Protein Aggregation in Electric Fields

Dr. K. Kang, in cooperation with Dr. Florian Platen (HHU-Düsseldorf)

DFG-GEPRIS Project (Feb. 2022-Feb. 2025)

We have observed the effects of an externally applied electric field on (lysozyme) protein crystallization and liquid−liquid phase separation (LLPS) and its crystallization kinetics. For a weak alternating current (AC) electric field, crystallization was found to occur in a wider region of the phase diagram in the presence of NaSCN-salt, while nucleation induction times were reduced, and crystal growth rates were enhanced.

First, the phase diagram of without and with the field is compared (in Figure 1), where the optical morphologies of phase behaviors for the protein crystals are discussed with possible effect of the field (in Figure 2). Second, the different shapes and orientations of protein crystals are found in terms of field variations of the frequency and amplitude (in Figure 3).

Figure 1: (a) Typical depolarized light-microscopy images. From left to right: homogeneous solution, protein crystals, and metastable LLPS. The scale bar is 200 μm. The phases are indicated by the symbols shown in the right-bottom corner of the images: blue circles, orange crosses, and pink crossed circles, respectively. The images refer to samples with a protein concentration c = 40 mg/mL and NaSCN concentrations of 0.05, 0.13, and 0.18 M, respectively. (b) Phase diagram (pH 4.5; 50 mM acetate buffer; at room temperature) in the protein versus salt concentration plane, without the electric field. The phase boundaries are indicated by dashed blue lines: the crystallization boundary between the homogeneous solution and the region in which crystal and the liquid phase coexist (lower line) and phase boundary between the crystal-solution coexistence and themetastable LLPS phase (upper line). (c) Phase diagram in the presence of the electric field (frequency 1 kHz and field strength 6 V/mm). The phase boundaries in the presence of the electric field are shown as red dash-dotted lines. The arrows indicate the shifts of phase boundaries due to the electric field. (published on J. Phys. Chem. Lett. 2024, 15, 8108−8113, https://doi.org/10.1021/acs.jpclett.4c01744)

Figure 2: Possible morphologies of a protein concentration of 40 mg/ml at the intermediate and final stage of crystallization from left to right. The field of view is 600 microns: (upper) for a NaSCN concentration of 0:10M, below the LLPS line, and (lower) for 0:18M, above the LLPS line, at the given field condition of electric field amplitude of 6 V/mm and the frequency of 1 kHz.(to be submitted in 2024)

Figure 3: The electric-field driven morphology of protein crystal for the middle protein concentration of 40 mg/ml and0.10M NaSCN salt: (a) in the large variation of AC field frequency (from ω = 10Hz to 10 kHz) for a given amplitudeof E0 = 3 V/mm, where the tendency of forming the flower-pXs are occurred in an increase of frequency. (b) Themorphology of protein crystal by varying the field amplitude (from E0 = 1.5 V/mm to 30 V/mm) at low frequencies (ofω = 10Hz to 1 kHz). These optical morphology of final stage of crystallization are somewhat ”ubiquitous” due to the fact that the nucleation of protein crystals depends on the way (or the process) with which field of view can be captured in the field condition. For instance, the E0 = 3 V/mm and ω = 1 kHz in (a) is different with the one in (b) for the condition of ω = 1 kHz and E0 = 3 V/mm. Overall, the single-pXs of protein crystals are found at lower frequency an amplitude, with some apparently increased colors with a light absorption on the surface of protein crystals.(to be submitted in 2024)