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. 1998 May 26;95(11):5929-34.
doi: 10.1073/pnas.95.11.5929.

Optimizing the stability of single-chain proteins by linker length and composition mutagenesis

C R Robinson  1 , R T Sauer
Affiliations

Optimizing the stability of single-chain proteins by linker length and composition mutagenesis

C R Robinson et al. Proc Natl Acad Sci U S A. .

Abstract

Linker length and composition were varied in libraries of single-chain Arc repressor, resulting in proteins with effective concentrations ranging over six orders of magnitude (10 microM-10 M). Linkers of 11 residues or more were required for biological activity. Equilibrium stability varied substantially with linker length, reaching a maximum for glycine-rich linkers containing 19 residues. The effects of linker length on equilibrium stability arise from significant and sometimes opposing changes in folding and unfolding kinetics. By fixing the linker length at 19 residues and varying the ratio of Ala/Gly or Ser/Gly in a 16-residue-randomized region, the effects of linker flexibility were examined. In these libraries, composition rather than sequence appears to determine stability. Maximum stability in the Ala/Gly library was observed for a protein containing 11 alanines and five glycines in the randomized region of the linker. In the Ser/Gly library, the most stable protein had seven serines and nine glycines in this region. Analysis of folding and unfolding rates suggests that alanine acts largely by accelerating folding, whereas serine acts predominantly to slow unfolding. These results demonstrate an important role for linker design in determining the stability and folding kinetics of single-chain proteins and suggest strategies for optimizing these parameters.

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Figures

Figure 1
Figure 1
(A) Tandem copies of the arc gene connected by DNA encoding a linker region comprise the gene for single-chain Arc repressor. (B) One model of how a linker might connect the two subunits (colored gray and white) of single-chain Arc. The positions of the N and C termini are indicated. Prepared using molscript (34) and coordinates of wild-type Arc (33).
Figure 2
Figure 2
Linker length has large effects on the stability of single-chain Arc to urea denaturation. The sequences of linkers LL9 (○), LL11 (Δ), LL17 (•), LL19 (□), LL31 (▴), and LL47 (▪) are listed in Fig. 3A. Fraction unfolded was calculated by fitting plots of CD ellipticity (234 nm) vs. urea concentration to a two-state-unfolding transition. The solid lines represent the best theoretical fits of the experimental data.
Figure 3
Figure 3
Properties of linker-length variants of single-chain Arc. (A) Linker sequences. (B) Equilibrium stability and effective concentration vary with linker length. Error bars indicate one SD from three independent experiments. (C) Folding rates in 2 M urea. (D) Unfolding rates in 7 M urea. Experimental conditions; protein 1–10 μM, 25°C, 50 mM Tris⋅HCl (pH 7.5), 250 mM KCl, and 0.1 mM EDTA.
Figure 4
Figure 4
Properties of ALX variants with 19-residue linkers and differing in Ala/Gly composition numbers of alanines and glycines. (A) Linker sequences. (B) Equilibrium stability and effective concentration vary with number of alanines. For compositional isomers, closed and open symbols represents “a” and “b” variants, respectively. Error bars indicate one SD from three independent experiments. (C) Folding rates in 4.5 M urea. (D) Unfolding rates in 9.1 M urea. See Fig. 3 for conditions.
Figure 5
Figure 5
Properties of SLX variants with 19-residue linkers differing in Ser/Gly composition. (A) Linker sequences. (B) Equilibrium stability and effective concentration vary with number of serines. For compositional isomers, closed and open symbols represents “a” and “b” variants, respectively. Error bars indicate one SD from three independent experiments. (C) Folding rates in 2.25 M urea. (D) Unfolding rates in 9.1 M urea. See Fig. 3 for conditions.
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