SLayer Proteins on Solid Supports

3.2.1. Preparation of Solid Supports

Silicon wafers were immersed in hot acetone followed by rinsing in propan-2-ol and finally washed with ethanol and Milli-Q water. The advancing contact angle of water on the clean silicon surface was 65°. To increase the hydrophilic-ity of the substrates, the silicon wafers were treated in an O2 plasma (20-s leaning time, 0.01-bar plasma pressure, 70% power density, high-purity-grade O2). The plasma-treated silicon substrates with an advancing contact angle of water of 5° were used immediately for the recrystallization studies. Other solid supports (e.g., metals, polymers, glass) were only rinsed with ethanol and Milli-Q water before use.

Silanization procedures (solution or vapor phase) using different silanes were applied to obtain silicon or glass substrates with more-hydrophobic surfaces (17,18). The substrates were cleaned in a solution containing 1:1:5 parts of ammonia (29%), hydrogen peroxide (30%), and Milli-Q water at 80°C for 10 min. Subsequently, the silicon or glass substrates were treated with 1:1:6 parts of concentrated hydrogen chloride (37%), hydrogen peroxide (30%), and deionized water at 80°C for 15 min. Finally, the substrates were rinsed thoroughly with Milli-Q water and dried in a stream of nitrogen gas. This procedure is known as RCA cleaning.

For silanization out of a solution, the substrates were further rinsed with acetone and dried with toluene. Subsequently, the supports were put into anhydrous toluene containing 1% silane. Silanization (e.g., with decyl-dimethylsilane), was carried out for 30 min to 2 h with mild shaking at room temperature. Finally, the silanized supports were rinsed with toluene, methanol, and Milli-Q water.

Silanization from vapor phase was performed with silanes of shorter chain lengths (e.g., hexamethyldisilane). Supports were baked with some drops of silane in an airtight glass vessel at 60°C for 2 h and finally rinsed with methanol.

3.2.2. S-Layer Protein Recrystallization on Solid Supports

For recrystallization of the S-layer protein SbsB of G. stearothermophilus PV72 and SbpA of B. sphaericus CCM 2177, buffer B and buffer C were used, respectively. The protein concentration in all experiments was 0.1 mg/mL. Recrystallization on solid supports was carried out either in rotating Eppendorf tubes that had been previously filled with the protein solution or in glass wells. In the latter case, the substrates were placed onto the air-liquid interface. After a recrystallization time of 4 h at room temperature, the supports were removed by tweezers, washed, and stored in Milli-Q water (4°C).

3.2.3. Atomic Force Microscopy

Scanning was carried out in contact mode in a liquid cell filled with a 100 mM NaCl solution (Fig. 4). The applied force was kept to a minimum during scanning to prevent modification of the sample surface by the tip. Scan speed was approx 6 Hz. Images were flattened line by line during recording using the microscope's software.

Fig. 4. Scanning force microscopic image of S-layer protein SbpA from B. sphaericus CCM 2177 recrystallized on a silicon wafer. The image was recorded in contact mode in a liquid cell (bar = 50 nm). (Reprinted from ref. 8 with permission from the publisher; © 2003, Wiley-VCH.)

Fig. 4. Scanning force microscopic image of S-layer protein SbpA from B. sphaericus CCM 2177 recrystallized on a silicon wafer. The image was recorded in contact mode in a liquid cell (bar = 50 nm). (Reprinted from ref. 8 with permission from the publisher; © 2003, Wiley-VCH.)

AFM studies showed crystalline domains with average diameters of 10 to 20 ^m for SbsB and 0.1 to 10 ^m for SbpA, when crystallized on a variety of solid supports (see Table 1). In particular, SbsB generated crystalline monolayers only on hydrophobic solid supports, whereas SbpA formed extended crystalline domains on hydrophilic surfaces, but only small patches on hydrophobic ones.

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