Promoting crystallization of intrinsic membrane proteins with conjugated micelles

A new technique for promoting nucleation and growth of membrane protein (MP) crystals from micellar environments is reported. It relies on the conjugation of micelles that sequester MPs in protein detergent complexes (PDCs). Conjugation via amphiphilic [metal:chelator] complexes presumably takes place at the micelle/water interface, thereby bringing the PDCs into proximity, promoting crystal nucleation and growth. We have successfully applied this approach to two light-driven proton pumps: bacteriorhodopsin (bR) and the recently discovered King Sejong 1–2 (KS1–2), using the amphiphilic 4,4′-dinonyl-2,2′-dipyridyl (Dinonyl) (0.7 mM) chelator in combination with Zn2+, Fe2+, or Ni2+ (0.1 mM). Crystal growth in the presence of the [metal–chelator] complexes leads to purple, hexagonal crystals (50–75 µm in size) of bR or pink, rectangular/square crystals (5–15 µm) of KS1–2. The effects of divalent cation identity and concentration, chelator structure and concentration, ionic strength and pH on crystal size, morphology and process kinetics, are described.

. Crystallization strategy. Spontaneous partitioning of an amphiphilic chelator into membrane protein detergent complexes (PDC) (Step I). Conjugation of the PDCs is accomplished by the addition of divalent metal cations capable of binding 2-3 chelators at the micelle/water interface (Step II, red arrow) Step III-growth of three-dimensional crystals.
Conjugating membrane protein-containing OTG/phospholipid mixed micelles. The conjugation of empty OTG micelles paved the way to assessing this approach with PDCs containing bR, native phospholipids and OTG (Fig. 3). The PDCs had been obtained by dissociating purple membranes with OTG (see "Methods" section) and validating that the characteristic bR absorption spectrum had been preserved (online Supplementary Information, Figure S1). With the availability of micellar preparations containing native bR, the effects of three divalent cations (Fe 2+ , Co 2+ , Cd 2+ ) on the efficiency of conjugation were studied (online Supplementary Information, Figure S2). We found that the [(Dinonyl) 3 :Zn 2+ ] complex successfully conjugated micellar aggregates and generated large purple oil-rich globules (20-100 µm) after 1 day of incubation in the dark at 19 °C (Fig. 3A). Control experiments in the absence of Zn 2+ , demonstrated the contribution of Zn 2+ ions to the conjugation process. Without Zn 2+ , globules reached a maximal size of ~ 30 µm and were significantly less purple, indicating a lower bR concentration within the globule (Fig. 3D, upper right image). The fact that globules form upon introduction of the Dinonyl chelator, but without zinc ions, is likely due to the presence of the ammonium sulfate (AS) precipitant. The latter, like many other precipitants commonly used in macromolecular crystallization protocols (e.g. PEGs), compete with solutes in the system for binding to water molecules and by so doing, reduce their water-solubility. This holds true also for hydrated PDCs which, upon introduction of AS, are dehydrated and this in turn leads to their clustering and fusion into stable oil-rich, purple globules as shown in Fig. 3D.   www.nature.com/scientificreports/ Chelator structure and growth of bR crystals. Other parameters that were expected to affect crystal growth included chelator structure and concentration, molarity of NaCl and pH. The chemical structure of the Dinonyl chelator is available in Fig. 3. Repetition of the crystallization protocol with a Dinonyl analog, 4,4′-diphenyl-2,2′-dipyridyl (Diphenyl), did promote crystallization of bR ( Supplementary Information, Figure S3). However, the crystals were smaller (max. 20 µm) and were not hexagonal (online Supplementary Information, Figure S3). Changing the Dinonyl concentration at constant Ni 2+ concentration (0.1 mM), revealed a dramatic effect (online Supplementary Information, Figure S4). With 0.175 mM Dinonyl, hexagonal purple www.nature.com/scientificreports/ crystals grew within 21 days, whereas with 1 mM Dinonyl, no crystals were observed at all, even after 60 days ( Figure S4, inset). An additional important parameter was found to be NaCl concentration. The largest crystals grew in the presence of 100 mM NaCl. With 20 mM NaCl, there were few crystals and they exhibited a globular morphology. With 50 mM NaCl, numerous small (5-15 µm) crystals appeared. However, with 500 mM NaCl, crystal growth was abolished (online Supplementary Information, Figure S5). The pH (5.2) of the hanging drop was a constant: higher or lower pH values failed to promote crystallization (not shown).

Crystallization of KS1-2.
Our protocol was also tested on KS1-2, a recently identified 255 amino acid membrane protein 26 (Fig. 5). KS1-2 functions as a light-driven proton pump rhodopsin bound covalently to the retinal chromophore. Since KS1-2 pigment absorbs in the green-blue region of the visible spectrum (λ max 517 nm) the proteins appear orange\purple-red in white light and pink crystals were accordingly expected. The MP was purified with DDM as the detergent (see "Methods" section) and pink rectangular/square crystals (5-15 µm) appeared only in the presence of the [(Dinonyl) 3 :Zn 2+ ] complex and with the same metal (0.1 mM) and chelator (0.7 mM) concentrations used for bR (Fig. 3). After 8 days, crystals reached their maximum size. They appear to be either fused to each other (Fig. 5A, left) or independent (Fig. 5A, right) but no crystals were seen in the absence of Zn 2+ ions (Fig. 5A, right, inset). Na citrate buffer, pH 5.2 was the same as used for bR; however, the precipitant was PEG-4000, rather than ammonium sulfate (AS). The [(Dinonyl) 3 :Fe 2+ ] complex also produced fused, square pink KS1-2 crystals (Fig. 5B, left) as well as crystals with a different morphology (Fig. 5B, right). Use of the [(Dinonyl) 3 :Ni 2+ ] complex led to significantly smaller, fused crystals (Fig. 5C, left), although a few diamond-shaped single crystals (5-10 µm) were also observed (Fig. 5C, right).

Discussion
Three-dimensional, high resolution crystal structures are seriously lacking for intrinsic membrane proteins (MPs). Detergent micelle environments for MPs during crystallization trials currently represent the most popular and convenient "playground" for structural biologists; nevertheless, crystallization of MPs, particularly those with small hydrophilic loop regions, remains an unpredictable, fine art. www.nature.com/scientificreports/ Two lines of evidence imply that observed crystals are comprised of the target membrane proteins: (a) No crystals of any type were observed when target membrane proteins (bR or KS1-2) were absent in the crystallization drops. (b) Interestingly, the observation of hexagonal purple bR crystals in this study was already observed with well-established crystallization strategies. Purple hexagonal bR crystals grew from lipid cubic phases 21,27 and from the "successive fusion vesicle" strategy 28 . However, diamond shaped purple bR crystals, grew from bicelles 29 .
Investigating the possibility of bringing protein detergent complexes (PDCs) into proximity via specific micellar conjugation led to two very encouraging findings. First, crystals of target proteins (bR or KS1-2) grew only in the presence of both the chelator (Dinonyl) and appropriate metal cations. The mandatory dependence of crystal growth on these two additives is consistent with our working hypothesis, demonstrates process specificity, and the existence of a simple chemical mechanism capable of promoting crystallization. That these crystals contain native, chromophore-containing, target membrane proteins is supported by the fact that no crystals of any type were observed when MPs (bR or KS1-2) were absent from the crystallization drops. Since it is known that a change in the chromophore conformation (or its hydrolysis from the protein) leads to the loss of color, color-preservation serves as an internal indicator for the presence of the protein's native state within the crystal. Second, once the optimal concentrations of chelator and metal had been successfully determined, crystallization was observed in > 80% of the hanging drops in each of > 10 independent trials. Such high process reproducibility may not be common in other MP crystallization protocols and demonstrates the efficiency and robust character of our approach.
Our strategy for crystallizing MPs, based on large-scale conjugation of PDCs, demonstrates several important advantages in comparison with the variety of procedures already in use by structural biologists [30][31][32] . These advantages include: (i) Covalent modifications, e.g. amino acid "tags", are not required; crystallization trials of PDCs can proceed directly from optimized expression and purification of the protein under study. (ii) Concentrations of cations (0.1 mM) and amphiphilic chelator (0.7 mM), adequate for producing micelle conjugation, are sufficiently low that the native states of the MPs studied (bR and KS1-2) appear to be preserved. (iii) PDCs may contain either non-ionic detergents (e.g. DDM in the case of KS1-2) or negatively charged mixed micelles (native phospholipids + OTG) as in the case of bR. (iv) Our strategy is sufficiently flexible to permit a wide range of working conditions. The Dinonyl chelator remains functional under both acidic or basic conditions. In this study, the chelator was found to be usable after 60 days at pH 5.2. (v). The low cost and commercial availability of all components, in particular the Dinonyl chelator, facilitate rational screening of diverse MPs. The chelator and divalent metal cation are simply added to the micelle dispersion as with any other additive. Furthermore, no special instrumentation is needed. Though the crystals obtained were too small to permit structure determination by X-ray diffraction, we are encouraged by the fact that color was preserved (purple for bR and pink for KS1-2), indicating that the MP's active site had not been distorted. Slow progress in obtaining high quality crystals of MPs remains a major bottleneck in drug design; to alleviate this problem, it will be necessary to apply as many innovative approaches as possible. We trust that our strategy will aid in overcoming this problem. Preparation of purple membranes. Halobacterium salinarum was grown from the S9 strain and purple membranes containing bacteriorhodopsin (bR) were isolated as previously described 25 . Expression and purification of King Sejong 1-2 KS1-2 were prepared according to Ghosh et al. 26 . Codon optimized KS1-2 was expressed in Luria-Bertani (LB) medium in Escherichia coli BL21 (DE3) cells. E. coli transformant was grown to OD (λ 600 ) = 0.8 in the presence of ampicillin (50 μg mL −1 ) at 37 °C. The cells were then induced with 0.1% IPTG (isopropyl β-d-1-thiogalactopyranoside) and 10 μM all-trans retinal for 8 h. Pink-colored cells were harvested by centrifugation at 4 °C, followed by resuspension with buffer I (50 mM 2-(N-morpholino) ethanesulfonic acid, (MES), 300 mM NaCl, 5 mM imidazole, 5 mM MgCl 2 ; pH 6) containing 1% DDM (n-dodecyl-β-d-maltoside) and lysed with lysozyme (0.1 mg mL −1 ) in the presence of DNase and a protease inhibitor. The mixture was stirred overnight at room temperature. The extracted protein was collected as a supernatant after centrifugation of the stirred solution at 18,000 rpm and 4 °C for 30 min. The protein was purified using a Ni 2+ NTA histidine-tagged agarose column. The histidine-tagged protein was washed with buffer II (50 mM MES, 300 mM NaCl, 50 mM imidazole, 0.06% DDM; pH 6) and eluted with buffer III (0.06% DDM, 50 mM Tris-HCl, 300 mM NaCl, 50 mM HCl, 150 mM imidazole; pH 7.5). The eluted protein was washed and concentrated using a 0.02% DDM solution using Amicon ultra centrifugal filter devices. The final KS1-2 was kept in 0.06% DDM. www.nature.com/scientificreports/ Solubilization of purple membranes with OTG. Into 15 µL of purple membranes (OD 14) in DDW, 8.5 µL of 100 mM octylthioglucoside (OTG), 7.1 µL of 0.5 M Na citrate (pH 5.2) and 40 µL of DDW were added to a total volume of 71 µL. The system was incubated for 3 h at 19 °C in the dark, followed by a short spin (15,000 rpm, 10 min, 19 °C). The resulting transparent purple supernatant was used immediately for crystallization trials.

Optical absorption of bR and KS1-2 with and without the [(Dinonyl) 3 :Ni 2+ ] complex.
The absorption spectrum of bR extracted from its native purple membrane with OTG (as described above) was measured ( Figure S6, A). The characteristic absorption of native bR at 565-568 nm was preserved when the amphiphilic chelator (Dinonyl), the metal (Ni 2+ ) or both were added and incubated for up to 24 h at 19 °C in the dark ( Figure S2, A). However, a decrease of ~ 20% in the OD of bR at 565-568 nm was observed after 1 day of incubation in the presence of the [(Dinonyl) 3 :Ni 2+ ] amphiphilic complex, presumably due to the ability of the latter to trigger bR aggregation. A similar decrease in the OD at 517 nm of KS1-2 in DDM micelles was observed in the presence of the same amphiphilic complex, after 1 day of incubation at 19 °C in the dark ( Figure S7, A). However, in this case a shoulder at ~ 476 nm was also observed after 8 h of incubation which did not further change following over-night incubation at 19 °C in the dark ( Figure S7, A).