A property fine-tuned sulfobetaine cholesterol derivative for membrane protein structural biology

Abstract

• Membrane proteins (MPs) play essential roles in biological and pharmacological processes. Many of them require cholesterol (CHOL) as their structure supportive lipid component and modulator for functionality. Unfortunately, commercially available CHOL and its derivatives have limitations on solubility or bio-activity.

• We report 4-((cholesteryloxy)-4-oxobut-2-enamido)ethyl) dimethylam-monio) propane-1-sulfonate (CHEAPS), a novel CHOL alternative for MP study. CHEAPS can be readily solubilized in both detergents and membrane-active polymers. Interestingly, it could stabilize and support a new discovered enzyme function of human mitochondrial TSPO. As such CHEAPS may have a broad application for MP structural biology.

Many modern drugs exert their functions through modulating membrane proteins (MPs), which play many crucial roles in living organisms [1]. Distinct from soluble proteins, the native structure and function of MP often rely on lipid environments. The dependency of the lipid environment of MPs coincides with the coevolution between MPs and their natural cell membrane environment [2]. Cholesterol (CHOL) is an essential component in the mammalian cell membrane system. Consequently, the natural structure and function of many mammalian MPs are CHOL-dependent.

Biochemical and biophysical characterization of MPs often require isolation from their natural cell membrane environments and purification afterward. Detergents work very well in extracting MPs from the cell membrane system. However, many of them are not stable and function once isolated with detergents, mainly due to delipidation [2]. Thus, CHOL derivatives are usually supplied in the purification buffer for mammalian MPs to maintain and mimic the protein-CHOL interactions. To some extent, this has been successful.

Thereby, many CHOL derivatives were developed, such as CHAPS, CHAPSO, CHAPSTEROL, CHOBIMALT and CHS to support for MP studies [3-6]. Despite owning propitious detergency properties, CHAPS, CHAPSO and CHAPSTEROL could not substitute CHOL in protein function study due to their structural differences from CHOL. CHOBIMALT has true CHOL as a non-polar portion, which makes it effectively retain the activity of a purified protein. Nevertheless, it serves as a detergent and its synthesis protocol requires eight linear steps to achieve an isolated product, with only 10% of the overall yield [3]. To date, cholesteryl hemisuccinate (CHS) is the most commonly used CHOL derivative for biochemical investigation. Despite its popularity in the purification of MPs, particularly G protein-coupled receptors, [7] some drawbacks are still present. CHS has no aqueous solubility and needs a relatively high concentration of detergents and strongly ultrasonic agitation to afford a homogeneous solution. At high pH, its solubility can be improved; however, a more abundant anionic form causes a difference in the interaction with a lipid bilayer than CHOL [8]. Besides, it cannot be solubilized in membrane-active polymers such as styrene-maleic acid (SMA) copolymers for MP functional study and high-resolution structure determination [9]. To overcome the limitations, we need non-detergent CHOL derivatives compatible with both detergents and membrane-active polymers. To our knowledge, currently, no such chemical is commercially available. This communication describes our design, synthesis, characterization, and application of CHEAPS, a property fine-tuned sulfobetaine CHOL derivative for enzyme activity measurement of a newly discovered CHOL-dependent human mitochondrial enzyme, HsTSPO.

CHEAPS possesses a CHOL portion linked to a zwitterionic sulfobetaine segment via an ester bond. Sulfobetaine has good compatibility with various pH conditions and divalent ions [10]. Three sequential modifications, including esterification, Steglich amidation, and sulfopropylation, are required to synthesize CHEAPS (Fig. 1A). Unlike other zwitterionic amphiphiles, this unique CHOL-derivative is non-hygroscopic and chemically stable.

Fig. 1.

Synthesis and characterizations of CHEAPS and its application in enzyme activity of HsTPSO. (A) A multiple-step preparation of CHEAPS. (B) ¹H-NMR spectrum recorded in CDCl₃ at room temperature. (C) FT-IR spectrum of isolated CHEAPS fully characterized by ATR mode. (D) Dose dependent of the degradation of HsTSPO in LMNG and CHEAPS. (E) The degradation of PpIX with activity of HsTSPO.

The structure of CHEAPS was confirmed by NMR analysis providing the characteristic signals of a pure sulfobetaine compound, thereby indicating the formation of the target CHEAPS (Fig. 1B). Further characterization was complemented with electrospray ionization mass spectrometry (ESI-MS), where the mass-to-charge ratios of CHEAPS ion (m/z 699.44) and its fragment (m/z 555.45) detected was in good agreement with the theoretical values (Fig. S1). Finally, Fourier-transform infrared (FT-IR) characterization went along with the previous analyses. It shows characteristic peaks of ester (C=O stretching, 1725 cm⁻¹), amide (C=O stretching, 1675 cm⁻¹ and N-H bending, 1550 cm⁻¹), quaternary ammonium cation (C-N stretching, 1210 cm⁻¹), and the strong signals at 1170 cm⁻¹ and 1037 cm⁻¹ assigned to the vibrations of sulfonate anion (Fig. 1C). Altogether, these results suggest that the CHOL was covalently bonded to the zwitterionic head group and not present in its free form.

Modification with more hydrophilic groups, such as amide, sulfonate and ammonium groups, significantly improves hydrophile-lipophile balance (HLB) of CHEAPS (HLBCHEAPS = 28.23 > HLBCHOL = 1.00, calculated based on Davies’ method). In theory, amphiphilic molecules with high HLB are miscible in water. However, our CHEAPS is insoluble in either water or saline/water mixture. Interestingly, it has good solubility in water containing detergents and amphiphilic polymers, such as LMNG, DDM and SMA copolymers, and slightly dissolves in chloroform under ultrasonic agitation. Non-aqueous solubility is assumed due to its lack of potential H-bonding formation with water. The absence of polar atoms in the steroid and hydrocarbon side chains cannot donate or accept hydrogens from water. Moreover, substantial intra- and intermolecular attractions between sulfonate anion and quaternary amine cation of polar fragment prevent the hydration. A similar tendency has also sought some polysulfobetaines that were solely swelling or soluble in the presence of inorganic salts or detergents [11]. Existing ionic molecules presumably screen zwitterionic charges that interrupt ionic bonds and bring profound changes in solvation. Because of having no interaction with pure water, CHEAPS does not absorb moisture. As expected, after long-term exposure to air, there is no weight loss at around 100 °C observed in the TGA thermograph of CHEAPS, indicating no water intake (Fig. S2). Non-hygroscopic character enables overcoming reported problems found for other steroid-based facile amphiphiles in storage, handling and scaled-up production.

CHEAPS is highly comparative to commercial CHOL-based amphiphiles as a new CHOL derivative employed in membrane chemistry. Containing sulfobetaine fragment, which is more hydrophilic than the carboxylic acid group, CHEAPS exhibits better solubility in detergents, ~44 mg/mL in 5% DDM and ~ 51 mg/mL in 5% LMNG, than CHS [12,13]. It can especially be solubilized up to ~62 mg/mL in SMA solution (5% w/v in water) without any addition of classical detergents. On the other hand, it weakly absorbs light at 280 nm (ε₂₈₀ ~ 0.86 × 10³ M⁻¹·cm⁻¹ in either DDM or LMNG solutions, Fig. S3). Thereby, CHEAPS will not seriously hamper the monitor of proteins by UV/Vis spectroscopy at this wavelength. On the other hand, despite containing ester and amide bonds, CHEAPS is highly stable at room temperature and even in alkaline and acidic solutions (Fig. S4 - S5). It provides an oppotunity to study MPs at various pH conditions.

Human mitochondrial TSPO (HsTSPO) is a newly discovered CHOL-dependent MP enzyme in the laboratory of Dr. Guo (Qiu., et al., 2021, to be published). The catalytic activity of HsTSPO strictly depends on steroid molecules. A previous study shows that TSPO purified with detergent can maintain its enzymatic functions to degrade the PpIX into bilindigin [14]. Thereby, we used CHEAPS instead of CHS to scrutinize enzyme activity of HsTSPO solubilized from its native membrane by LMNG. A single-step Nickle column purification resulted in relatively pure HsTSPO (Fig. S6), enabling the investigation of enzyme assay (Fig. 1D and E). Fluorescence analysis of PpIX degradation showed fresh purified HsTSPO with CHEAPS has strong enzyme activity (Fig. 1D -E). Success in protein purification and enzyme characterization suggests that CHEAPS effectively binds to HsTSPO protein and maintains its natural structure.

As a novel steroid analog, which membrane-active polymers can solubilize, CHEAPS may be incorporated into the emerging detergent-free systems [2] as a useful tool for analyzing steroid-dependent MPs. When MPs proteins are overexpressed, the natural CHOL on the cell membrane may not be sufficient to satisfy the requirement of overexpressed MPs to maintain the natural structure and function. In this case, CHEAPS might be introduced with membrane-active polymers in the course of membrane solubilization as in the case CHS is often supplied with detergents for MPs for which the CHOL molecules are crucial for their structural and functional integrity. Hence, CHEAPS may have broad applications for many CHOL-dependent MPs. CHEAPS can substitute CHS in the detergent-based method and it is easier to be synthesized and solubilized in detergents than CHS. Besides, this compound is not hygroscopic and hence stable to be stored than CHS. More importantly, CHEAPS is compatible with membrane-active polymers, make it a novel molecular tool for the investigation of MP-CHOL interactions using the emerging detergent-free systems.

In summary, we have designed and synthesized a novel CHOL derivative (CHEAPS). CHEAPS has many advantages relative to the popular CHS on the market, such as solubility, stability, compatibility with a broader range of pH conditions and, most importantly, compatibility with membrane-active polymers [15]. We have demonstrated that CHEAPS interacts with HsTSPO, a strict CHOL-dependent PpIX oxygenase and preserves its enzyme activity. CHEAPS may have a broad application for MP structural biology and related drug discovery and development.