journal menu
![[Figure 1]](cb5061fig1mag.jpg)
|
|
Figure 1
BurrH recognizes its target DNA with high affinity and specificity in an endothermic reaction. (a) Scheme of the BurrH domain structure. The central DNA-binding domain contains the BuD repeats with the residues involved in DNA recognition (BSRs; Supplementary Figs. S1 and S2). The sequence of the coding (the strand defined by the single amino acid-to-nucleotide correspondence) and noncoding (the complement of the coding strand) strands of the oligonucleotide used in the biophysical characterization and crystallization is depicted below. The BurrH target sequence is shown in bold. (b) ITC binding curves of BurrH. The protein specifically recognizes its double-strand (ds) DNA target. BurrH is not able to bind DNA duplexes with other sequences or single-strand (ss) DNAs containing its target sequence. (c) ITC binding curves of BurrH using DNA-RNA hybrids and RNA duplexes as targets. (d) ITC binding curves of BurrH-based variants display the same thermodynamic behaviour as the wild-type protein (see Supporting Information and Supplementary Fig. S4). (e) Table summarizing the Kd values of the ITC analysis. The affinities of the redesigned variants are similar to the wild-type protein except for Var2. (f) SPR analysis of BurrH target binding compared with AvrBs3 TALE. The BuD array presents a fast association and low dissociation behaviour (see Supplementary Fig. S5). In both cases 12.5 nM protein was flowed over the chip for 95 s. Mono exponential fits are shown in black for the curves (Supplementary Fig. S5). (g) On–off rate map showing the values of the association and dissociation rate constants and the resulting affinity as obtained from SPR. Dashed diagonals represent different Kd values (indicated on the upper and right axes). Positions along the same diagonal have the same Kd values but different kon and koff values. |
![[Figure 1]](cb5061fig1.jpg)
 | STRUCTURAL BIOLOGY |
ISSN: 2059-7983
access
