Engineering subunit association of multisubunit proteins: a dimeric streptavidin

Abstract

A dimeric streptavidin has been designed by molecular modeling using effective binding free energy calculations that decompose the binding free energy into electrostatic, desolvation, and side chain entropy loss terms. A histidine-127 --> aspartic acid (H127D) mutation was sufficient to introduce electrostatic repulsion between subunits that prevents the formation of the natural tetramer. However, the high hydrophobicity of the dimer-dimer interface, which would be exposed to solvent in a dimeric streptavidin, suggests that the resulting molecule would have very low solubility in aqueous media. In agreement with the calculations, a streptavidin containing the H127D mutation formed insoluble aggregates. Thus, the major design goal was to reduce the hydrophobicity of the dimer-dimer interface while maintaining the fundamental structure. Free energy calculations suggested that the hydrophobicity of the dimer-dimer interface could be reduced significantly by deleting a loop from G113 through W120 that should have no apparent contact with biotin in a dimeric molecule. The resulting protein, containing both the H127D mutation and the loop deletion, formed a soluble dimeric streptavidin in the presence of biotin.

Figure 3

Gel filtration chromatography of Stv-43. (A) Purified Stv-43 (approximately 3 μg) with excess d-[carbonyl-¹⁴C]biotin was applied to a Superdex 75 HR 10/30 column (1.0 × 30 cm) that had been equilibrated with TTBS. Proteins were eluted at room temperature (22°C) with TTBS, and the absorbance at 280 nm was monitored (solid line). The eluate was collected in 320-μl fractions, and the radioactivity of each fraction was determined (○). The minimum-sized core streptavidin (50.4 kDa) (26) was also analyzed, and the absorbance at 280 nm is shown by a dashed line. (B) Stv-43, which had been stored without bound biotin at 4°C for 7 days, was mixed with excess d-[carbonyl-¹⁴C]biotin. The mixture was analyzed by gel filtration chromatography by the same procedure as in A.

Figure 1

(A) Backbone structure of a tetrameric streptavidin. Also shown are the side chains of W120 and H127; the latter is converted to D127 in Stv-33 and Stv-43. (B) Stereo view of the local three-dimensional structure (T106–D128) around the loop region (G113–W120) which has been deleted in Stv-43 (shaded), superimposed on the same sequence of natural streptavidin. These are drawn based on the x-ray structure of natural streptavidin (16, 17) by using molscript (32).

Figure 2

(A) Expression of Stv-43 in E. coli carrying expression vector pTSA-43. Total cell protein of BL21(DE3)(pLysE), with or without pTSA-43, was analyzed by SDS/PAGE (31). Lanes: a, BL21(DE3)(pLysE); b, BL21(DE3)(pLysE)(pTSA-43); and M, molecular mass standard proteins. The number above each lane is the time in hours after induction. The position where Stv-43 migrates is shown by an arrow. Each lane contains the total cell protein from the following volume of culture: at 5 hr for a and at 3 hr and 5 hr for b, 25 μl; at 0 hr and 1 hr for b, 83 μl. (B) SDS/PAGE analysis of purified Stv-43. Approximately 1 μg of purified Stv-43 was analyzed. The right lane contains molecular mass standard proteins.