Membrane protein crystallization

2.4.1.1

Detergents in crystallization of TOM core complex

Twenty to thirty-five percent of the proteins encoded by an organism`s genome are integral membrane proteins. Membrane proteins such as channels, transporters, and receptors are critical components of many fundamental biological processes242. Most of the successful experimental methods for obtaining membrane proteins crystals suitable for structure determination by X-ray crystallography are identical to those used for soluble proteins. The major difference is the necessity for inclusion of detergents above the critical micelle concentration (CMC) in the membrane protein solution. The most typical use of a detergent is to maintain a target membrane protein in a functional, folded state in the absence of a membrane. Eukaryotic multisubunit membrane proteins such as the TOM core complex cannot be overexpressed in E. coli and have to be isolated directly from its native environment, for example the outer mitochondrial membrane. When the membrane is removed during the solubilization of the protein, it must be replaced with a detergent as solvent. Despite the large number of detergents that are commercially available, no single “universal detergent” is ideally suited to all biochemical applications. As a result, the choice of detergent is one of the most fundamental decisions in isolation and crystallization procedures of membrane proteins. With essentially no exception, membrane proteins purified for structural studies are isolated in alkyl-chain detergents with generally 7-12 carbon in length and with headgroups that are typically uncharged or zwitterionic, but conceivably could also be charged. However, most membrane proteins have been crystallized from maltosides, glucosides, dimethyl N-amine oxides (e.g. LDAO) and CnEm polyoxyethylene detergents242.

The detergent n-dodecyl-β-D-maltoside (DDM, the alkyl-chain is 12 carbons in length) is used for the isolation of TOM core complex from mitochondria which results in highly pure and stable protein. The TOM core complex in DDM is functional: it has the characteristics of the general insertion pore, contains high-conductance channels and binds preprotein in a targeting sequence-dependent manner. In contrast to the holo complex, it forms a homogeneous double ring structure in electron microscopy231. DDM is one of the gentler detergents, and has very favourable properties for maintaining the functionality of more aggregation-prone membrane proteins in solution. Its main drawback is the formation of large

micelles making crystallization difficult. This may be responsible for the limited diffraction quality of TOM core complex crystals. The crystal lattice of a membrane protein crystal has to accommodate three components: protein, detergent and aqueous solution. Protein-protein contacts from the principal scaffolding of the lattice, and proteins with large extra- membranous domains are often favoured in crystallization trials. Small-micelle detergents such as octyl glycoside (OG) and lauryldimethylamine oxide (LDAO) form smaller belts around the transmembrane region of a protein. Potentially, this allows more contacts between exposed polar surface of the protein, and hence stronger lattices and better diffracting crystals243. Unfortunately, these detergents are often destabilizing and their application results in partial dissociation of TOM complex233. The use of different detergents can yield different crystal forms, as in the case of the bacterial photosynthetic reaction centre from Rhodobacter sphaeroides244 and often, the best-quality crystals may be obtained only in one or a small number of detergents. Due to the limited quality of TOM core complex crystals in DDM, detergent exchange was performed in this study by size exclusion chromatography to screen for more suitable detergents. The low CMC of DDM makes it considerably easier to remove DDM by dialysis or gel filtration. Membrane protein detergent exchange can be accomplished by a variety of methods. However, the size exclusion approach allows monitoring the stability of TOM core complex in various detergents by observation of the peak shape and the retention time. Thereby, aggregation and oligomerization are easily detectable. A very successful strategy for improving poorly-diffracting membrane protein crystals is to first optimize a lead condition by testing closely related detergents (for example, undecylmaltoside versus dodecylmaltoside). In general, the head group has a strong influence on the interactions of detergents with proteins, while the length of the alkyl chain affects the detergent CMC and aggregation number. Upon variation of alkyl chain-length the physical properties remain very similar, whereas the slight but significant differences in micelle size can result in improved packing within the crystal lattice243_{. For example, good-diffracting} crystals of bovine mitochondrial cytochrome c oxidase were obtained with the detergent decylmaltoside and varying of the detegent`s chain-length degraded crystal quality245. In case of TOM core complex maltosides with longer alkyl chain length like tridecylmaltoside238 and shortened maltosides like undecyl- and decylmaltoside were exchanged but could not improve crystallization. The α-anomer of DDM (n-dodecyl-α-D-maltoside) is generally less soluble and not commonly used. Although it was essential for the crystallization of the NhaA Na+/H+ antiporter239, no crystals of TOM core complex were obtained in α-DDM. A next level of optimization might involve the use of detergent mixtures as in case of the crystallization of bacterial outer membrane protein TolC246.

In addition to the choice of a suitable detergent its concentration in the protein-detergent complex (PDC) may be critical. Too much detergent can denature the protein or impede crystallization. Too little and the protein can become insoluble. Detergents self-assemble into relatively small, well-defined micelles that typically contain several hundred molecules243_. The number of molecules in the micellar particle is termed the aggregation number, a characteristic of each detergent. Every detergent possesses a critical micelle concentration (CMC). At concentrations above the CMC, there is equilibrium between monomers and an increasing concentration of micelles.

After the final purification step, the protein is often concentrated many-fold by centrifugation which can increase the detergent concentration, with possible negative consequences. This is especially true in case of a final gel filtration polishing step, which dilutes the protein. Excess detergent can be denaturing and can lead to a large amount of unwanted phase separation in crystallization experiments. Existing methods to decrease detergent concentration include dialysis, the use of absorbing materials such as BioBeads247,248 or other detergent removing gels (e.g., Extracti-Gel D, Pierce). However, for low CMC detergents possessing large micelles (such as dodecylmaltoside) dialysis fails. The application of the largest possible molecular mass cut-off filter will reduce the amount of detergent that is concentrated along with the protein. Due to the large size of TOM core complex (~400 kDa), centrifugal devices with 100 kDa cut-off can be used for concentration and even the large DDM micelles (~60 kDa) should pass the filter.

Controlling the detergent concentration may be important for crystal reproducibility. A variety of methods exist for determination of detergent concentration. For example, for detergents that contain a sugar headgroup (such as maltosides or glucosides), phenol/sulphuric acid hydrolysis and reaction with molybdate is a valuable colorimetric assay. Furthermore attenuated total reflection Fourier transformation infrared spectroscopy (ATR-FTIR) and quantitative thin-layer chromatography (TLC) can be used to determine the amount of detergent. It might be important for crystallization of TOM core complex to monitor the detergent concentration during the purification and crystallization procedures by one of these methods.

2.4.1.2

Lipid requirements of TOM core complex

Membrane proteins are isolated as protein-lipid-detergent particles and in addition to the detergent influence the lipid composition is often a critical aspect for preparing samples suitable for structural analysis. Indeed, specific lipids are sometimes required to maintain the

structural stability of membrane proteins. The detergent choice correlates with the presence of a particular amount of lipids in the PDCs as detergents can directly remove tightly bound lipids that may be important for the native structure of the protein. Ordered lipids have been observed in several high-resolution membrane protein crystal structures249,250_{, and excessive} detergent concentrations may lead to protein aggregation by stripping away these essential bound lipids. The presence of excess lipids in the PDC can also reduce the chances for crystallization since they reduce monodispersity251, but lipid/detergent mixtures may be favourable for maintaining protein function. For example, the first successful functional reconstitution of the lactose permease required a mixture of OG and lipids to preserve protein function during purification252. Without added phospholipids the protein did not survive OG solubilization. Twenty-five years later, the addition of a mixture of E. coli phospholipids was found to improve crystals of lac permease purified in DDM253. It can be very difficult to adjust the appropriate lipid levels in a protein-detergent complex. For instance, suitable crystals of the glycerol 3-phosphate transporter (Glp3T) were not obtained if too much lipid was removed during purification254, whereas optimal crystals of the bacterial cytochrome b6f complex were only obtained when lipids were added to the purified protein255.

Characterization of PDCs in respect to their lipid content is critical for the optimization of downstream sample preparation and crystallization. FTIR and TLC can both determine the amounts and specific types of lipids present. From experiments where lipids were added to the crystallization trials of TOM core complex no crystals were obtained, but determination of the lipid composition in the isolated complex might give hints about its lipid requirements and for optimization of its crystallization.

2.4.1.3

Additive approach in TOM core complex crystallization

The use of additives has played a significant role in membrane protein crystallization. Due to the high solvent content of protein crystals, soaking of chemicals into the crystal is possible. Soaking or co-crystallization with additives can improve the diffraction quality of crystals. Therefore, crystals were subjected to crystallisation additives such as detergents, heavy metal compounds and lipids. For instance, the use of amphiphilic additives (such as heptane-triol) has been shown to assist in the formation of highly ordered crystals256. Small amphiphiles can act to effectively reduce the size of the detergent region with subsequent improvement of crystal quality257,258. Heavy metal additives can deliver initial phases, form and stabilize crystal contacts, and have led to some spectacular improvements in resolution259,260.

In case of TOM core complex mainly FOS-choline12 and CuCl2 showed an effect on the crystallization. Addition of the detergent FOS-choline12 shifted the crystallisation condition to higher PEG concentrations and slightly improved crystal quality, whereas addition of CuCl2, which was reported to block the TOM channel221, increased the crystal size.

2.4.2 Antibody-fragment mediated crystallization of TOM