Study Human Chemokine Receptor Figure 5. Systematic detergent screens. 96 detergents were systematically screened for their ability to solubilize the Trx-hCRs protein preparation. 96 detergents were selected from the commercial Solution Master Detergent Kit and from literature. Detergents generally are divided into 4 groups depending on their chemical properties, namely, anionic (A), cationic (C), non-ionic (N) and zwitter-ionic (Z). For the majority of detergents, 1% was thus chosen as a standard concentration during the extraction trials, except for detergents with higher CMC, where ,2% was used. As can be seen from the figure, several clusters of detergents are most effective, particularly phosphocholine (FC) series. doi:10.1371/journal.pone.0004509.g005 phosphocholine (Fos-) series are most effective for solubilizing and stabilizing human olfactory receptors hOR17-4 that was large- scale produced from mammalian cell [46, Cook, unpublished results]. Cook subsequently found FC14 is optimal for all the hOR17-4 study. Likewise, Liselotte Kaiser and Johanna Gravel- and-Bikker also found that FC14 is optimal for stabilizing several olfactory receptors produced in wheat germ cell-free system [51] and in yeast Pichia production [unpublished results]. However, it is unknown if FC14 would work equally well in human chemokine receptors. We again carried out a systematic search for optimal detergents Each Trx-hCR expressed by pBAD-hCRs in Top10 was simultaneously solubilized in each of the 96 detergents in a 96- well plate and the extract was examined using Dot blot detection. The extraction of Trx-hCR proteins by detergents was tested on the whole cell lysate (see Method section). The images of dot blots are shown in Figure 6. The higher the solubility it has, the darker the dots are. The average intensity of spots was determined by scanning using FluorChem software. It is presented as a 2D column in Figure 5, with the numbers a1-h12 corresponding to the detergents 1–96. We carried out analyses by comparing the solubilizing effectiveness of various detergents for Trx-hCR proteins. We identified the most effective detergents (Fig. 5, Fig. 6,). We found two series of zwitter-ionic (Z) detergents, CycloFos series (#26–29) and Fos-Choline series (#37–44) to be most effective of solubilizing all of the Trx-hCRs. Another zwitter-ionic detergents n-Dodecyl-b-iminodipropionic acid, monosodium salt (#79) was also reasonably effective to solubilize Trx-hCRs. Anapoe-35 (#3, termed Brij-35), Anapoe-58 (#4, termed Brij-58) and Anzergent series (#16–18) showed specificity in solubilizing Trx-hCCR5. Maltosides (#62–70), glucosides (#60, 61) and thio-maltosides (#71–76) series were moderately effective for solubilization of Trx- hCRs. Cymal series (#31–34) showed a higher solubilization of Trx-hCCR5 than of other hCRs, including the Cymal-5, which has been used for solubilization of functional CCR5 [61]. Digitonin, CHAPS, DDM and some detergent mixtures, which are of rather general use for GPCR solubilization, show certain capability of isolation of hCRs. Zwitter-ionic detergents were the most effective for solubilizing hCRs in this system, as they were for solubilizing other GPCRs. For example, DDAO was used for structural studies on rhodopsin, CHAPSO is able to maintain the native structures of CCR5 and CXCR4, and LDAO has been used for solubilizing human leukotriene B4 receptor [62,63,64,65]. The Fos-Choline series shares the same hydrophilic phospho- choline (Fos-) head group with CycloFos series, but possess a simple hydrophobic tails that likely makes them more effective than CycloFos series. Recently, some members of Fos-Choline series have been used to solubilizing GPCRs, for example, Fos- Choline-16 (FC16) was selected as the suitable detergent for human NK1 receptor (hNK1R) [66]; Fos-Choline 10, 11, 12 (FC10, FC11, FC12) were tested in a high-throughput expression system for membrane proteins, and revealed excellent capabilities for solubilizing membrane proteins, however, at a similar level with maltosides series, NM, DM, UDM and DDM [67,68]; Fos- choline 14 was used to solubilize human CCR5, but was less effective than Cymal-5 [61]. Fos-cholines 12–16 (#40–44) were the most effective detergents for solubilizing hCRs in our study. Stability of Trx-hCRs (Trx-hCCR5 shown in Figure 7E) was tested in the presence of each of the 16 optimal detergents. Fos- choline14 (#42, FC14) was the most effective of all the tested detergents for stabilizing TRX-CCR5. For the remaining 3 Trx- hCRs, FC13, FC14 and FC15 were effective as well. It is possible that n-Dodecyl-b-iminodipropionic acid, monosodium salt (#79) is also a good choice for CCR5 and CXCR4, and CX3CR1. It is as effective as Fos-choline series, particularly FC14, but it was less effective for Trx-hCCR3. We used Western blot detection to study the solubilization of Trx- hCRs. Samples of Trx-hCCR3 and hCX3CR1 were solubilized using FC14 and FC12 respectively. FC14 proved more effective for solubilization of Trx-hCCR3 and hCX3CR1 than FC12 (Figure 6F). FC14 was previously found to solubilize and to purify several olfactory receptors including human hOR17-4, mouse mOR23, mS51 and I7 [46]. Furthermore, Fos-choline-14 was used in the crystallization of the E. Coli mechanosensitive ion channel MscS for a successful structure [69]. One purified E.coli membrane protein in our laboratory has also been crystallized using FC14 and diffracted to 3.6 A˚ resolution (unpublished results). Based on current and the previous detergent optimization, FC14 detergent was selected as standard detergent in all subsequent hCR purifications. High-level production of Trx-hCRs Each of the pBAD-hCRs produced in Top10 was scaled up to 1 liter for high-level production of Trx-hCRs. Washed cell membrane fractions were dissolved in the Solubilization buffer containing 1% FC14 and subjected to ultracentrifugation at 100,0006g for one hour to remove the non-solubilized proteins and collect the supernatant for purification. The purification of Trx-hCCR3 is a simple 2-step process (Fig. 7A). First, the protein was purified by Ni2+ chelation chromatography (I), then further purified using size exclusion gel filtration (II). All protein fractions were monitored by SDS-PAGE and Western blot detection (Figure 7B). The proteins purified from the first step include monomer, dimer and higher oligomer of Trx-hCCR3. These species could be further resolved in the gel filtration step. Trx-hCCR5, Trx-hCXCR4 and Trx-hCX3CR1 were also scaled up and purified, but with a lower yields than for Trx- hCCR3 (Figure 7D). The yields of purified Trx-hCRs finally achieved in this system were ,1–3 mg/L for Trx-hCCR3, 0.1– 0.3 mg/L for Trx-hCCR5, 0.05–0.1 mg/L for hCXCR4 and 0.5– 1 mg/L for Trx-hCX3CR1. Individual membrane protein behaves differently, hCCR5 and hCXCR4 showed higher cell toxicity than hCCR3 and hCX3CR1, which may explain the tenfold lower yield of these proteins. Secondary structure analysis Circular dichroism (CD) is one of the best methods to rapidly study secondary structures of proteins, and the far-UV CD spectroscopy from 200 nm to 250 nm can be used to estimate contents of secondary structures. We used the purified Trx- hCCR3 for CD study. The CD profile displayed a typical a-helical curve with ,40% helical content (Figure 7C). This value is somewhat lower than the theoretical ,45% (NNPredict at http:// PLoS ONE | www.plosone.org 8 February 2009 | Volume 4 | Issue 2 | e4509