Diagnósticos de la dislexia

To assess if the B. burgdorferi ortholog of Pur-α is indeed a functional Pur protein, its nucleic-acid binding properties were probed. The oligonucleotides previously employed for human Pur-α were used in filter binding assays with B. burgdorferi Pur-α full length (amino acids 1 to 122).

Filter binding assays

In filter binding assays, the binding of B. burgdorferi Pur-α to ssDNA was confirmed (JCVupTAR ssDNA KD = 480 nM and hTel12 ssDNA KD = 413 nM) (Table 2-13). The

affinities were similar to those obtained with human Pur-α (KD = 200-400 nM) (Table 2-

12 and Figure 2-31, A).

The binding seems to be specific, as no significant signal was observed for control ssDNA (Cntrl1) lacking the consensus sequence (Table 5-2). This indicated that the

B. burgdorferi ortholog shares not only a sequence similarity, but also a functional

Figure 2-29. Representative filter binding curves for human Pur-α. A: Binding of Pur-α 56-287 C272S to hTel12 DNA, KD = 438 nM. B: Binding of Pur-α 56-287 C272S to TAR14GC RNA, KD = 85 nM.

Oligonucleotide Type Length KD in nM n

JCVupTAR ssDNA 12 nt 480 ± 81 4

hTel12 ssDNA 12 nt 413 ± 18 4

TAR14AU Stem loop RNA 16 nt no saturation 3 TAR14GC Stem loop RNA 16 nt no saturation 3

Cntrl1 ssDNA 12 nt no binding 3

MS2 Hairpin RNA 19 nt no saturation 3

PP7 Hairpin RNA 25 nt no saturation 3

Table 2-13. Filter binding assays with B. burgdorferi Pur-α full length. KD = equilibrium

dissociation constant, n = number of experiments.

Binding to the RNA oligonucleotides could be not confirmed. The signal of bound RNA increased with higher protein concentration, but no saturation was observed even with protein concentrations as high as 8 µM (Table 2-13). This indicates a very weak affinity or even an unspecific mode of binding.

Crystal structure determination of B. burgdorferi Pur-α revealed a structural similarity to bacteriophage coat proteins from Pseudomonas phage pp7 (Chao et al., 2008) and from enterobacterio phage ms2 (Ni et al., 1995) (section 2.1.1.2). These are RNA binding proteins, which bind a specific hairpin RNA (PP7 and MS2, respectively). It was speculated whether B. burgdorferi Pur-α binds to these hairpin RNA, therefore filter binding experiments with the respective oligonucleotides were performed.

B. burgdorferi Pur-α full length showed some binding to the PP7 RNA and MS2 RNA oligonucleotides, as the signal of retained radioactivity increased with higher protein concentrations. Alas, no saturation of the signal was obtained with a protein concentration as high as 8 µM (Table 2-14). A KD would be even higher for this

interaction. The binding is therefore very weak and/or unspecific (Table 2-13).

In summary, B. burgdorferi Pur-α binds specifically to the PUR consensus sequence in DNA oligonucleotides, but only very weakly and/or unspecifically to the RNA targets tested. It is possible that species-specific RNA targets for B. burgdorferi Pur-α exist, but none are reported.

Filter binding assays with mutant Pur-α

To map the binding surface on the crystal structure of B. burgdorferi Pur-α, mutational studies were performed. Surface assessment of the crystal structure of B. burgdorferi Pur-

α led to the identification of candidate amino acids that might be involved in nucleic-acid binding. Surface-exposed candidate amino acids were selected by their positive charge (preferably arginines) and their degree of conservation between species, as the DNA binding properties are conserved. The respective arginines were replaced by alanines (section 5.6.1).

One point mutation (R18A) was introduced in the second β-strand of the β−sheet in the

Borrelia PUR repeat. Two adjacent arginines (R28A, R29A) were mutated in the connector between the second and third β-strand. One mutation was introduced in the fourth strand of the β-sheet (R49A) (Figure 2-30).

The B. burgdorferi Pur-α full length mutant proteins were tested in filter binding assays and the results were compared to the binding affinities of the wild type protein (Table 2- 13 and Table 2-14).

Figure 2-30. Position of the mutations which affect nuleic acid binding in B. burgdorferi Pur-α. Backbone Ribbon model of the crystal structure, one monomer is

Protein Oligonucleotide Type Length KD n bbPurA1-122 wt JCV upTAR ssDNA 12 nt 480 ± 81 3 bbPurA1-122 wt hTel12 ssDNA 12 nt 413 ± 18 3 bbPurA1-122 R18A JCV upTAR ssDNA 12 nt Appr. 1 µM 3 bbPurA1-122 R18A hTel12 ssDNA 12 nt No saturation 3 bbPurA1-122 R28A hTel12 ssDNA 12 nt No saturation 3 bbPurA1-122 R28A, R29A hTel12 ssDNA 12 nt no binding 3 bbPurA1-122 R49A JCV upTAR ssDNA 12 nt no binding 3 bbPurA1-122 R49A hTel12 ssDNA 12 nt No binding 3

Table 2-14. Filter binding assays with mutants of B. burgdorferi Pur-α. KD = equilibrium

dissociation constant, n = number of experiments, wt = wild type. No saturation at 8 µM.

The B. burgdorferei Pur-α 1-22 R18A bound JCVupTAR ssDNA with an approximate KD of 1 µM (Table 2-14), thus the mutation R18A only weakly affected binding. The

affinity to hTel12 ssDNA was more strongly impaired. The signal of retained oligonucleotide increased with higher protein concentrations, but no saturation of the signal was obtained with a protein concentration as high as 8 µM (Table 2-14). A KD

would be even higher for this interaction.

The same effect was observed for the mutation R28A, as for the binding of

B. burgdorferi Pur-α 1-122 R28A to hTel12 ssDNA, no saturation was achieved at a protein concentration of 8 µM (Table 2-14).

Because of their immediate vicinity, arginine 29 was also mutated and the double mutant was assessed. B. burgdorferei Pur-α 1-22 R28A R29A showed a total loss of affinity to both ssDNA oligomers tested (Table 2-14).

An abolishment of ssDNA binding was also seen for B. burgdorferei Pur-α 1-22 R49A In summary, the effects of the point mutations support the hypothesis that the β-sheets are the nucleic-acid binding surfaces of B. burgdorferi Pur-α.