The identity of the peptides was confirmed by matrix-assisted laser desorption
ionization – time of flight (MALDI-TOF) mass spectrometry. Upon confirmation of the
peptides, purification was carried out on reverse phase high performance liquid
chromatography (RP-HPLC) to greater than 95% purity. The crude yield of peptide
synthesis, molecular formula, calculated [MH+], and observed m/z of GCN4 peptides
are shown in Table 2-1.
H2N O
OH
R Fmoc-OSu, THF
Na2CO3 (aq), ice bath, 3.5 hrs
R= Allo Ile Cpa Nle
Fmoc-Allo Ile-OH, 90.1% yield Fmoc-Cpa-OH, 86.9% yield Fmoc-Nle-OH, 78.5% yield
O N
H O
OH O R
!
Table 2-1. Crude Yield of Peptide Synthesis, Molecular Formula, Calculated [MH+], and Observed m/z of GCN4 Peptides
Peptide Crude
Yield (%)
Molecular Formula Calculated [MH+]
aObserved m/z and calculated [MH+] of GCN4-Leu and GCN4-Phe were given as average molecular weight. For the rest of the peptides, exact mass was given.
UV-Visible Spectroscopy (UV-vis) of GCN4-Derived Peptides
The concentration of the peptide stock solutions was determined by the tyrosine
absorbance in 6 M guanidinium chloride as described by Edelhoch.36, 37 UV data were
obtained using 1 mm pathlength cells. Absorbance at 276 nm, 278 nm, 280 nm and 282
nm were measured at different concentrations of peptide. Linear regression was
performed to obtain the concentration. Data with correlation coefficients (R) less than
0.99 were discarded. Concentrations and regression coefficients for the GCN4 peptides
are shown in Table 2-2.
Table 2-2. Concentrations and Regression Coefficients of GCN4 Peptides Peptide Concentration (mM) Regression Coefficient
GCN4-Abu 5.11±0.05 0.99914
GCN4-Allo Ile 4.72±0.03 0.99950
GCN4-Asn 5.01±0.03 0.99950
GCN4-Asp 5.53±0.06 0.99856
GCN4-Cha 4.9±0.1 0.99488
GCN4-Cpa 5.05±0.04 0.99921
GCN4-Gln 4.46±0.03 0.99940
GCN4-Glu 4.32±0.04 0.99911
GCN4-Ile 5.23±0.05 0.99914
GCN4-Leu 4.90±0.04 0.99908
GCN4-Nle 4.50±0.03 0.99954
GCN4-Nva 5.40±0.04 0.99926
GCN4-Pff 5.14±0.07 0.99781
GCN4-Phe 6.65±0.04 0.99959
GCN4-Tba 4.54±0.04 0.99920
GCN4-Val 3.73±0.03 0.99906
Circular Dichroism (CD) Spectroscopy of GCN4-Derived Peptides
CD data was acquired at 30 µM peptide concentration in 50 mM phosphate and 150
mM NaCl buffer at pH 7 and 4 °C.18 The magnitude of the CD signal at 222 nm reflects
!
the helical content of a coiled coil.38 The CD spectra of peptides are shown in Figure
2-7, and the MRE values at 222 nm ([θ]222) are listed in Table 2-3.
(a) (b)
(c) (d)
Figure 2-7. CD spectra of GCN4-Xaa peptides at 30 µM peptide concentration in 50 mM phosphate and 150 mM NaCl buffer at pH 7 and 4 °C. (a) CD spectra of
GCN4-Xaa peptides in which Xaa are residues with linear chains. (b) CD spectra of GCN4-Xaa peptides in which Xaa are residues with β-branched side chains. (c) CD spectra of GCN4-Xaa peptides in which Xaa are residues with γ-branched side chains.
(d) CD spectra of GCN4-Xaa peptides in which Xaa are residues with polar side chains.
Table 2-3. [θ]222 at 0 M Guanidinium, Melting Concentrations ([C]m), m values, and
GCN4-Abu -12800±300 0.45 -1.26±0.04 3.46±0.02
GCN4-Nva -22900±400 1.35 -1.023±0.008 4.25±0.01
GCN4-Nle -28900±500 1.15 -1.167±0.009 4.21±0.01
GCN4-Ile -18500±400 0.85 -1.14±0.01 3.85±0.01
GCN4-Allo Ile -24700±400 0.56 -1.241±0.005 3.557±0.003
GCN4-Val -21900±400 0.66 -1.21±0.03 3.69±0.02
GCN4-Leu -24800±400 2.44 -1.04±0.02 5.42±0.04
GCN4-Tba -28100±600 2.67 -0.923±0.008 5.33±0.02
GCN4-Cpa -26400±500 1.60 -1.15±0.02 4.71±0.03
GCN4-Cha -24300±600 1.25 -1.033±0.008 4.15±0.01
GCN4-Phe -25400±400 0.45 -1.34±0.02 3.478±0.009
GCN4-Pff -9700±400 0.05 -1.4±0.1 2.92±0.03
GCN4-Asp -3600±300 --d --d --d
GCN4-Asn -5900±400 --d --d --d
GCN4-Glu -20900±400 0.05 -1.13±0.01 2.922±0.004
GCN4-Gln -18500±500 0.15 -1.25±0.02 3.053±0.009
a[C]m is the concentration of guanidinium chloride at which 50% of the total peptide is unfolded. bm value is the slope of the regression line for fitting of ΔGunfold, H2O. cΔGunfold, H2O is the free energy required for the coiled coil to unfold at 0 M guanidinium chloride. dThese peptides barely fold even at 0 M of
guanidinium chloride. Therefore, [C]m, m value, and ΔGunfold, H2O cannot be deduced.
The helical content of GCN4-Xaa with linear Xaa residues (Abu, Nva, and Nle)
increased with increasing side chain length. This indicates that a longer linear side chain
at the coiled coil interface reinforces the coiled coil helical structure. When side chains
of Xaa are β-branched, the helical content of the peptides followed the trend: GCN4-Ile
!
this trend. Regardless, Ile and Allo Ile are diastereomers with opposite chirality at the
Cβ. Surprisingly, the unnatural side chain structure yielded a higher helical content in
the coiled coil. For γ-branched amino acids, the helical content followed the trend:
GCN4-Pff < GCN4-Cha < GCN4-Leu < GCN4-Phe < GCN4-Cpa < GCN4-Tba. Still,
no simple obvious structural explanation can be deduced from this trend. Regardless,
the helical content of GCN4-Pff was significantly lower than the rest of the group,
indicating that the bulky and very hydrophobic pentafluorophenyl group imposes
adverse effects onto the coiled coil structure. For the polar amino acids, the helical
content followed the trend: GCN4-Asp < GCN4-Asn < GCN4-Gln < GCN4-Glu.
Apparently, the longer the side chain, the higher the helical content. However, the
contribution of the carboxylate (-COO-) and amide (-CONH2) groups to the coiled coil
structure remains unclear.
The θ222 signal is a measure of the helical content of a peptide. It does not provide
information on the stability of a coiled coil. In other words, a peptide with a higher
helical content does not guarantee that the peptide is also more stable. Therefore,
guanidinium titration was performed to assess the stability of the coiled coils.
Guanidinium Denaturation of GCN4-Derived Peptides
Guanidinium denaturation experiments were performed at 30 µM peptide in 50
mM phosphate, 150 mM NaCl, and 0 M to 6 M (with 0.1 M intervals) guanidinium
chloride at pH 7 and 4 °C.18 CD was used to monitor the denaturation process and the
signal at 222 nm was acquired at various guanidinium chloride concentrations. The
coiled coil structure gradually unfolded upon addition of guanidinium chloride, leading
to the decrease in CD signal. Suitable CD signals were chosen to derive the folded and
unfolded baselines.39 These baselines describe the expected CD signal at different
guanidinium concentrations for a fully folded dimer and a fully unfolded monomer
(Figure 2-8). The fraction unfolded of a given peptide at each guanidinium
concentration can be derived using the unfolded and folded baselines.39 The melting
concentrations ([C]m, concentration of guanidinium chloride at which 50% of the total
peptides is unfolded) of the GCN4-based peptides are shown in Table 2-3. The
denaturation curves and graphs showing the fraction unfolded are shown in Figures 2-9
and 2-10.
!
Figure 2-8. A typical guanidinium titration curve with the proper folded and unfolded baselines depicted. The denaturation curve for GCN4-Tba is shown.
Peptide GCN4-Nva and GCN4-Nle exhibited different θ222 at 0 M guanidinium
(Figure 2-9a). However, the denaturation curves gradually overlapped as the
guanidinium concentration increased. This also happened for GCN4-Ile, GCN4-Allo Ile,
and GCN4-Val (Figure 2-9b). On the other hand, GCN4-Cha and GCN4-Leu exhibited
similar θ222 at 0 M guanidinium, but the denaturation curves diverged as guanidinium
increased in concentration (Figure 2-9c). As mentioned above, θ222 represents the helical
content of a peptide but provide no information on the stability of a coiled coil.
Therefore, it is not surprising for some coiled coils to exhibit different starting θ222 but
gradually overlapping denaturation curves, or to exhibit similar θ222 but gradually
diverging denaturation curves.
A peptide with a higher [C]m means that a higher concentration of guanidinium is
(a) (b)
(c) (d)
Figure 2-9. Guanidinium denaturation curves for GCN4-Xaa peptides at 30 µM peptide in 50 mM phosphate, 150 mM NaCl, and 0 M to 6 M (with 0.1 M intervals)
guanidinium chloride at pH 7 and 4 °C as monitored by CD at 222 nm reported in mean residue ellipticity. (a) Guanidinium denaturation curves of GCN4-Xaa peptides in which Xaa are residues with linear side chains. (b) Guanidinium denaturation curves of
GCN4-Xaa peptides in which Xaa are residues with β-branched side chains. (c) Guanidinium denaturation curves of GCN4-Xaa peptides in which Xaa are residues with γ-branched side chains. (d) Guanidinium denaturation curves of GCN4-Xaa peptides in which Xaa are residues with polar side chains.
!
(a) (b)
(c) (d)
Figure 2-10. Fraction unfolded as a function of guanidinium concentration for
GCN4-Xaa peptides as derived from the guanidinium denaturation curves. (a) Fraction unfolded plots for GCN4-Xaa peptides in which Xaa are residues with linear side chains.
(b) Fraction unfolded plots for GCN4-Xaa peptides in which Xaa are residues with β-branched side chains. (c) Fraction unfolded plots for GCN4-Xaa peptides in which Xaa are residues with γ-branched side chains. (d) Fraction unfolded plots for
GCN4-Xaa peptides in which Xaa are residues with polar side chains.
needed to denature half of the peptides. In other words, the peptide is more resilient
towards guanidinium denaturation. For the linear residues, [C]m followed the trend:
GCN4-Abu < GCN4-Nle < GCN4-Nva (Table 2-3). There does not appear to be any
structural reason for this trend. For β-branched amino acids, [C]m followed the trend:
GCN4-Allo Ile < GCN4-Val < GCN4-Ile (Table 2-3). Peptide GCN4-Ile is more stable
than GCN4-Val, consistent with the hydrophobicity trend Ile > Val. Allo Ile and Ile
share the same stereochemistry at the Cα, but have opposite chirality at the Cβ. This
may impair the packing of the Allo Ile side chain at the coiled coil interface, making
GCN4-Allo Ile more susceptible to guanidinium denaturation compared to GCN4-Ile.
For the aliphatic γ-branched residues, the [C]m followed the trend: GCN4-Cha <
GCN4-Cpa < GCN4-Leu < GCN4-Tba (Table 2-3). The difference between GCN4-Leu
and GCN4-Tba can be attributed to the hydrophobicity of Leu versus Tba. However,
Cpa and Cha exhibit higher hydrophobicity, and are larger in size and higher in rigidity
compared to Leu and Tba. The larger and more rigid side chains may introduce
unfavorable steric clashes at the coiled coil interface, overwhelming the stabilization
effects from the increase in hydrophobicity. The Cha, Phe, and Pff residues all bear a
six-membered ring. The [C]m followed the trend: GCN4-Pff < GCN4-Phe < GCN4-Cha
!
(Table 2-3). The side chain of Cha and Phe are similar in size. Nevertheless, the
benzene ring of Phe possesses a quadrupole whereas Cha is simply hydrophobic. The
quadrupole may interfere with the packing of side chains in the interface, making the
coiled coil more prone to be denatured by guanidinium. The superb hydrophobicity of
Pff may disrupt the packing at the coiled coil interface in such a way that the coiled coil
is to some extent distorted to allow for the maximum fluorous effect,25 which describes
the superior affinity between fluorocarbons such that fluorocarbons form a fluorous
phase distinct from organic phase,25 between the pentafluorophenyl groups. This can
lead to the very low [C]m for GCN4-Pff. For the polar residues, GCN4-Asn and
GCN4-Asp did not fold, whereas GCN4-Gln and GCN4-Glu were more well folded.
The side chain of Asn and Asp are similar in structure compared to Leu. Therefore, Asn
and Asp may pack in a similar pattern as Leu. While such a packing pattern can create a
stabilizing hydrophobic contact for GCN4-Leu, the same packing pattern for
GCN4-Asn and GCN4-Asp would place the polar entities at the hydrophobic interface,
thereby destabilizing the coiled coil structure. On the other hand, one-methylene-longer
side chains of Gln and Glu not only increased the side chain hydrophobicity, more side
chain rotamers with the longer side chains can place the polar entities toward the
aqueous environment instead of at the interface, resulting in the relatively higher
stability of the coiled coil structure.
ΔGunfold, H2O of GCN4-Derived Peptides
The equilibrium constant (Keq) of “folded dimer!⇌ 2 unfolded monomer” was
derived from the denaturation curves using the folded baseline and unfolded baseline.
The ΔGunfold for each guanidinium concentration was derived from ΔGunfold = -RT ln Keq.
The ΔGunfold was plotted as a function of the concentration of guaninidium chloride
(Figure 2-11a). Data points near the melting concentration were fit linearly and
extrapolated to 0 M guanidinium to obtain the ΔGunfold, H2O (Figure 2-11b). The slope of
the line of the fit (m value) and the free energy of unfolding of GCN4 peptides are listed
in Table 2-3 and graphed in Figure 2-12.
A higher ΔGunfold, H2O indicates that it is more unfavorable to unfold the coiled coil,
and thus the coiled coil is more stable. The trend for ΔGunfold, H2O is generally the same
as the trend for [C]m except for peptides GCN4-Leu and GCN4-Tba. The [C]m of
!
(a) (b)
Figure 2-11. The plot of ΔGunfold against guanidinium concentration for GCN4-Tba. (a) The plot of ΔGunfold against guanidinium concentration using all ΔGunfold data points. (b) The plot of ΔGunfold against guanidinium concentration using data points near the
melting concentration. Line of fit was also shown.
Figure 2-12. The bar graph of ΔGunfold, H2O of GCN4 peptides.
GCN4-Tba is greater than that of GCN4-Leu, whereas the order is reversed for ΔGunfold,
H2O. This originates from the different slopes (m values) in the ΔGunfold - guanidinium concentration plots for the two peptides (Figure 2-10c, Figure 2-13). The slope is related
to the number of the denaturant binding sites of a peptide.40-42 The steeper the slope, the
more the denaturant binding sites, the more the surface area burial of a peptide.40-42 A
larger surface area burial means that it is more difficult to denature the peptide, because
the denaturant binding sites are buried.40-42 However, once the denaturation begins, the
peptide unfolds readily because the numerous denaturant binding sites are now
exposed.40-42 In other words, the peptide exhibits a higher cooperativity when the slope
is steeper.40-42 A peptide with a higher ΔGunfold, H2O, a lower [C]m and a steeper slope (as
in GCN4-Leu) suggests that it is more difficult to initiate the denaturation (a higher
ΔGunfold, H2O), but denaturation occurs more readily (a lower [C]m and a steeper slope)
once the process is initiated. On the other hand, a peptide with lower ΔGunfold, H2O, a
higher [C]m and a flatter slope (as in GCN4-Tba) suggests that it is easier to initiate the
denaturation (a lower ΔGunfold, H2O), but is more difficult for the denaturation process to
complete (a higher [C]m and a flatter slope).
!
Figure 2-13. ΔGunfold of GCN4-Leu and GCN4-Tba.
The ΔGunfold,!H2O generally followed the trend γ-branched > linear > β-branched for the
peptides with aliphatic Xaa. More specifically, the ΔGunfold,!H2O followed the trend:
GCN4-Leu > GCN4-Nle > GCN4-Ile > GCN4-Allo Ile (Figure 2-12). The
hydrophobicity of the four residues Leu, Nle, Ile, and Allo Ile are similar. As such, the
side chain structures must play a role in coiled coil stability, leading to the significant
differences in ΔGunfold,!H2O.!
Although Cha bears a γ-branched side chain and is more hydrophobic compared to
Nle and Nva, the ΔGunfold,!H2O!is lower for GCN4-Cha compared to GCN4-Nle and
GCN4-Nva. As mention earlier, Cha bears a large (and bulky) side chain, which may
cause unfavorable steric clashes at the coiled coil interface. The stability gained from
hydrophobicity and the γ-branched structure may not be sufficient to compensate for the
unfavorable steric clashes. This indicates that hydrophobicity, side chain shape, and side
chain size all affect coiled coil stability. Therefore, as a first step to further elucidate the
factors contributing to coiled coil stability, namely coiled coil propensity,
hydrophobicity, and side chain size and shape, the coiled coil propensity for various
amino acids were measured.
Design of IaLd-Derived Peptides
Coiled coil propensity of the residues at the guest site affects inherent coiled coil
stability. To measure coiled coil propensities, homodimeric parallel coiled coils were
used (Figure 2-14). The sequence was based on DeGrado's23 and Hu's design.43
Isoleucines were placed at the a positions, since Ile-Ile provides very stable a-a'
interactions.43, 44 One asparagine was placed at the 4th a position to control the
oligomeric state and orientation of the coiled coil.43 The d positions were occupied by
leucines, the most favored residue at this position.27 Glutamic acid and lysine were
placed at e and g positions respectively, providing interhelical Coulombic attractions
and controlling coiled coil orientation. Many alanines were used because alanine has the
highest coiled coil propensity.23 Tyrosine (Tyr) was incorporated to facilitate
!
concentration determination by UV-vis,36 and the Gly-Gly intervening sequence was
included to minimize interference in the CD signal by the Tyr chromophore.45 Studies
on IaLd-Allo Ile, IaLd-Ile, IaLd-Leu, IaLd-Tba and IaLd-Val were previously
performed by Hsien-Po Chiu in our lab. These peptides, along with IaLd-Abu,
IaLd-Cha, IaLd-Cpa, IaLd-Nle, IaLd-Nva, IaLd-Pff and IaLd-Phe, are reported in this
chapter. IaLd-Asp, IaLd-Asn, IaLd-Glu and IaLd Gln were not included due to the
following reasons. First, GCN4-Asn and GCN4-Asp barely folded. It would be difficult
to discuss factors affecting the stabilities of these peptides. Second, the packing of polar
residues in the coiled coil interface often involves buried water molecules and the
formation of species of higher oligomeric state,12, 17 which complicates the discussion.
Therefore, polar substituents were excluded in these experiments.
IaLd-Xaa:
Ac-YGGE IEALEKK IAALEXK IQALEKK NEALEKK IAAL - NH2
Figure 2-14. Sequence and helical wheel of IaLd. X denotes the mutation sites.
a
Peptide Synthesis of IaLd-Derived Peptides
Peptides were synthesized using commercially available reagents by Fmoc-based
solid peptide synthesis (SPPS). Nle and Cpa were Fmoc protected as described ealier
(Scheme 1). The identity of the peptides was confirmed by MALDI-TOF mass
spectrometry. Upon confirmation of the peptides, purification was carried out on reverse
RP-HPLC to greater than 95% purity. The crude yield of peptide synthesis, molecular
formula, calculated [MH+], and observed m/z of GCN4 peptides are shown in Table 2-4.
Table 2-4. Crude Yield of Peptide Synthesis, Molecular Formula, Calculated [MH+], and Observed m/z of IaLd Peptides
Peptide Crude
aApproximate yield is given here. These three peptides were initially synthesized together. Upon coupling of Xaa, the resin was divided into three portions, one portion for each peptide. The resin was not
lyophilized, therefore, exact yield cannot be calculated. bObsreved m/z and calculated [MH+] were given as exact mass for IaLd-Abu, IaLd-Cha, and IaLd-Cpa. For other peptides, average molecular weights were given.
!
UV-Visible Spectroscopy (UV-vis) of IaLd-Derived Peptides
The concentration of the peptide stock solutions was determined by the tyrosine
absorbance in 6 M guanidinium chloride as described by Edelhoch.36, 37 UV data were
obtained using 1 mm pathlength cells. Absorbance at 276 nm, 278 nm, 280 nm and 282
nm were measured at different concentrations of peptide. Linear regression was
performed to obtain the concentration. Data with correlation coefficients (R) less than
0.99 were discarded. Concentrations and regression coefficients of IaLd peptides are
shown in Table 2-5.
Table 2-5. Concentrations and Regression Coefficients of IaLd Peptides Peptide Concentration (mM) Regression Coefficient
IaLd-Abu 2.97±0.06 0.99502
IaLd-Cha 3.57±0.05 0.998
IaLd-Cpa iaIaLd
5.52±0.09 0.99685
IaLd-Nle 2.73±0.04 0.99799
IaLd-Nva 2.06±0.06 0.99208
IaLd-Pff 8.7±0.1 0.99772
IaLd-Phe 5.14±0.04 0.99920
Circular Dichroism (CD) Spectroscopy of IaLd-Derived Peptides
CD measurements were performed at 20 µM peptide in 10 mM 3-(N-morpholino)
-propanesulfonic acid (MOPS) at pH 7.5 and 25 °C.23 The magnitude of the CD signal
at 222 nm reflects the helical content of a coiled coil.38 The CD spectra of the peptides
are shown in Figure 2-15, and the MRE values at 222 nm ([θ]222) are listed in Table 2-6.
(a) (b)
(c) (d)
Figure 2-15. CD spectra of IaLd-Xaa at 20 µM peptide in 10 mM MOPS at pH 7.5 and 25 °C. (a) CD spectra of IaLd-Xaa peptides in which Xaa are residues with linear side chains. (b) CD spectra of IaLd-Xaa peptides in which Xaa are residues with β-branched side chains. (c) CD spectra of IaLd-Xaa peptides in which Xaa are residues with
γ-branched aliphatic side chains. (d) CD spectra of IaLd-Xaa peptides in which Xaa are residues with γ-branched aromatic side chains.
!
Table 2-6. [θ]222 at 0 M Guanidinium, Melting Concentrations ([C]m), m values, and ΔGunfold, H2O of IaLd Peptides
Peptides [θ]222
(deg cm2 dmol-1)
[C]m (M) m Value ΔGunfold, H2O
(kcal/mole)
IaLd-Abu -19300±500 3.49 -1.06±0.02 6.97±0.07
IaLd-Nva -27500±900 3.64 -1.02±0.02 6.92±0.06
IaLd-Nle -27100±800 3.58 -1.02±0.02 6.86±0.06
IaLd-Ile -24100±300 3.31 -1.146±0.005 6.99±0.02
IaLd-Allo Ile -25700±300 3.00 -1.08±0.01 6.46±0.03
IaLd-Val -20800±400 3.15 -1.126±0.006 6.75±0.02
IaLd-Leu -26700±300 3.73 -1.155±0.009 7.52±0.03
IaLd-Tba -25700±200 3.53 -1.11±0.02 7.15±0.09
IaLd-Cpa -25600±800 3.56 -1.08±0.02 7.05±0.08
IaLd-Cha -26400±800 3.55 -1.05±0.02 6.96±0.08
IaLd-Phe -22500±400 3.16 -1.08±0.02 6.61±0.06
IaLd-Pff -10200±300 2.84 -0.94±0.02 5.88±0.06
The helical content of the the IaLd peptides with linear Xaa side chains followed the
trend: IaLd-Abu < IaLd-Nva IaLd-Nle (Figure 2-15a). For β-branched amino acids,
the helical content of the IaLd peptides followed the trend: IaLd-Val < IaLd-Ile
IaLd-All Ile (Figure 2-15b). For these two groups, the longer side chain resulted in a
higher helical content, as observed in monomeric helices.46 Furthermore, the IaLd
higher helical content, as observed in monomeric helices.46 Furthermore, the IaLd