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. 2017 Aug 1;89(15):8013-8020.
doi: 10.1021/acs.analchem.7b01339. Epub 2017 Jul 10.

Interrogating Detergent Desolvation of Nanopore-Forming Proteins by Fluorescence Polarization Spectroscopy

Aaron J Wolfe  1   2 , Yi-Ching Hsueh  1 , Adam R Blanden  3 , Mohammad M Mohammad  1 , Bach Pham  4 , Avinash K Thakur  1   2 , Stewart N Loh  3 , Min Chen  4 , Liviu Movileanu  1   2   5
Affiliations

Interrogating Detergent Desolvation of Nanopore-Forming Proteins by Fluorescence Polarization Spectroscopy

Aaron J Wolfe et al. Anal Chem. .

Abstract

Understanding how membrane proteins interact with detergents is of fundamental and practical significance in structural and chemical biology as well as in nanobiotechnology. Current methods for inspecting protein-detergent complex (PDC) interfaces require high concentrations of protein and are of low throughput. Here, we describe a scalable, spectroscopic approach that uses nanomolar protein concentrations in native solutions. This approach, which is based on steady-state fluorescence polarization (FP) spectroscopy, kinetically resolves the dissociation of detergents from membrane proteins and protein unfolding. For satisfactorily solubilizing detergents, at concentrations much greater than the critical micelle concentration (CMC), the fluorescence anisotropy was independent of detergent concentration. In contrast, at detergent concentrations comparable with or below the CMC, the anisotropy readout underwent a time-dependent decrease, showing a specific and sensitive protein unfolding signature. Functionally reconstituted membrane proteins into a bilayer membrane confirmed predictions made by these FP-based determinations with respect to varying refolding conditions. From a practical point of view, this 96-well analytical approach will facilitate a massively parallel assessment of the PDC interfacial interactions under a fairly broad range of micellar and environmental conditions. We expect that these studies will potentially accelerate research in membrane proteins pertaining to their extraction, solubilization, stabilization, and crystallization, as well as reconstitution into bilayer membranes.

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Figures

Figure 1
Figure 1. Side view of the FhuA ΔC/Δ5L protein, illustrating five truncated extracellular loops, L3, L4, L5, L10, and L11 of FhuA by top arrows.
The bottom arrow indicates the T7 β turn and site for protein labeling with Texas Red, which is marked by a red sphere.
Figure 2
Figure 2. Time-dependent anisotropy showing the protein-detergent complex (PDC) interfacial dynamics of FhuA ΔC/Δ5L
The anisotropy data were acquired by adding overnight refolded protein to a bath of varying detergent concentration. All anisotropy measurements were carried out at room temperature in 200 mM NaCl, 50 mM HEPES, pH 7.4. The starting detergent concentrations were as follows: (A) 20 mM LysoFos; (B) 5 and 25 mM LD; and (C) 0.2, 0.5, and 1 mM LPPG. In (D), it is shown concentration-response anisotropy changes as a result of the PDC dissociation. The horizontal axis indicates individual dilutions of detergent concentrations, while keeping the final protein concentration constant at 28 nM (see Experimental Section). The anisotropy values on the vertical axis were collected 24 hours later for equilibrium determinations. The LPPG data points belonging to the maximum state (rmax = ~0.342) were obtained when the protein was refolded in either 0.5 or 1 mM LPPG. The orange horizontal line on the LPPG data points corresponds to a secondary maximum anisotropy value, rmax = 0.31, when the protein was refolded in 200 µM LPPG; (E) This panel shows a low anisotropy value, r1, which was recorded either in the presence of 40 mM SDS or 6 M Gdm-HCl. The top of each panel or vertical bars indicate the CMC (Table S1).
Figure 3
Figure 3. Concentration-response anisotropy changes recorded with protein nanopores of varying isoelectric point pI
(A) Side views of the four protein nanopores inspected in this work, OmpG, FhuA ΔC/Δ5L, FhuA ΔC/Δ5L_25N, and FhuA ΔC/Δ7L_30N. Locations of fluorophore attachment are marked in yellow. Negative charge neutralizations with respect to FhuA ΔC/Δ5L are marked in red. For FhuA ΔC/Δ7L_30N, there are three additional lysine mutations in the β turns (marked in blue), out of which two are negative-to-positive charge reversals; The top of each cartoon indicates the nanopore abbreviated name and its respective isoelectric point. (B) Dose-response of the LysoFos depletion in the well; (C) Dose-response of OG depletion in the well. Vertical bars indicate the CMC (Table S1). The horizontal axis indicates individual dilutions of detergent concentrations, while keeping the final protein concentration constant at 28 nM (see Experimental Section). The anisotropy values on the vertical axis were collected 24 hours later for equilibrium determinations. All the other experimental conditions were the same as in Fig. 2.
Figure 4
Figure 4. Unusual thermostability of LPPG-refolded protein nanopores and functional reconstitution of LysoFos-, DDM-, and OG-refolded protein nanopores
(A) Wavelength circular dichroism scans of ~1 µM FhuA ΔC/Δ5L in 200 mM NaCl, 50 mM potassium phosphate, pH 7.4 with 20 mM of the specified detergent. In the negative control experiment, we used a buffer solution containing 6 M Gdm-HCl; (B) Temperature-dependent ellipticity θ225 of FhuA ΔC/Δ5L in either 20 mM LPPG or in 20 mM LysoFos. For DDM, UM, and DM, we could not achieve a sufficiently high aggregation-free protein concentration; (C) Representative step-wise insertions of single nanopores, over at least 6 distinct experiments, after the addition of DDM- (blue), OG- (black), or LysoFos-refolded (red) FhuA ΔC/Δ5L at an applied transmembrane potential of +40 mV. 40 µl pure and denatured 6×His+-tagged FhuA ΔC/Δ5L was 50-fold diluted into 29 mM DDM, 85 mM OG, or 16 mM LysoFos, containing 200 mM NaCl, 50 mM Tris.HCl, 1 mM EDTA, pH 8.0. The dilution ratio of the refolded protein within the bilayer chamber was ~ 1:1000. Therefore, the presence of detergent within the bilayer chamber did not affect the stability of the membrane; (D) The unitary-conductance histograms of DDM-, OG-, and LysoFos-refolded FhuA ΔC/Δ5L. The electrical recordings were collected using 1M KCl, 10 mM potassium phosphate, pH 7.4.
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