Figure 1.
Combinatorial fragment exchange.
(1) Regions known to play an important role in the protein structure-function relationship are chosen and defined based on alignment of known structures or homology models. (2) Appropriate oligonucleotide primers are designed to fragment the gene encoding the core protein scaffold based on the structural alignment determined in (1). (3) The oligonucleotides generated in (2) are used to fragment the core gene. (4) Oligonucleotides encoding the selected regions from the selected donor sequence elements (red arrows) are used to splice together the core gene fragments generated either by PCR (white blocks; labelled Fn) or bridging oligonucleotides (blue arrows; labelled fn). This generates the library of variants with different combinations of sequences at each of the selected regions. The example given in the figure represents the fragmentation and reassembly approach used in this study.
Figure 2.
Sequence identity and region definition of chosen subtilisins.
Region marked with a + contains an additional calcium binding site and the S49D mutation to Sav core. The regions marked with * a Cys-X-Cys disulphide bond.
Figure 3.
Structural features of selected donor subtilisins.
(A) The additional calcium binding site observed in R2 of Ther and AK1. Calcium ion is shown as blue sphere, R2 is green with calcium coordinating residues shown as sticks. The catalytic triad is shown as grey sticks. Diagram created using PDB file 1THM [41]. (B) The Cys-X-Cys disulphide bridge in the R4 region of AK1 (PDB code 1DBI [31]). R4 is coloured cyan, with the disulphide bridge shown as sticks. The catalytic triad is shown as grey sticks. (C) Structure of ISP from B. clausii (PDB code 2X8J [33]). The two protomers are coloured grey and green respectively. The catalytic triad (space-fill) and the 6 selected regions (coloured red) are shown on the left, grey protomer. The homology model of ISP generated as described in the material and methods is coloured yellow and overlaid on the right, green protomer of the experimentally determined structure of ISP.
Figure 4.
Selected Sav regions for ComFrEx.
The structure of Sav (1SVN; [37] with each of the six regions highlighted as shown in the top panel of figure. The catalytic triad is shown as space-fill and calcium ions shown as green spheres. The right hand panel defines each of the regions in terms of their placement within Sav primary and secondary structure. The sequence of each region is shown and coloured as indicated in the diagram. Letters in bold and coloured black indicate residues contributing to the catalytic triad and anoxyion hole.
Figure 5.
Activity of rationally constructed Sav-hybrid variants.
The donor fragments at each of the 6 regions derives from one of the subtilisins, which is indicated in superscript in the figure. An active variant is indicated by the production of a clearing zone or halo around the B. subtilis colony due to digestion of casein embedded in the agar growth medium.
Table 1.
Activity of rationally constructed variants
Figure 6.
Characterisation of LibR34 variants.
Variants are blocked according to their FAAF:AAPF ratio as described in the main text. Variant v1F3 (labelled with *) has an additional N43L mutation. The sequence of R3 or R4 hybrid regions are described in Table S2. Data for variants labelled with + sharing the same sequence identified from different transformants is presented in Table S3.
Figure 7.
Characterisation of LibRall variants.
Variants are blocked according to their FAAF:AAPF ratio class as described in the main text. The colour code for regions R1 to R6 are: black, Sav; blue, BPN; yellow, Alc; green, SbE; orange, AK1; purple, Ther; cyan, ISP. Sequence of hybrid regions described in Table S4. Variants with additional mutations are labelled with a +: G206S (vaF4), N221I (vaB4), T23L (vaG5).