Nanocrystallites Modulate Intermolecular Interactions in Cryoprotected Protein Solutions

Studying protein interactions at low temperatures has important implications for optimizing cryostorage processes of biological tissue, food, and protein-based drugs. One of the major issues is related to the formation of ice nanocrystals, which can occur even in the presence of cryoprotectants and can lead to protein denaturation. The presence of ice nanocrystals in protein solutions poses several challenges since, contrary to microscopic ice crystals, they can be difficult to resolve and can complicate the interpretation of experimental data. Here, using a combination of small- and wide-angle X-ray scattering (SAXS and WAXS), we investigate the structural evolution of concentrated lysozyme solutions in a cryoprotected glycerol–water mixture from room temperature (T = 300 K) down to cryogenic temperatures (T = 195 K). Upon cooling, we observe a transition near the melting temperature of the solution (T ≈ 245 K), which manifests both in the temperature dependence of the scattering intensity peak position reflecting protein–protein length scales (SAXS) and the interatomic distances within the solvent (WAXS). Upon thermal cycling, a hysteresis is observed in the scattering intensity, which is attributed to the formation of nanocrystallites in the order of 10 nm. The experimental data are well described by the two-Yukawa model, which indicates temperature-dependent changes in the short-range attraction of the protein–protein interaction potential. Our results demonstrate that the nanocrystal growth yields effectively stronger protein–protein attraction and influences the protein pair distribution function beyond the first coordination shell.


Measurement protocol
illustrates an example of the measurement protocol. Panels A and B represent cooling and heating which together comprise one full temperature cycle. For cooling, the temperature was varied with the rate of 4 K/min from T = 300 K to the target temperature T = 195 K. SAXS/WAXS scattering data were acquired simultaneously with an exposure time of 1 s followed by a 9 s dead time until the next measurement, i.e. every 10 s. In total, 165 scans were taken during cooling equally spanned over the entire temperature range, hence limiting the total exposure time of the sample to 165 s. The measurements during heating were started right away and the same protocol was employed. Thus, for the whole temperature cycle the sample was exposed to X-rays for 330 s. Figure S1: The measurement protocol for the entire temperature cycle including cooling (panel A) and heating (panel B). The temperature was varied with a constant rate of 4 K/min (black solid line) while the SAXS/WAXS scattering data (blue markers) were acquired simultaneously every 10 seconds, i.e. with the exposure time of 1 s and the dead time of 9 s. The insets show a zoomed-in region demonstrating the time structure of the measurements along the temperature variation line.

Radiation damage assessment
The data discussed in the main text were measured for all samples using the X-ray flux F = 1.25 × 10 10 ph/s, where no X-ray-induced damage to the system was observed in S2 contrast to higher fluxes (Fig. S2). Figure S2: The SAXS intensity, I(Q, t), measured using the flux F = 1.25 × 10 9 ph/s (panel A) and F = 2 × 10 11 ph/s (panel B) for 200 mg/ml lysozyme in 23 mol% glycerol-water solution at room temperature (T = 300 K). The color indicates the measurement time, t. (C) Porod invariant, Q p (t), calculated from the data displayed in A and B and two additional fluxes as a function of measurement time, t, i.e. the time the sample is exposed to X-rays. The vertical dashed line shows the total exposure time needed for the full temperature cycle. (D) The WAXS intensity measured using the flux F = 1.25 × 10 10 ph/s for lysozyme in 23 mol% glycerol-water solution at room temperature (T = 300 K). The inset shows the absent variation of the Q-value of the first peak over the measurement time. Note that the oscillations of the integrated intensity visible in panel C for higher fluxes arise from the top-up mode of the storage ring. Fig. S2 A evolution of the SAXS signal measured over the time span of 500 seconds using the X-ray flux F = 1.25 × 10 10 ph/s does not exhibit any change in the Qrange of interest. On the contrary, when measured using a much higher flux (F = 2 × 10 11 ph/s) a clear increase of I(Q, t) at Q-values below 1 nm −1 is seen in Fig. S2 B. Here, the Porod invariant, calculated as Q p (t) = Qmax Q min Q 2 I(Q, t) dQ, where Q min = 0.5 nm −1 and S3 Q max = 1 nm −1 is used to quantify the X-ray-induced changes in SAXS. The variation of the Porod invariant as a function of measurement time, t, is shown in Fig. S2 C for the different X-ray fluxes. It is evident that already at F = 2.5 × 10 10 ph/s, the X-ray-induced changes in SAXS are negligible over the time span required for the full temperature cycle (330 s, dashed vertical line), discussed in the main text. Figure S2D presents the evolution of the WAXS signal measured using F = 1.25 × 10 10 ph/s over 500 s time span, where no indication of the X-ray-induced effect can be seen.

Shown in
Neither the lineshape of the scattering intensity peak, nor the position of the first maximum, Temperature dependent WAXS for dilute lysozyme solution Figure S3 shows the variation of the WAXS scattering intensities measured for 10 mg/ml lysozyme in glycerol-water solution upon cooling down to 195 K (panel A) and warming back up to 300 K (panel B). The overall behavior is similar to that observed for the con- Furthermore, the temperature dependence of the first WAXS peak Q position for the dilute lysozyme solution is qualitatively similar to that observed for the concentrated one, shown for comparison in Figs. S4 A,B. A change of the linear slope at T ≈ 245 K upon cooling as well as a thermal hysteresis upon reheating are observed for 10 mg/ml lysozyme Figure S3: WAXS intensity measured for 10 mg/ml lysozyme in 23 mol% glycerol-water solution as a function of temperature while cooling down from T = 300 K to T = 195 K (panel A) and warming back up to room temperature (panel B). The insets in both panels represent the data in contour plots to emphasize that the ice peaks are absent in the cool down and manifest in the warm up. The black arrows in panel B indicate the peaks matching some of the Bragg peaks of hexagonal ice. (C, D) representative 2D scattering patterns measured upon heating at T = 195 K and T = 245 K, where ice peaks appear in the former case. The white rectangle highlights the ice peak which profile along the radial direction is plotted in panels E and F upon cooling and warming, respectively. In panels E and F, an offset has been added to facilitate the comparison between temperatures.
S5 in 23 mol% glycerol-water solution. The similar behavior independent of the concentration of proteins in the system suggests that it mainly originates from the interplay of water and glycerol in the solvent itself.

Nanocrystallites in pure glycerol-water solution
As mentioned in the main text, the low temperature behavior in glycerol-water mixtures with concentrations of 15-28 mol% of glycerol likely stems from the formation of ice nanocrystallites occurring due to demixing of the solution components. [1][2][3][4] In Fig. S5, we present the measurement on a pure 23 mol% glycerol-water solution without proteins, where the formation of ice nanocrystallites can be expected. Indeed, as seen in panel A, upon reheating after cooling down to T = 195 K several small ice Bragg peaks are developed and can be resolved in the I(Q) (Fig. S5 D). Figure S5: (A, B) representative 2D scattering patterns in the WAXS range measured on 23 mol% glycerol-water solution upon heating at T = 195 K and T = 245 K, where ice peaks appear in the former case. The white rectangle highlights the ice peak whose profile along the radial direction is plotted in panels C and D upon cooling and warming, respectively. In panels C and D, an offset has been added to facilitate the comparison between temperatures.