Secondary structure assessment of the Zf3 mutants

The modified zinc finger constructs described here were compared to the unmodified analogue in order to evaluate similarities or oppositions in terms of secondary structure formation. The CD spectrum of the native reference peptide showed all aforementioned characteristics, which indicate the correct formation of the -structure upon metal complexation. The measurement under metal-free conditions (dashed line) revealed a strong negative band at 200 nm and a shallow shoulder around 222 nm as expected for the high random-coil content present in unstructured zinc fingers (Figure 2.9). After metal addition (solid line), the band at 200 nm decreased and showed a pronounced red-shift to 205.5 nm.

In addition, the shoulder around 222 nm increased significantly indicating the increase of -helical content in the peptide. The results are very well in line with the data published for the native Zf3 peptide found in literature.^[83]

Figure 2.9 CD spectrum of the native Zf3 peptide 24 in absence (dashed line) and in presence (solid line) of Zn(II).

Afterwards, the secondary structures of the peptides 25a (blue) and 25b (red) with the building blocks incorporated at position 70 (Figure 2.10) were examined in a similar experiment and were compared to the reference peptide (black). The CD spectra were recorded in absence (dashed lines) and in presence (solid lines) of Zn(II). The results clearly indicate that both mutants showed similar negative maxima around 200 nm under metal-free conditions. Nevertheless, the latter are slightly increased in comparison to the reference spectrum, whereas the local negative maxima around 222 nm are almost identical. Upon Zn(II) addition, the negative bands at 200 nm decreased similarly when compared to the reference but showing a slightly less pronounced bathochromic shift. However, the negative maxima were found at 204.7 nm for peptide 25a and 204.4 nm for peptide 25b being well in line with the value obtained for the native peptide (205.5 nm). In addition, the spectral overlap of the bands at 222 nm indicated a comparable formation of -helical content in all three samples. In general, the CD spectra of both mutants with the building blocks incorporated at position 70 showed all important characteristics, which indicate correct secondary structure formation and are in line with the reference spectra. Thus, it can be assumed that the exchange of the arginine residue was well tolerated by the peptides and resulted in the essential -structure required for DNA binding.

Figure 2.10 CD spectra of mutant 25a (blue), mutant 25b (red) and native Zf3 (black) in absence (dashed lines) and in presence (solid lines) of Zn(II).

In contrast, the CD spectra recorded for the peptides 26a (blue) and 26b (red) with the building blocks incorporated at position 75 (Figure 2.11) showed significant differences when compared to the reference spectra (black). In the unfolded state, both mutants showed increased bands around 200 nm, which were accompanied by slight bathochromic shifts to 199.1 nm for peptide 26a and 199.3 nm for peptide 26b. The shoulders around 222 nm were

considerably less pronounced especially for peptide 26a. Consequently, a negative influence on the peptide structure induced by the building blocks incorporated at position 75 can be generally stated even under metal-free conditions. This situation was further confirmed by the corresponding spectra recorded after the addition of Zn(II). It has been found that the negative maxima just slightly decreased after metal complexation but more importantly, the bands were much less bathochromically shifted in comparison to the reference sample.

Whereas the band of the native peptide was eminently red-shifted to 205.5 nm, the band of peptide 26a shifted just to 203.1 nm and, moreover, the band of peptide 26b even only shifted to 201.9 nm. In addition, the shoulder around 222 nm observed for peptide 26a remained at a similar level when compared to its unfolded state, and therefore, clearly indicated a lack of -helical content upon Zn(II) addition. In summary, the relatively high deviations observed in the CD spectra of the peptides with the building blocks incorporated at position 75 concluded a disturbed secondary structure formation. It is to note that similar differences were also observed under metal-free conditions implying a negative impact of the building blocks on the overall peptide sequence even in the unfolded state. This might be seen as a consequence of the incorporation site of the building block at position 75. It is conceivable that the bulky residues might interact with the neighboring amino acids, Arg74 and Asp76, which negatively affected the peptide folding.

Figure 2.11 CD spectra of mutant 26a (blue), mutant 26b (red) and native Zf3 (black) in absence (dashed lines) and in presence (solid lines) of Zn(II).

In conclusion, the CD spectra of the zinc finger mutants 26a and 26b with the building blocks incorporated at position 75 were recorded and compared to the unmodified reference peptide. It was clearly demonstrated that both spectra significantly differ from the reference implying an incorrect peptide folding. In contrast, the findings for the mutants 25a and 25b

having the building block incorporated at position 70 were in line with the reference sample.

This allows the assumption that the structural discrepancies of 26a and 26b derived from unsuitable intrinsic conditions for the building blocks when incorporated at position 75, which prevent correct peptide folding. These might originate from the neighboring amino acids being influenced by the bulky dinuclear complex in close proximity.^[10] The arginine residue at position 74 could orientate its relatively long and nitrogen-rich side chain in the direction of the metal complex that may induce a bending of the peptide with reduced helix formation (Figure 2.12a). The same considerations are valid for the aspartic acid residue at position 76.

Furthermore, both neighboring amino acids make direct nucleobase contacts, whereby a deviated orientation of their side chains would have a substantial adverse effect on DNA recognition and binding.

This hypothesis was further supported with respect to the good results obtained for the incorporated building blocks at position 70. The building blocks themselves are located in the junction region between the -sheets just as the neighboring amino acid residues Gly69 and Lys71 (Figure 2.12b).^[10] This region is known to provide more space and flexibility in comparison to the rigid helical region.^[69] Moreover, the lysine residue and in particular the glycine residue are much less sterically demanding in comparison to the aforementioned residues, and therefore, allow the evasion of steric repulsions.

Due to the results of the CD spectroscopic measurements for both building blocks that have been incorporated at the two most promising positions of the zinc finger, it can be concluded that position 70 turned out to be most beneficial. This assumption is based on the fact that the modifications done at this position just marginally influence the secondary structure that

Figure 2.12 Molecular models of 26b (a) and 25b (b) showing the incorporated dinuclear building block as well as their neighboring amino acids. The second neighboring amino acid residue in (b) is a glycine residue without a side chain, which could be highlighted in the model. The models were generated with UCSF Chimera (PDB code 1AAY).

(a) (b)

is vital for maintaining the DNA-binding ability of the zinc finger. However, this does not seem to be the case for the serine 75 position, which revealed significant differences in the recorded CD spectra indicating a problematic secondary structure formation. The question to what extent this fact may have an impact on DNA binding and hydrolysis is covered in section 2.7.