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2.3.5.2.1 Experiments to improve crystallization

Since the resolution limit of 7-8 Å abolished elucidation of the three-dimensional protein structure of TOM core complex, the diffraction properties of the crystals had to be improved considerably. All additives from the detergent screens from Hampton Research were tested in the standard TOM crystallization condition and used at CMC (critical micelle concentration). For all additives, where crystal growth occurred (Table 2.2), fine screens were performed to find the optimal concentration of PEG400. Optimized crystals, which were large enough for measuring, were examined using synchrotron radiation. For most of the additives, the diffraction quality of the crystals became worse and the resolution limit was significantly higher than 8 Å (Table 2.2). For maltosides with shorter carbon chain length than n-Dodecyl-

β-D-maltoside, such as n-Undecyl-β-D-maltoside and n-Nonyl-β-D-maltoside (Figure 2.12), the resolution limit was comparable to DDM, although crystals in n-Undecyl-β-D-maltoside were smaller.

Detergent MM (g/mol) CMC (mM) Type Synchrotron max. Resolution

C12E9 583.1 0.08 N Y >7 Ǻ C12E8 539.1 0.11 N Y >7 Ǻ n-Dodecyl-ß-D-maltoside 510.6 0.17 N Y 7 Ǻ Sucrose monolaurate 524.6 0.2 N N CYMAL®-6 508.5 0.56 N Y >7 Ǻ n-Decyl-ß-D-maltoside 482.6 1.8 N N ZWITTERGENT® 3-12 335.6 4.0 Z Y >7 Ǻ n-Hexadecyl-ß-D-maltoside 566.6 0.0006 N N n-Tetradecyl-ß-D-maltoside 538.6 0.01 N N n-Tridecyl-ß-D-maltoside 524.6 0.033 N N n-Undecyl-ß-D-maltoside 496.6 0.59 N Y 7 Ǻ n-Decyl-ß-D-thiomaltoside 498.6 0.9 N Y >7 Ǻ FOS-Choline®-12 315.5 1.5 Z Y <7 Ǻ 1-s-Nonyl-ß-D-thioglucoside 322.4 2.9 N Y >7 Ǻ n-Nonyl-ß-D-thiomaltoside 484.6 3.2 N N n-Nonyl-ß-D-maltoside 468.4 6.0 N Y 7 Ǻ

Table 2.2: List of detergent additives in which crystals grew still under the typical TOM core complex crystallization condition. N=nonionic, Z=zwitterionic. Yes and No in the column “Synchrotron” means, if the crystals were measured or not.

In case of the detergents sucrose monolaurate, n-Decyl-ß-D-maltoside and the maltosides with longer carbon chains than DDM, crystals were too small for measuring. FOS-Choline®-12

seemed to be the only detergent of the additive screen, which slightly improved the diffraction quality of the TOM core complex crystals to a maximal resolution of ~6.5 Å (Table 2.2, Figure 2.12).

Figure 1.12: Crystals of TOM core complex with different additives (a) C12E8. (b) FOS-

Choline-12. (c) n-Nonyl-ß-D-maltoside.

Since it was shown that copper ions can block channel activity of reconstituted TOM complex in conductivity measurements221, CuCl2 was tried as an additive for crystallization. A fraction of TOM core complex (10-14 mg/ml) mixed with CuCl2 is precipitating and if the mixture is used for crystallization, crystal grew to bigger size, but without any significant alteration of diffraction properties.

All crystallized membrane protein complexes show defined lipid-protein contacts and lipid requirement may also be necessary for stabilization of membrane proteins. Therefore, different lipids were used as additives to enhance ordered crystallization of TOM core complex, but addition of the lipids L-α-phosphatidylcholine, L-α-phosphatidylethanolamine, L-α-phosphatidylinositol and cardiolipin prevented crystal growth of the protein under the original crystallization condition.

Due to the thermostability of TOM core complex, another trial to improve crystallization was performed by incubation of the protein at 45°C for 20 min and subsequent crystallization, but no amelioration of the crystal order could be achieved by this experiment.

2.3.5.2.2 Manipulation of the crystals

The direct manipulation of already grown crystals represents an alternative option to the optimization of the crystallization conditions. It is known, that glutaraldehyde, a frequently used amine-reactive homobifunctional crosslinker reagent, can link protein molecules in a crystal and thereby improve the order of a crystal (Prof. Cramer, LMU Munich, personal communication). Such a protein crystal, in which the molecules are sufficiently crosslinked, will not dissolve in water anymore. Crosslinking of the protein molecules in the TOM core

complex crystal with this reagent was possible, but this treatment appeared to disturb the crystal order as the diffraction became even worse.

As typical for membrane proteins, it is expected that TOM core complex crystals possess a high solvent content, since crystals turned out to be very soft. An alternative to improve crystal order might be the stepwise transfer of TOM core complex crystals to increasing PEG400 concentration, thereby reducing the solvent content and possibly transforming the crystals to higher order. However, the crystals could not be transformed using this method.

2.3.5.2.3 Heavy metal atom soaks and cocrystallization

Owing to the high solvent content of protein crystals, it is possible to soak chemicals and in some cases even small proteins into crystals which bind at defined positions of the protein. Heavy metal atoms or heavy metal atom clusters are able to bind specifically to sites of a protein and are therefore essential for determining the crystallographic phases to solve the three-dimensional structure. Soaking of crystals with heavy metal atoms can not only deliver initial phases, but sometimes also improve the diffraction properties of the crystals by occupation of specific symmetry plies or alteration of crystal contacts. For this experiments, the Heavy Atom Screen kits (Hampton Research) and the collection of heavy metal atom clusters like W6Br12, W18, K2Ta6Br12, Mo6Cl14 and K6Mo7O22(O2)2 (kindly provided by Prof. Patrick Cramer, Gene Center, Munich and Prof. Huber, Max-Planck-Institute of Biochemistry, Munich) served as the platform to try various different heavy metal compounds. Additionally, heavy metal atoms were used for co-crystallization trials with TOM core complex. In case of Ta6Br122-, which shows a green colour in solution, the soaked crystals adopted the colour, indicating the presence of the cluster in the crystals (Figure 2.13). However, the diffraction quality of the crystals suffered for each employed heavy metal and consequently, determination of initial phases was not possible.

Figure 2.13: TOM core complex crystals, soaked with the TaBr heavy metal atom cluster.