Assembling a Correctly Folded and Functional Heptahelical Membrane Protein by Protein Trans-splicing

Abstract

Protein trans-splicing using split inteins is well established as a useful tool for protein engineering. Here we show, for the first time, that this method can be applied to a membrane protein under native conditions. We provide compelling evidence that the heptahelical proteorhodopsin can be assembled from two separate fragments consisting of helical bundles A and B and C, D, E, F, and G via a splicing site located in the BC loop. The procedure presented here is on the basis of dual expression and ligation in vivo. Global fold, stability, and photodynamics were analyzed in detergent by CD, stationary, as well as time-resolved optical spectroscopy. The fold within lipid bilayers has been probed by high field and dynamic nuclear polarization-enhanced solid-state NMR utilizing a (13)C-labeled retinal cofactor and extensively (13)C-(15)N-labeled protein. Our data show unambiguously that the ligation product is identical to its non-ligated counterpart. Furthermore, our data highlight the effects of BC loop modifications onto the photocycle kinetics of proteorhodopsin. Our data demonstrate that a correctly folded and functionally intact protein can be produced in this artificial way. Our findings are of high relevance for a general understanding of the assembly of membrane proteins for elucidating intramolecular interactions, and they offer the possibility of developing novel labeling schemes for spectroscopic applications.

Keywords: intein; membrane protein; photoreceptor; protein folding; protein splicing; proton pump; seven-helix receptor; solid-state NMR.

FIGURE 1.

A, in vivo protein trans-splicing of two α-helical fragments of pR on the basis of split inteins. B, design of the pR-intein segments. The N-terminal precursor contains helices A and B of pR, followed by DnaE_N102 and a Strep tag. The C-terminal precursor consists of a Strep tag, an Smt3 solubility tag, Npu_Δ36, and helices C, D, E, F, and G of pR, followed by an HSV-His₆ tag. By protein trans-splicing, the solubility and both Strep tags are excised, resulting in full-length HSV-His₆-tagged pR (pR_LIG). C, topology plot of pR_WT and pR_LIG/pR_SCFNG. To ensure an efficient splicing reaction, the BC loop amino acid sequence was modified as depicted. pSB, protonated Schiff base.

FIGURE 2.

A–C, Western blots (A and B) and SDS-PAGE (C) of the protein trans-splicing reaction. 4 h after the first and 1 h after the second induction, the presence of N and C precursors as well as ligated protein, pR_LIG, was detected in the anti-Strep (A) and anti-His₆ blot (B). After 20 h, a C-terminal cleavage product (C-precursorΔSmt3) and the excised intein (inteinΔSmt3) became visible. After 24 h, precursors and pR_LIG were also detectable in SDS-PAGE (C).

FIGURE 3.

A, stationary light absorption spectrum of pR_LIG at pH 7. The ratio of the absorption at 280 nm was twice as strong compared with 520 nm, demonstrating high sample purity. The values of λ_max of pR_SCFNG and pR_LIG were nearly identical, but they were blue-shifted by 5 nm with respect to pR_WT. a.u., arbitrary units. B, pH titration of _max for pR_LIG, pR_SCFNG, pR_WT, and pR_InsNG in the range of pH 4–11. The pK_a values of the primary proton acceptor Asp⁹⁷ for each sample were determined from the inflections points of the curve. C, pH titration of λ_max for pR_D88S, pR_S89C, and pR_P90F. The pK_a of Asp⁹⁷ increases upon introduction of D88S and P90F but is reduced with the S89C mutation. The observation that the pK_a is not altered in pR_LIG and pR_SCFNG might be explained by compensating effects when replacing the residue triplet Asp⁸⁸-Ser⁸⁹-Pro⁹⁰ with Ser⁸⁸-Cys⁸⁹-Phe⁹⁰.

FIGURE 4.

A, CD spectra of pR_WT, pR_SCFNG, and pR_LIG showing similarly shaped spectra, indicating a primarily α-helical fold. B, the temperature dependence of the α-helical band was followed at 222 nm. A melting temperature of 100 °C was determined for pR_WT, which is slightly lower for pR_SCFNG (88 °C) and pR_LIG (80 °C).

FIGURE 5.

Flash photolysis experiments on pR_LIG, pR_SCFNG and pR_WT. The formation and decay of the M state were monitored at 400 nm, the ground-state bleach at 500 nm, and the decay of the K state and buildup of late intermediates were observed at 590 nm. pR_LIG and pR_SCFNG are indistinguishable from each other but show an accelerated photocycle compared with pR_WT. a.u., arbitrary units.

FIGURE 6.

A, isotope labeling of the retinal-Schiff base complex. B, DNP-enhanced ¹³C MAS NMR spectrum of 14,15-¹³C-all-trans-retinal bound to pR_WT and pR_LIG. The natural abundance of ¹³C signals was suppressed by a double quantum filter so that only signals C14 and C15 of the retinal cofactor remained. The chemical shift of resonances in pR_LIG was identical to pR_WT. C, ¹⁵N MAS NMR spectra of [U-¹⁵N]pR_WT, pR_SCFNG, and pR_LIG as well as of [¹⁵N-Lys]pR_SCFNG and of the mutants [U-¹⁵N]pR_S89C, pR_P90F, and pR_InsNG. The signal of His⁷⁵ was shifted from 162 ppm in pR_WT to 172 ppm in pR_LIG and pR_SCFNG. The signal of the protonated Schiff base (pSB) was broadened but not shifted in comparison with pR_WT. The spectrum of [¹⁵N-Lys]pR_SCFNG confirmed the assignment of both Schiff base and His⁷⁵ resonances. The spectrum of the pR_S89C mutant was identical to that of pR_WT. In contrast, the P90F mutant also showed a broad peak at 182 ppm and two broad peaks at 172 and 162 ppm. The spectrum of pR_InsNG was very similar to the ligation product. It also showed two broad peaks at 182 and 172 ppm. The spectra in A were recorded at 400 MHz/263 GHz at 115 K. The spectra in B were acquired at 850 MHz at 275 K. The proteins were reconstituted into l-α-dimyristoylphosphatidylcholine/1,2-dimyristoyl-sn-glycero-3-phosphate lipid bilayers. The pH was adjusted to 9.

FIGURE 7.

A and B, comparison of (A) ¹⁵N-¹³C (NCA) and (B) ¹³C-¹³C (proton-driven spin diffusion) correlation spectra of pR_WT, pR_SCFNG, and pR_LIG. Resolution and line width were comparable between all three samples. Most peak positions were conserved, as shown for some selected resonances, illustrating that assembling a membrane protein via protein trans-splicing does not alter its structure. Because of the alteration in the BC loop, some resonances disappeared in pR_LIG and pR_SCFNG compared with pR_WT (Asp⁸⁸ in Aii/Bi, Ser⁸⁹ in Aii, and Pro⁹⁰ in Bii). A few other peaks in pR_WT were shifted or broadened in pR_SCFNG and pR_LIG (Thr²⁹, Thr⁸⁶, Asp⁹⁷, Glu¹⁴², Lys¹⁷², Asn²²⁴, and an unassigned cross peak (asterisk)). The signal-to-noise ratio for the pR_LIG spectra was slightly reduced because of the lower amount of available protein. As for Fig. 6C, spectra were acquired at 850 MHz at 270 K. The proteins were reconstituted into l-α-dimyristoylphosphatidylcholine/1,2-dimyristoyl-sn-glycero-3-phosphate lipid bilayers. The pH was adjusted to 9.