Interrogating Encapsulated Protein Structure within Metal-Organic Frameworks at Elevated Temperature

Abstract

Encapsulating biomacromolecules within metal-organic frameworks (MOFs) can confer thermostability to entrapped guests. It has been hypothesized that the confinement of guest molecules within a rigid MOF scaffold results in heightened stability of the guests, but no direct evidence of this mechanism has been shown. Here, we present a novel analytical method using small-angle X-ray scattering (SAXS) to solve the structure of bovine serum albumin (BSA) while encapsulated within two zeolitic imidazolate frameworks (ZIF-67 and ZIF-8). Our approach comprises subtracting the scaled SAXS spectrum of the ZIF from that of the biocomposite BSA@ZIF to determine the radius of gyration of encapsulated BSA through Guinier, Kratky, and pair distance distribution function analyses. While native BSA exposed to 70 °C became denatured, in situ SAXS analysis showed that encapsulated BSA retained its size and folded state at 70 °C when encapsulated within a ZIF scaffold, suggesting that entrapment within MOF cavities inhibited protein unfolding and thus denaturation. This method of SAXS analysis not only provides insight into biomolecular stabilization in MOFs but may also offer a new approach to study the structure of other conformationally labile molecules in rigid matrices.

Figure 1

Characterization of BSA@ZIF-67/ZIF-8 biocomposites. Representative XRD spectra of (a) BSA@ZIF-67 and (d) BSA@ZIF-8 showing the biocomposite (red) and MOF (black). Representative FTIR spectra of (b) BSA@ZIF-67 and (e) BSA@ZIF-8, showing the biocomposite (red), lyophilized native BSA protein (blue), and MOF (black). Representative SEM images of (c) BSA@ZIF-67 and (f) BSA@ZIF-8.

Figure 2

Schematic of (a) scaled spectral subtraction to reveal encapsulated BSA in ZIF biocomposites. Log–log scale SAXS spectra are shown for (b) BSA@ZIF-67, (c) ZIF-67, (d) native or encapsulated BSA in ZIF-67, (e) BSA@ZIF-8, (f) ZIF-8, and (g) native or encapsulated BSA in ZIF-8. Spectra for encapsulated BSA (d, g) were generated by subtracting the ZIF (c, f) spectra from the BSA@ZIF (b, e) spectra and compared to native BSA in buffer solution. Before subtracting MOF spectra from biocomposite spectra, solvent background subtraction was completed for all spectra. ZIF-8 and ZIF-67 spectra were subtracted from their corresponding biocomposite spectra using scaling subtraction factors of 5 and 20, respectively. The Q-range is shown from 0.01 to 0.20 Å^–1. BSA@ZIF-8 and BSA@ZIF-67 were prepared at BSA/MOF ratios of 3:1 in HEPES buffer. The native BSA was prepared at a concentration of 4 mg/mL in HEPES buffer.

Figure 3

Representative PDDFs for BSA, BSA@ZIF-67/ZIF-8, and a physical mixture of BSA and ZIF-67 prepared at a BSA/MOF ratio of 1:4 (20% BSA). PDDFs shown for (a) 4 mg/mL native BSA in HEPES buffer at 23 °C, (b) calculated spectra of BSA encapsulated in ZIF-67 at 23 °C, (c) calculated spectra of BSA encapsulated in ZIF-8 at 23 °C, (d) 4 mg/mL native BSA in HEPES buffer at 70 °C, (e) calculated spectra of BSA encapsulated in ZIF-67 at 70 °C, and (f) calculated spectra of BSA from a physical mixture of lyophilized BSA and ZIF-67. R_g values calculated from PDDFs were (a) 29.53 ± 0.18 Å for native BSA at 23 °C, (b) 32.21 ± 0.06 Å for ZIF-67 encapsulated BSA at 23 °C, (c) 32.57 ± 0.46 Å for ZIF-8 encapsulated BSA at 23 °C, (d) 133.0 Å for native BSA at 70 °C, (e) 33.34 ± 0.04 Å for ZIF-67 encapsulated BSA at 70 °C, and (f) 29.9 Å for the physical mixture at 23 °C. Corresponding SAXS spectra are shown in Figure 2.

Figure 4

Representative Kratky plots of encapsulated and native BSA at 23 and 70 °C. Kratky plots from the calculated spectra of (a) BSA@ZIF-67 as a dry powder and (b) native BSA in HEPES buffer at a concentration of 4 mg/mL heated to 70 °C for 3 h. Corresponding PDDFs at 23 °C are shown in Figure 3a,b, and those at 70 °C are shown in Figure 3d,e.