Comparison of backbone modification in protein β-sheets by α→γ residue replacement and α-residue methylation

Article abstract of DOI:10.1039/c4ob00886c

The mimicry of protein tertiary structure by oligomers with unnatural backbones is a significant contemporary research challenge. Among common elements of secondary structure found in natural proteins, sheets have proven the most difficult to address. Here, we report the systematic comparison of different strategies for peptide backbone modification in β-sheets with the goal of identifying the best method for replacing a multi-stranded sheet in a protein tertiary fold. The most effective sheet modifications examined led to native-like tertiary folding behavior with a thermodynamic folded stability comparable to the prototype protein on which the modified backbones are based. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4ob00886c

Volume 12 Number 29 7 August 2014 Pages 5313–5546
Organic &
Biomolecular
Chemistry
www.rsc.org/obc
ISSN 1477-0520
PAPER
W. Seth Horne et al.
Comparison of backbone modification in protein β-sheets by α→γ residue
replacement and α-residue methylation

Organic &
Biomolecular Chemistry
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PAPER
Comparison of backbone modification in
protein β-sheets by α→γ residue replacement
and α-residue methylation†
Cite this: Org. Biomol. Chem., 2014,
12, 5375
George A. Lengyel, Zachary E. Reinert, Brian D. Griffith and W. Seth Horne*
The mimicry of protein tertiary structure by oligomers with unnatural backbones is a significant contem-
porary research challenge. Among common elements of secondary structure found in natural proteins,
sheets have proven the most difficult to address. Here, we report the systematic comparison of different
strategies for peptide backbone modification in β-sheets with the goal of identifying the best method for
replacing a multi-stranded sheet in a protein tertiary fold. The most effective sheet modifications
examined led to native-like tertiary folding behavior with a thermodynamic folded stability comparable to
the prototype protein on which the modified backbones are based.
Received 30th April 2014,
Accepted 29th May 2014
DOI: 10.1039/c4ob00886c
www.rsc.org/obc
isolated α-helix5 and β-sheet6 secondary structures has recently
been leveraged to simultaneously modify all the secondary
Introduction
Synthetic oligomers with the capacity to adopt discrete folded structures in a small protein tertiary fold.7 A fundamental
structures (“foldamers”)1 have received significant research question that must be addressed for sequence-guided back-
attention,2 due in part to their ability to mimic natural peptide bone alteration to be effective for the widest array of target
folding patterns. In more than two decades of work showing folds is how to best apply chemical modification without dis-
increasingly sophisticated structures from unnatural back- rupting sequence-encoded folding.
bones, tertiary folds like those commonly found in proteins
Among common protein secondary structures, sheet folds
have proven difficult to recreate. Although significant progress have proved more challenging targets than helices or turns for
has been made with helix-turn-helix targets,3 these represent mimicry by unnatural oligomers. Building on pioneering work
only a small fraction of the diverse array of folds found in carried out largely in organic solvents,8 we have recently
nature. Reproducing a wider selection of natural protein struc- focused on developing strategies for the design of hetero-
tural motifs with unnatural oligomers is an important goal geneous-backbone β-sheet mimics that fold in water.6 Hairpin
because it opens the door to reproducing the full repertoire of model systems, widely used in fundamental studies on β-sheet
functions enabled by those folds.
formation in proteins,9 have proved valuable in assessing
One design concept that shows promise in addressing the sheet propensity of unnatural building blocks. Unfortunately,
challenge of tertiary structure mimicry is the systematic back- the lessons learned in the hairpin context are not always appli-
bone alteration of natural sequences. Folded proteins can tol- cable in a more complex protein tertiary fold. As an example,
erate diverse backbone modifications without compromising when incorporated in each strand of a hairpin-forming
sequence-encoded folding.4 Bridging the gap between these peptide, appropriately substituted β-amino acid residues
observations and precedent on de novo foldamer design2 (homologated analogues of α-residues) can maintain native-
suggests an approach toward protein mimicry in which a like folding,6a,b but the same modifications abolish folding
number of α-residues in a sequence with known folding behav- entirely when made in a four-stranded β-sheet in a small
ior are replaced with various unnatural building blocks to protein.7
generate heterogeneous backbones capable of adopting native-
Here, we report the side-by-side comparison of several
like folds. The versatility of the above method for mimicry of different strategies for peptide backbone modification in
β-sheet secondary structures with the goal of identifying the
best method for replacing a multi-stranded sheet in a protein
tertiary fold. The most effective sheet modifications examined
led to native-like tertiary folding behavior with thermodynamic
fold stability comparable to the prototype protein on which
Department of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260, USA.
E-mail: horne@pitt.edu
†Electronic supplementary information (ESI) available: Fig. S1–S3, Tables S1–S8,
and experimental methods for the synthesis of unnatural amino acid mono-
the modified backbones are based.
mers. See DOI: 10.1039/c4ob00886c
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actually more thermodynamically stable than that of the proto-
type α-peptide on which it was based.6c A second open ques-
tion we wanted to address in this study is whether such α→γcyc
Results and discussion
Strategies examined for sheet backbone alteration
We compared three strategies for peptide backbone alteration substitutions are tolerated in the complex structural environ-
in two different β-sheet forming host sequences – a two- ment of a multi-stranded sheet in a protein tertiary fold.
stranded hairpin peptide and a four-stranded sheet in a small
A third strategy examined for backbone modification in
protein tertiary fold (Fig. 1). α-Residues in each prototype β-sheets involved the replacement of α-residues with γ-residues
sequence were replaced with N-methylated analogues (α→N- bearing a side chain at Cγ and an (E)-double bond between Cα
Me-α), (E)-vinylogous γ4-residues (α→γ4), or the cyclically and Cβ. These vinylogous γ4-residue building blocks are known to
constrained γ-residue Acc (α→γcyc). These three backbone modi- be compatible with hairpin formation in organic solvent,8a,f but
fications and some of the unanswered questions we sought to their impact on folding in aqueous environments has not been
address about each are discussed in more detail below.
reported. γ4-Residues offer an advantage over the γcyc residue Acc
Methylation of backbone amide nitrogen atoms has been in that they can retain protein-derived side chains when they
widely applied in small peptides10 and can be used to modify replace α-residues in a native sequence. We incorporated vinylo-
capping strands of sheet-forming sequences.11 We recently gous γ4-residues (α→γ4 substitution in each strand) into both
showed α→N-Me-α residue substitution is accommodated in a peptide hairpin and protein sheet contexts in order to ascertain
small bacterial protein, but it resulted in a degree of destabili- the compatibility of these residues with the native folds.
zation that was surprising given the subtle nature of the
chemical change.7 One goal in the present work was to
elucidate the molecular basis of this destabilization by system-
Backbone alteration in a peptide β-hairpin host sequence
Peptide hairpin 1 (Fig. 2), derived from the C-terminal
atically examining the site-dependent structural and thermo-
segment of the B1 domain of Streptococcal protein G,12 has
dynamic effects of N-methylation on the folding of a small
proven a useful host sequence for exploring the sheet folding
propensities of modified peptide backbones in aqueous solu-
hairpin sequence.
In another effort toward sheet mimetics based on systema-
tion.6b,c Sequences 2–7 are variants of peptide 1 designed to
tic modification of natural sequences, we recently reported
systematically compare the impact of the different strategies
for backbone alteration described above on the structure and
stability of the sequence-encoded hairpin fold.
that the cyclically constrained γ-residue (1R,3S)-3-aminocyclo-
hexane carboxylic acid (Acc) can be incorporated into a
hairpin-forming α-peptide sequence (α→γcyc substitution in
In peptide 2, α-residues Ala4 and Ala13 in 1 are replaced by
each strand) without significantly altering the folded struc-
the constrained γcyc-residue Acc (α→γcyc substitution). We have
ture.6c Interestingly, the fold of the chimeric α/γcyc-peptide was
previously reported the synthesis and biophysical analysis of 2,
and it is included here as a point of comparison.6c Peptide 3
has the same sites of backbone modification as 2, but the
unnatural building blocks are vinylogous γ4-residues bearing
Fig. 2 Sequences of peptides 1–7, key to α-residue replacements (Xxx
Fig. 1 Summary of strategies examined for peptide backbone modifi-
cation in β-sheets. The impact of three different types of α-residue
replacement on folding was evaluated in two different structural con-
texts, a β-hairpin peptide and a four-stranded β-sheet in a small protein.
indicates the side chain on the unnatural monomer is the same as the
corresponding α-residue), and minimized average coordinates from the
NMR solution structure of prototype peptide 1 in pH 6.3 phosphate
buffer.
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the side chain of the replaced α-residues in 1 (α→γ4 substi- NMR-derived distance restraints and compared the resulting
tution). In peptides 4–7, α-residues Trp3, Tyr5, Phe11, or Val13 coordinates to the previously determined structure of γcyc
-
from host sequence 1 are individually modified by N-methyl- residue-containing variant 2. Due to the low folded population
ation (α→N-Me-α). These four sites, all at non-hydrogen- of 3, we synthesized a cyclic derivative for NMR structural ana-
bonding positions in the hairpin, were modified separately to lysis (3cyc), which has Cys residues appended to each terminus
determine how sequence context influences the thermo- and linked together via a disulfide bond. α/γ4-Peptide 3cyc
dynamic impact of N-methylation on hairpin folded stability.
forms a β-hairpin fold very similar to that of α/γcyc-peptide 2
Peptides 1–7 were synthesized by Fmoc solid-phase peptide (Fig. 3). The similarity among the solution structures of 2 and
methods, purified by reverse-phase HPLC, and the identities 3cyc suggest the different number of freely rotatable bonds in
of the purified oligomers confirmed by mass spectrometry. We the two γ-residue classes (three for each unsaturated γ4 vs. two
compared the folding behavior of 1–7 by a series of multi- for each γcyc) is likely responsible for the different folded stabi-
dimensional NMR experiments carried out in pH 6.3 phos- lities of the hairpins containing them.
1
phate buffer at 5 °C. Homonuclear H–1H COSY, TOCSY, and
Analysis of the NMR data for peptides 4–7 reveals two
NOESY spectra were sufficient to enable full resonance assign- important issues with respect to backbone N-methylation in
ment of each oligomer. As described in prior work, we used sheet-forming sequences. First and most pronounced are com-
the chemical shift separation of the two diastereotopic Hα’s in plications arising from cis/trans amide isomerization.10,14 Each
Gly10 to quantify folded population and estimate folding free N-methyl hairpin showed signals for two distinct species by
energy in the modified hairpins (Table 1).6b,c,12,13
NMR, which we attributed to a mixture of cis and trans
Comparison of the NMR data for peptides 2 and 3 suggests isomers at the tertiary amide introduced upon N-methylation.
that the connectivity of the γ-residue incorporated into each We calculated the isomer ratio by integrating well-resolved
strand of the hairpin has a significant effect on folding ener- peaks in the TOCSY spectra. Population ratios varied among
getics. We previously showed that peptide 2, which contains the four oligomers, but the trans amide was predominant in
two α→γcyc substitutions relative to 1, forms a more stable each case. We made this assignment based on analysis of the
hairpin than the wild-type backbone.6c In contrast, the α→γ4 NOESY data, which showed close contacts between the back-
substitutions in peptide 3 measurably destabilized the fold bone methyl group in the trans isomer and both the side-chain
(∼0.6 kcal mol−1 relative to prototype 1 and ∼1.1 kcal mol−1 and Hα protons of the preceding residue. The consistently
relative to variant 2 with the Acc residues).
lower Gly Hα/Hα′ chemical shift separation indicates the pres-
In considering the different impact of γcyc and γ4 residues ence of a cis amide in the chain destabilizes the hairpin fold
on hairpin folded stability, we saw two possible origins: a sig- considerably. This is reasonable considering how such a
nificant change in the folded structure or altered backbone change would disrupt backbone direction and side chain con-
flexibility. In order to test the former hypothesis, we pursued a tacts that enable parent sequence 1 to fold.
solution structure of
3
by simulated annealing with
Separate from the issue of amide isomerization in N-Me-
α-peptides 4–7 is the question of how the folded stability of the
all-trans isomers compare to α-peptide 1. The answer depends
on the positioning of the backbone methyl group relative to
the hairpin turn. The presence of an N-methyl amide in a trans
configuration at either Trp3 (peptide 4) or Val14 (peptide 7) led
to a folded population identical within error to that of proto-
type sequence 1. By contrast, when the site of backbone
Table 1 Folding thermodynamics of peptides 1–7 from NMR
measurementsa
Δδ Gly10
Hα/Hα′
(ppm)
Fraction
folded
(%)
ΔGfold
(kcal
ΔΔGfold vs. 1
Peptide
mol−1
)
(kcal mol−1
)
1
2
3
4
0.20
0.26
0.12
65
83
39
37b
61
−0.3
−0.9
+0.2
+0.3
−0.3
−0.6
+0.5
+0.6
+0.0
4trans (60%)
4cis (40%)
0.19
0.09
5
6
7
19b
30
+0.8
+0.5
+1.1
+0.8
5trans (76%)
5cis (24%)
0.09
0.00
29b
38
+0.5
+0.3
+0.8
+0.6
6trans (65%)
6cis (35%)
0.12
0.00
55b
63
−0.1
−0.3
+0.2
+0.0
7trans (87%)
7cis (13%)
0.20
0.12
a NMR carried out at 5 °C in pH 6.3 phosphate buffer. Assuming a
0.01 ppm uncertainty in measured Gly Hα/Hα′ separation, error
propagation estimates uncertainties of 5% for fraction folded and
Fig. 3 Minimized average coordinates from NMR solution structures of
α,γcyc-peptide
2 6 phosphate buffer.
and α/γ4-peptide 3cyc in pH
∼0.2 kcal mol−1 for ΔGfold and ΔΔGfold b Overall folded population
.
Carbons are colored green for γ-residues and yellow for α-residues.
Most side chains are omitted for clarity.
calculated as product of the fraction of peptide in the trans amide
configuration and fraction folded for trans isomer.
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methylation was closer to the turn (peptides 5 and 6), the
We compared the folding behavior of proteins 8–13 by cir-
folded state was measurably destabilized – even after taking cular dichroism (CD) spectroscopy in pH 7 phosphate buffer.
into account the detrimental contribution of the cis isomer. CD scans (Fig. 5A) for three of the four modified backbones
Computational studies have shown that the energetically (proteins 9, 11, and 13) showed shapes and magnitudes
accessible backbone conformations of N-Me-α-residues are similar to wild-type GB1. These results suggest α→N-Me-α sub-
more restricted than their non-methylated analogues.15 Notably, stitution and two different patterns of α→γcyc substitution are
one region of the Ramachandran plot that becomes signifi- well tolerated in the tertiary fold. Protein 10, bearing four
cantly disfavored energetically upon N-methylation corresponds α→γ4 residue replacements, had a CD spectrum qualitatively
to typical backbone dihedrals for strands from an antiparallel different from all the other GB1 analogues examined. Its dis-
β-sheet.16 The above observations help to rationalize the similarity to typical random coil signatures and dependence
observed destabilization of the hairpin folds of trans-amide on temperature (vide infra) argue against 10 existing as an
isomers of N-Me-α-peptides 5 and 6 relative to α-peptide 1.
unstructured chain. We cannot definitively say whether the
spectrum of 10 is a result of an altered folded state or a change
in the CD signature of the native-like tertiary fold due to the
presence of four α,β-unsaturated amides in the backbone. In
an effort to clarify this point, we attempted to obtain diffrac-
tion-quality crystals of protein 10 but were unsuccessful.
We performed thermal denaturation experiments to deter-
mine the impact of backbone substitutions on folded stability,
monitoring the CD minima at 220 nm as a function of temp-
erature (Fig. 5B). All the proteins showed sharp sigmoidal
unfolding transitions with cooperativities similar to wild-type
GB1. The steep thermal unfolding transitions suggest the
modified oligomers have well-ordered folded states. Compar-
ing the midpoints of the thermal unfolding transitions (Tm)
provides an estimate of the energetic impact of various back-
bone alterations on folding (Table 2).
As reported previously, protein 9 bearing two α→N-Me-α
substitutions has a stability only slightly lower than wild-type
GB1.7 In contrast, α→γ residue replacements in proteins 10 and
11 destabilize their folded states considerably. Consistent with
their relative folding propensities in the hairpin host sequence,
the γcyc Acc residues (protein 11) are superior to vinylogous γ4
residues (protein 10) in supporting the tertiary structure when
incorporated at identical positions it the host sequence.
Backbone alteration in a protein β-sheet host sequence
As a host sequence to examine sheet backbone modification in
the context of a tertiary structure, we employed the full-length
56-residue B1 domain of Streptococcal protein G (8, Fig. 4).
This sequence, from which hairpin peptide 1 is derived,
adopts a compact tertiary fold consisting of an α-helix packed
against a four-stranded β-sheet.17 Of the three backbone altera-
tion strategies above, one has previously been examined in GB1:
α→N-Me-α substitution at terminal strands in the sheet
(protein 9).7 We include data for protein 9 here for comparison.
In proteins 10 and 11, α→γ residue substitutions are made in
place of Ile6, Glu15, Thr44, and Thr53. Protein 10 incorporates
constrained γcyc residues at these positions, while variant 11
bears vinylogous γ4-residues with side chains derived from the
natural GB1 sequence. In both 10 and 11, the positioning of
γ-residues is designed to create a stripe of unnatural residues
along the center of the sheet if the modified backbones adopt a
native-like tertiary fold. Protein 12 is a variant of GB1 with a
completely natural backbone but mutations that remove three
polar side-chain functional groups that are lost upon incorpor-
ation of Acc residues in protein 11. Finally, protein 13 is a
variant of 11 with the same number of γcyc residues but incor-
porated at positions intended shift the stripe of unnatural
monomers to a different region of the sheet.
In considering the data for α/γ-hybrid protein 11, we found
it striking that the γcyc residue Acc, which stabilized the
Fig. 4 Sequences of proteins 8–13, key to α-residue replacements (Xxx indicates the side chain on the unnatural monomer is the same as the
corresponding α-residue), crystal structure of wild-type GB1 8 (PDB 2QMT), and schematics showing the placement of unnatural residues in 9, 10,
11 and 13.
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tution. When found in the core of the protein as in 10 and 11,
this change may disrupt critical hydrophobic contacts between
the sheet and helix necessary for folding. In order to test this
hypothesis, we examined an analogue of GB1 (protein 13),
bearing four Acc residues in a pattern that would create a stripe
of γ-residues in the sheet as in 10 and 11 but further removed
from the hydrophobic core of the protein in the folded state.
We reasoned that the alteration in backbone length of the sheet
would be better tolerated if it was not located in close proximity
to key tertiary contacts. Supporting the above hypothesis,
protein 13 showed a dramatically improved thermal stability
compared to closely related analogue 11.
Conclusions
In summary, we have reported here the systematic comparison
of three different strategies for peptide backbone modification
in β-sheet secondary structures using two different host
systems – a hairpin peptide and a small protein with a defined
tertiary fold. Our results provide new insights into the design of
heterogeneous backbones based on natural peptide sequences
that encode for β-sheet folds. In the peptide hairpin host
sequence, α→γcyc substitution was superior to α→γ4, which was
better than backbone methylation (α→N-Me-α). Destabilization
of the sheet fold by α→N-Me-α substitution appears to result pri-
marily from population of an unproductive cis tertiary amide
isomer at the methylation site, though the fold of the trans
isomer is also destabilized relative to native due to local stereo-
electronic effects. In the best case for hairpin modification
(α→γcyc), the heterogeneous backbone had a more stable fold
than the prototype α-peptide on which it was based.
When substitutions are applied to a central stripe of strand
residues in the protein tertiary structure, the trend was signifi-
cantly different than the hairpin host sequence: N-Me-α
residue incorporation at capping strands of the sheet was best
tolerated, followed by γcyc and vinylogous γ4 substitutions in
all four strands. Optimization of the placement of γcyc resi-
dues, however, had a dramatic effect on the thermodynamic
consequences of the modification. Shifting the position of the
backbone expansion resulting from α→γ residue substitution
away from the hydrophobic core of the protein led to a hetero-
geneous backbone with near wild-type folded stability. We
anticipate these results will aid in ongoing efforts to recreate
β-sheet folding patterns from heterogeneous backbones and
open the way toward design of protein-mimetic oligomers with
increasingly diverse tertiary folding topologies.
Fig. 5 Circular dichroism scans at 25 °C (A) and thermal melts moni-
tored at 220 nm (B) for proteins 8–13. Experiments were carried out on
40 μM concentration protein samples in 20 mM phosphate buffer, pH 7.
Table 2 Folding thermodynamics of proteins 8–13 from circular
dichroism measurementsa
ΔΔGfold per
ΔΔGfold vs. 8
Substitutions
vs. 8
substitution
Protein
Tm a (°C)
(kcal mol−1
)
(kcal mol−1
)
8
9
10
11
12
13
82.1
75.6
43.5
46.7
78.0
74.3
+1.1
+6.3
+5.9
+0.7
+1.3
2 α→N-Me-α
4 α→γ4
0.6
1.6
1.5
0.2
0.3
4 α→γcyc
4 side chains
4 α→γcyc
a CD experiments carried out in pH 7 phosphate buffer.
hairpin secondary structure, was so destabilizing to the tertiary
fold. One difference between protein 11 and wild-type 8
besides the altered backbone in the former is the loss of three
functionalized side chains upon substitution of α-residues
Glu15, Thr44, and Thr53 with Acc. Inspection of the crystal
structure of wild-type GB1 shows three of these side chains are
involved in inter-strand polar contacts that potentially stabilize
the tertiary fold. The CD thermal stability observed for protein
12, which has a natural backbone but lacks polar groups
necessary for these contacts, indicate that the lost side-chain
functionality is at most a very small contributor to the differ-
ence in folding behavior between 11 and 8.
Experimental
Peptide and protein synthesis
Protected γ4-amino acids were prepared via the corresponding
α-amino aldehydes18 according to published methods.19
Fmoc-Acc was prepared as previously described.6c Full experi-
mental details and characterization data for new compounds
are given in the ESI.†
Another factor we considered as potentially responsible for
destabilization of the γ-residue modified proteins is the increase
in backbone length by two atoms with each α→γ residue substi-
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β-Hairpin peptides were synthesized using microwave- tration of 0.8–3 mM. 3-(Trimethylsilyl)-1-propanesulfonic acid
assisted Fmoc solid-phase synthesis techniques on a MARS sodium salt (DSS, 50 mM in water) was added to a final con-
microwave reactor (CEM) using NovaPEG Rink Amide resin. centration of 0.2 mM. Each solution was passed through a
Couplings were carried out in NMP at 70 °C for 4 min using 0.2 µm syringe filter, and transferred to an NMR tube. The
4 equiv. of Fmoc-protected amino acid, 4 equiv. of HCTU, and NMR tube headspace was purged with a stream of nitrogen
6 equiv. DIEA. PyAOP was used in place of HCTU for the coup- prior to capping.
ling of N-methylated residues and residues immediately fol-
NMR experiments were performed on a Bruker Avance-700
lowing them. Deprotections were performed using an excess of spectrometer. Chemical shifts are reported relative to DSS
20% 4-methylpiperidine in DMF at 80 °C for 2 min. After each (0 ppm). TOCSY, NOESY, and COSY pulse programs used exci-
coupling or deprotection cycle, the resin was washed three tation-sculpted gradient-pulse solvent suppression. For all 2D
times with DMF. Double couplings were performed at experiments, 2048 data points were collected in the direct
sequence positions following proline or N-methylated residues. dimension and 512 data points in the indirect dimension. The
Prior to cleavage, the resin was washed three times each with mixing times for TOCSY and NOESY were 80 ms and 200 ms,
DMF, dichloromethane, and methanol, and then dried. respectively. NMR measurements were performed at a tempera-
Peptide cleavage was accomplished using 95% trifluoroacetic ture of 278 K for hairpin peptides 3–7 and at 293 K for
acid (TFA), 2.5% triisopropylsilane (TIS), and 2.5% water.
cyclized hairpin peptide 3cyc. The Sparky software package
Peptide was precipitated from the cleavage solution by (T. D. Goddard and D. G. Kneller, SPARKY 3, University of
addition of diethyl ether and purified by preparative HPLC on California, San Francisco) was used to analyze 2D NMR data.
a C18 column using gradients between 0.1% TFA in water and Backbone chemical shift assignments for peptides 3–7 are
0.1% TFA in acetonitrile. After purification, the linear precur- reported in Tables S2–7.† Analysis of NMR data for 3–7 and
sor to peptide 3cyc was dissolved in 10 mM pH 8.9 phosphate estimation of folded populations followed previously pub-
buffer with 5% v/v DMSO, stirred until analytical HPLC and lished methods.6b,c Tabulated NOEs for peptide 3cyc are
MS showed complete conversion to the cyclic disulfide (2 d), reported in Table S8.† These data were applied to calculate an
and then purified by HPLC to obtain 3cyc
.
NMR solution structure of 3cyc using the Crystallography and
Protein GB1 and variants were synthesized on a PTI Tribute NMR system (CNS) software package20 according to published
synthesizer using NovaPEG Rink Amide resin (70 μmol scale). methods.6b,c
Coupling reactions were performed by combining 3 mL of
Circular dichroism spectroscopy
0.4 M N-methylmorpholine in DMF with 7 equiv. Fmoc-amino
acid and 7 equiv. HCTU. Following a two minute preactivation,
the activated amino acid was added to the resin and vortexed
for 45 min. Deprotection reactions were carried out twice with
3 mL of a 20% v/v solution of 4-methylpiperidine in DMF for
4 min. The resin was washed three times with 3 mL of DMF
for 40 s between each cycle. After the final deprotection step,
the resin was washed with 3 mL of dichloromethane followed
by 3 mL of methanol. Resin was dried and subjected to clea-
vage by treatment with a solution of 94% TFA, 1% TIS, 2.5%
water, and 2.5% ethanedithiol. Crude protein was precipitated
by addition of cold diethyl ether. The solid was pelleted by
centrifugation and dissolved in 6 M guanidinium chloride,
25 mM sodium phosphate, pH 6. This solution was subjected
to purification by preparative C18 reverse-phase HPLC using
gradients between 0.1% TFA in water and 0.1% TFA in aceto-
nitrile. Each protein was subjected to a second round of purifi-
CD measurements were performed on an Olis DSM17 Circular
Dichroism Spectrometer in 2 mm quartz cells. Samples con-
sisted of 40 μM protein in 20 mM sodium phosphate buffer,
pH 7. Scans were carried out at 25 °C over the range of
200–260 nm in 1 nm increments with a 2 nm bandwidth. Scan
data were smoothed by the Savitzky–Golay method. Melts were
monitored at 220 nm over the range of 4 °C to 98 °C with 2 °C
increments, a dead band of 0.5 °C, and a 2 min equilibration
time at each temperature. All measurements were baseline cor-
rected for blank buffer. Temperature-dependent CD data were
fit to a two-state unfolding model to obtain melting tempera-
ture (Tm). The change in free energy of folding for each mutant
relative to wild-type (ΔΔGfold) was estimated from the change
in Tm (ΔTm) using the enthalpy of folding determined for GB1
by differential scanning calorimetry.21
cation by anion-exchange chromatography on
a monoQ
5/50GL column (GE Healthcare) using 0.02 M Tris buffer at
pH 8 and eluting with increasing concentrations of KCl.
All peptides and proteins used for biophysical analysis were
>95% pure as determined by analytical HPLC on a C18
column. Identities were confirmed by mass spectrometry
using a Voyager DE Pro MALDI-TOF instrument (Table S1†).
Acknowledgements
Funding for this work was provided by the University of
Pittsburgh and the National Institutes of Health (R01GM107161).
Notes and references
NMR sample preparation, data collection, and analysis
NMR samples were prepared by dissolving peptide in
750–850 µL of degassed 50 mM phosphate, 9 : 1 H2O–D2O,
pH 6.3 (uncorrected for the presence of D2O) to a final concen-
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