DATOS INFORMATIVOS

The binding between E_GW and K_GW was studied using CD spectroscopy. The UV-CD spectrum of E_GW/K_GW at 25 °C is typical for peptides with high α-helical content,

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Chapter IV

Figure 1. Helical wheel projection of the 18 C-terminal amino acids of K_GW (left) and E_GW (right).1

200 210 220 230 240 250 −40 −20 0 20 40 60 λ / nm [ θ ] / 10 3 deg cm 2 dmol −1 K_{G W} E_{G W} E_{G W} / K_{G W} E / K_{G W} E_{G W} 0 25 50 75 100 −30 −20 −10 0 T / ° C [ θ ] 222nm / 10 3 deg cm 2 dmol −1

A

B

Figure 2. (A) CD spectra of E_GW, K_GW, and, the coiled coil complexes E_GW/K_GW, E/K_GW and K_GW/E at 50 µM total peptide concentrations and 25°C. Values in parenthesis are the helicities calculated

from [θ]_222nm (B) CD thermal denaturation curves of E_GW/K_GW at [50 µM; 25 µM; 12.5 µM; 6.25 µM]

total peptide concentrations (squares) and best fitting binding model (straight lines) and its baselines

(dashed lines). The apparent T_M increases with the total peptide concentration.

showing two minima around 208 and 222 nm, while the spectra of the isolated peptides E_GW and K_GW show spectra typical for less helical structures (Figure 2A).21-23 _{The helical}

content can be estimated from the ellipticity at 222 nm ([θ]_222nm).21,22 _{The helicity was} found to increase from 21% and 33% for E_GW and K_GW respectively, to 83% for the E_GW/K_GW complex (Table 1). Also the formation of E_GW/K and K_GW/E complexes could be observed based on spectra representing high helical content with 74% and 75%, respectively (Figure 2A). Under the same conditions E, K, and E/K showed helicities of 21%, 23%, and 72% respectively (Table 1, spectra not shown). The increasing helical contents prove the interactions between the peptide chains that are accompanied by folding into more helical structures, i.e. formation of the coiled-coil complexes.

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Chapter IV

The thermodynamics of folding for the E_GW/K_GW complex were studied by mea- suring the temperature and concentra- tion dependencies of [θ]_222nm (Figure 2B). With increasing temperature [θ]_222nm in-

creases, indicating that the coiled-coil complex unfolds. The shift of the apparent melting temperature T_M with increasing

Table 1. Helicities of studied peptides and pep- tide mixtures

CHAPTER II 5

Coiled coil binding

The binding between EGWand KGWwas studied using CD spectroscopy. The UV-CD spectrum

of EGW/KGWat 25 °C is typical for peptides with high -helical content, showing two minima

around 208 and 222 nm, while the spectra of the isolated peptides EGWand KGW show spectra

typical for less helical structures (Figure 2A).21-23 _{The helical content can be estimated from}

the ellipticity at 222 nm ([]222nm).21,22The helicity was found to increase from 21% and 33%

for EGWand KGW respectively, to 83% for the EGW/KGW complex (Table 1). Also the formation

of EGW/K and KGW/E complexes could be observed based on spectra representing high helical

content with 74% and 75%, respectively (Figure 2A). Under the same conditions E, K, and E/K showed helicities of 21%, 23%, and 72% respectively (Table 1, spectra not shown). The

increasing helical contents prove the interactions between the peptide chains that are

accompanied by folding into more helical structures, i.e. formation of the coiled-coil

complexes.

The thermodynamics of folding for the EGW/KGW complex were studied by measuring

the temperature and concentration dependencies of []222nm (Figure 2B). With increasing

temperature []222nm increases, indicating that the coiled-coil complex unfolds. The shift of the

apparent melting temperature TM with increasing peptide concentration proves that the

unfolding process involves more than one peptide chain (Chapter II).24,25 _{The data was fitted}

by a dimeric A1B1 binding model as described in Chapter II and the folding constant Kfat 20°C

was found to be ~3.2 107 _M-1 _{which is slightly higher than Kf= 5.5 10}6 _M-1 _{of the unlabeled}

complex E/K (Chapter VI). In terms of Gibbs energy of folding (Gf) this corresponds to -42.1

and -37.8 kJ/mol respectively. Litowski and Hodges reported Gf = -40.2 kJ/mol for the

original sequence of E/K, which is in line with the results reported here.20_{The slightly higher}

folding constant of EGW/KGW, might be caused by the increase of hydrophobic interactions due

to an extended hydrophobic face or -- stacking of the C-terminal tryptophan residues.

Taken together these results show, that the C-terminal elongation of the peptides E and

K by a glycine and a tryptophan residue did not perturb the oligomeric state of the coiled coil

complex and had little influence on the binding

strength between the two peptides. For the further

investigation of the interactions of the peptides with lipid vesicles these small changes are

considered to be negligible.

Table 1. Helicities of studied peptides and

peptide mixtures KGW 33 % E/KGW 75 % EGW 21 % EGW/K 74 % K 23 % EGW/KGW 83 % E 21 % E/K 72 % [total Peptide]=50 µM

peptide concentration proves that the unfolding process involves more than one peptide chain (Chapter II).24,25 _{The data was fitted by a dimeric A}

1B1 binding model as described in Chapter II and the folding constant K_f at 20 °C was found to be ~3.2 107 _M-1 _{which is slightly higher than K}

f = 5.5 106 M-1 of the unlabeled complex E/K (Chapter VI). In terms of Gibbs energy of folding (∆G_f) this corresponds to -42.1 and -37.8 kJ / mol respectively. Litowski and Hodges reported ∆G_f = -40.2 kJ / mol

for the original sequence of E/K, which is in line with the results reported here.20 The slightly higher folding constant of E_GW/K_GW, might be caused by the increase of hydrophobic interactions due to an extended hydrophobic face or π-π- stacking of the C-terminal tryptophan residues.

Taken together these results show, that the C-terminal elongation of the peptides E and K by a glycine and a tryptophan residue did not perturb the oligomeric state of the coiled coil complex and had little influence on the binding strength between the two peptides. For the further investigation of the interactions of the peptides with lipid vesicles these small changes are considered to be negligible.