Re-engineering a beta-lactamase using prototype peptides from a library of local structural motifs

Abstract

B. licheniformis exo-small beta-lactamase (ESBL) has a complex architecture with twelve alpha helices and a five-stranded beta sheet. We replaced, separately or simultaneously, three of the ESBL alpha helices with prototype amphiphatic helices from a catalog of secondary structure elements. Although the substitutes bear no sequence similarity to the originals and pertain to unrelated protein families, all the engineered ESBL variants were found able to fold in native like structures with in vitro and in vivo enzymic activity. The triple substituted variant resembles a primitive protein, with folding defects such as a strong tendency to oligomerization and very low stability; however it mimics a non homologous recombinant abandoning the family sequence space while preserving fold. The results test protein folding and evolution theories.

Figure 1

The structure of ESBL. The cartoon depicts the α (left) and α+β (right) domains and the location of α helices 9, 10, and 12 (in black).

Figure 2

Features of ESBL helices 9, 10, and 12. The original and substitute sequence are shown in upper case. The E value represents the number of sequences expected by chance to yield scores as those calculated for the ungapped alignment (in parenthesis are the E values for perfect matches). The engineered helices are shown in a wheel representation with ESBL and prototype residues placed in the outer and inner layer, respectively. Hydrophobic residues are highlighted by a gray circular area.

Figure 3

Far (Panel A) and near (Panel B) UV CD spectra. Full line, wild-type ESBL; dots, α₉ ESBL_; short dashes, α₁₀ ESBL; dots/dashes, α₁₂ ESBL; and short/long dashes, α₉,_10,12 ESBL.

Figure 4

Fluorescence spectra. Full line, wild-type ESBL; dots, α₉ ESBL_; short dashes, α₁₀ ESBL; dots/dashes, α₁₂ ESBL; short/long dashes, α₉,_10,12 ESBL; double dots/dashes, N-acetyl tryptophanamide. All samples were dissolved in Buffer A.

Figure 5

Urea induced unfolding equilibrium. From top to bottom, the curves for wild type, α₉, α₁₀, α₁₂, and α_9,10,12, respectively are shown. Panels on the left show the conformational changes monitored by far UV CD (circles), fluorescence emission (squares), and enzymic activity (triangles). A three-state unfolding mechanism was assumed to fit the data for the three probes simultaneously [see Eqs. (5)–(8)], except for the triple mutant for which only CD and fluorescence data were globally fit. Right panels show the changes in the equilibrium fraction of native (dashed line), partially folded (full line), and unfolded state (dotted line) derived from the fit.

Figure 6

Thermal unfolding. Panel A shows the unfolding curves for the ESBL variants wild type (squares), α₉ (triangles), α_10, (diamond), and α₁₂ (circles). For clarity, arbitrary constants were added to each set of experimental data to displace the curves along the ordinate axis. Lines are the best fit of Eqs. (3) and (4) (see Materials and Methods). Only the pH 7.0 data and fit are shown. However, the experiment was performed at pH 6.0, 7.0, and 8.0 (not shown), and the fit was global to the whole set of data. The thermodynamic parameters derived from the fit are shown in Table 4 (pH 7.0) and in Supplementary Material (pH 6.0 and 8.0). Panel B shows the free energy of unfolding as a function of temperature, as calculated from the thermodynamic parameters listed in Table 4. Full, dot, dash, and dot-dash correspond to wild type, α₉, α_10, α₁₂ ESBL.