Detergent Desorption of Membrane Proteins Exhibits Two Kinetic Phases

Abstract

Gradual dissociation of detergent molecules from water-insoluble membrane proteins culminates in protein aggregation. However, the time-dependent trajectory of this process remains ambiguous because the signal-to-noise ratio of most spectroscopic and calorimetric techniques is drastically declined by the presence of protein aggregates in solution. We show that by using steady-state fluorescence polarization (FP) spectroscopy the dissociation of the protein-detergent complex (PDC) can be inspected in real time at detergent concentrations below the critical micelle concentration. This article provides experimental evidence of the coexistence of two distinct phases of the dissociations of detergent monomers from membrane proteins. We first noted a slow detergent predesolvation process, which was accompanied by a relatively modest change in the FP anisotropy, suggesting a small number of dissociated detergent monomers from the proteomicelles. This predesolvation phase was followed by a fast detergent desolvation process, which was highlighted by a major alteration in the FP anisotropy. The durations and rates of these phases were dependent on both the detergent concentration and the interfacial PDC interactions. Further development of this approach might lead to the creation of a new semiquantitative method for the assessment of the kinetics of association and dissociation of proteomicelles.

Figure 1. Side-view of the molecular structures of OmpG and truncation FhuA mutants

(A) OmpG; (B) FhuA ΔC/Δ5L_25N; (C) ΔC/Δ7L_30N. The positions of the fluorophore were marked in yellow. For OmpG, Texas Red was tethered at position D224C on loop L6. For the two truncation FhuA derivatives, Texas Red was attached to an engineered GS-rich, cysteine-containing loop on the T7 β turn. FhuA ΔC/Δ5L_25N and FhuA ΔC/Δ7L_30N show charge neutralizations, which are marked in red with respect to the native FhuA. For the latter FhuA mutant, there are three additional lysine mutations within the β turns, which are marked in blue, out of which two are negative-to-positive charge reversals.^– The arrows indicate molecular dimensions, as inferred from C_α to C_α, which were obtained from the X-ray crystal structure of both proteins.^– Cartoons show proteomicelles in a prolate geometrical packing. The homology structure of truncation FhuA derivatives was accomplished using Swiss-model and FhuA PDB ID:1FI1.

Figure 2. Time-dependent alterations in the FP anisotropy when the membrane proteins were incubated at different concentrations of LysoFos, a zwitterionic detergent

(A) OmpG; (B) FhuA ΔC/Δ5L_25N; (C) FhuA ΔC/Δ7L_30N. The solubilized protein concentration was 28 nM. The buffer solution contained 200 mM NaCl, 50 mM HEPES, pH 7.4. The experimental FP anisotropy data were presented as average ± SD over a number of at least three distinct acquisitions.

Figure 3. Time-dependent alterations in the FP anisotropy when the membrane proteins were incubated at different concentrations of UM, a neutral maltoside-containing detergent

(A) OmpG; (B) FhuA ΔC/Δ5L_25N; (C) FhuA ΔC/Δ7L_30N. The other experimental conditions were the same as those in Fig. 2.

Figure 4. Time-dependent alterations in the FP anisotropy when the membrane proteins were incubated at different concentrations of DM, a neutral maltoside-containing detergent

(A) OmpG; (B) FhuA ΔC/Δ5L_25N; (C) FhuA ΔC/Δ7L_30N. The other experimental conditions were the same as those in Fig. 2.

Figure 5. Time-dependent alterations in the FP anisotropy when the membrane proteins were incubated at different concentrations of CYMAL-4, a neutral maltoside-containing detergent

(A) OmpG; (B) FhuA ΔC/Δ5L_25N; (C) FhuA ΔC/Δ7L_30N. The other experimental conditions were the same as those in Fig. 2.

Figure 6. Cartoon showing different FP anisotropy-based trajectories of the proteodemicellization

The predesolvation phase underwent a linear change in the FP anisotropy, whereas the desolvation phase followed either a linear regime, which was marked in green, or an exponential decay, which was marked in red. In the case of weak PDC interactions and low incubating detergent concentration, there is no a predesolvation phase, whereas the desolvation phase, which is marked in blue, is rapid.