic labeling.

Figure 1

Microfluidic capillary flow set-up. (a) The portable set-up consists of a syringe pump (bottom left), water-cooled sample mount on a motorized stage (center) and photodiode (behind the sample mount). The sample mount is assembled close to the ultrahigh-vacuum beampipe which is capped with a beryllium window. (b) Front view of the temperature-controlled sample mount. The horizontal slits hold the capillary horizontal to the beam. The photodiode allows detection of attenuated beam through the aluminium sample mount, and the alignment of the capillary tube is carried out by horizontal and vertical slits.

Figure 2

Beam profile and alignment. Horizontal and vertical profile of the focused beam at 5.3.1 is obtained by vertical and horizontal scans, respectively, using a single slit of the sample mount. The derivative of the photodiode signal is used to obtain the beam profile. The data (black line) are fitted to a Gaussian function (red line) to obtain the FWHM.

Figure 3

Schematics of major reactions for X-ray radiolysis in dilute protein samples. Radiolysis of bulk water starts with the ionization of water under 10⁻¹⁶ s (spur). The key product, , diffuses (10⁻⁷ s) out in the bulk and undergoes reactions with the buffer and protein side chains. The former reaction, as well as various recombination reactions, scavenge and reduce the concentration of in the bulk. Sufficient X-ray dose is needed to maintain a steady-state concentration of .

Figure 4

Microsecond irradiation of Alexa 488 solution. Dose response plot of 10 mM Alexa in 10 mM phosphate buffer, pH 7, using the 106 µm × 164 µm focused beam at ALS beamline 5.3.1. A 200 µL volume of Alexa solution was passed through the 100 µm ID capillary tube on the sample mount at different speeds (Table 1 ▶) to obtain irradiation times in the range 15–120 µs. Fluorescence analysis was carried out as previously described (Gupta et al., 2007a ▶). The solid line represents a single exponential fit with a rate constant k = 13764 s⁻¹, with individual points representing the mean of three independent measurements with standard error. The inset shows the dose-response plot of the same sample using a larger beam to irradiate samples homogeneously inside a 535 µm ID capillary tube, with an exponential fit rate constant of k = 6410 s⁻¹.

Figure 5

Dose response of residue M80 of cyt c. 10 µM of cyt c sample in 10 mM phosphate buffer, pH 7.0, was irradiated using the flow set-up at beamlines ALS 5.3.1, NSLS X28C and ALS 3.2.1. The deliverable dose on the sample was adjusted to be the same at 5.3.1 and X28C as determined by the standard Alexa assay. The fraction of unmodified peptide at a given exposure was calculated as the unmodified peak area divided by the sum of the unmodified and modified peak areas. The peak areas were calculated from the extracted ion chromatogram, and the site of modification was identified by MS/MS. The solid lines represent single exponential fits with rate constants k = 360 s⁻¹, 330 s⁻¹ and 14 s⁻¹ for radiolysis carried out at 5.3.1 (black), X28C (red) and 3.2.1 (blue), respectively, with individual points representing the mean of three independent measurements with standard error.

Figure 6

Sub-millisecond irradiation of a megaDalton protein assembly. (a) Extracted ion chromatogram showing the increase of the doubly protonated +16 Da modified product of peptide 496–511, TQAIQSAAESTEMLLR (eluted at 4.0 min, 882.95 m/z) with the increase in irradiation time on the sample solution containing 10 µM mmcpn in 10 mM phosphate buffer, pH 7, containing 1 mM ATP, 1 mM TCEP, ∼5% glycerol and 150 mM NaCl. For easy visualization, the abundances of doubly protonated native peaks (eluted at 4.3 min, 874.95 m/z) are made equal for all the irradiation time points and the corresponding modified peak abundances are adjusted accordingly by multiplication factors. The peak areas are calculated from the extracted ion chromatograms of raw data. The fraction of unmodified peptide at a given exposure is calculated as the unmodified peak area divided by the sum of unmodified and modified peak areas. The ∼3.5% background modification for peptide 496–511 is normalized in the dose-response plot (inset). The solid line represents a single exponential fit with rate constants k = 253.7 s⁻¹, with individual points representing the mean of three independent measurement with standard error. (b) The sites of modification are identified by the MS/MS of the double protonated modified precursor ion of 882.95 m/z. The signature +16 m/z shift on y and b fragment ions shown in red indicate modification at M508. (c) Pictorial representation of modified Met residues on two adjacent subunits out of the 16 stacked subunits in the protein assembly (3kfb) (Pereira et al., 2010 ▶).

Figure 7

Effect of N₂O saturation on the dose response in cyt c. Comparative site-specific dose response plots between N₂O–O₂ (4:1 v/v) saturated (red) and aerated (black) samples of 10 µM cyt c in 10 mM phosphate buffer, pH 7.0, irradiated at beamline 5.3.1. Solid lines represent single exponential fits to provide rate constants (k; s⁻¹), which are used to calculate the ratio R = (k, N₂O)/(k, air). The individual points represent the mean of three independent measurements with standard error.