-
Do guanidinium and tetrapropylammonium ionsspecifically interact
with aromatic amino acidside chains?Bei Dinga,b, Debopreeti
Mukherjeea, Jianxin Chena,b, and Feng Gaia,b,1
aDepartment of Chemistry, University of Pennsylvania,
Philadelphia, PA 19104; and bUltrafast Optical Processes
Laboratory, University of Pennsylvania,Philadelphia, PA 19104
Edited by Michael L. Klein, Temple University, Philadelphia, PA,
and approved December 16, 2016 (received for review November 1,
2016)
Many ions are known to affect the activity, stability, and
structuralintegrity of proteins. Although this effect can be
generally attributedto ion-induced changes in forces that govern
protein folding,delineating the underlying mechanism of action
still remains chal-lenging because it requires assessment of all
relevant interactions,such as ion–protein, ion–water, and ion–ion
interactions. Herein, weuse two unnatural aromatic amino acids and
several spectroscopictechniques to examine whether guanidinium
chloride, one of themost commonly used protein denaturants, and
tetrapropylammo-nium chloride can specifically interact with
aromatic side chains.Our results show that tetrapropylammonium, but
not guanidinium,can preferentially accumulate around aromatic
residues and that tet-rapropylammonium undergoes a transition at
∼1.3 M to form aggre-gates. We find that similar to ionic micelles,
on one hand, suchaggregates can disrupt native hydrophobic
interactions, and on theother hand, they can promote α-helix
formation in certain peptides.
guanidinium | tetrapropylammonium | Hofmeister ions |unnatural
amino acid | 2D IR
The stability of a protein, or more precisely, the free
energydifference between its folded and unfolded states, can
bemodulated by various solution properties. For example, addition
ofanother solute to a protein solution can result in either a
decreaseor an increase in this protein’s stability (1–3). The most
noticeableexample in this regard is the Hofmeister series (4–6), a
group ofions that are ranked based on their protein-denaturing
abilities.Among this series, the guanidinium ion (Gdm+) is the most
widelyused chemical agent in protein denaturation due to its
strongdestabilizing effect and high solubility in water.
Consequently, itsmechanism of action has been subjected to
extensive studies (7–14).Although different interpretations have
been put forth (15–17), thegenerally accepted notion is that Gdm+
denatures a protein bypreferentially interacting with its peptide
groups (7, 11, 18), in-cluding certain side chains (11, 19–22). In
particular, it has beenhypothesized that Gdm+, which exists in
aqueous solution as a rigid,flat object (12, 18, 19), can engage in
stacking interactions withamino acids consisting of planar side
chains, such as arginine (Arg)(19), asparagine (Asn) (11, 20),
glutamine (Gln) (11, 20), and ar-omatic residues (20–22). However,
to the best of our knowledge,the only experimental evidence that
supports this hypothesis comesfrom crystallographic data (20–22),
which show that the guanidi-nium group of Arg is more frequently
found to be stacked againstthe side chain of tyrosine (Tyr) or
tryptophan (Trp) in proteins.Therefore, additional experimental
studies that can directly probesuch stack interactions in solution
are needed.Another ion in the Hofmeister series that has a similar
protein-
denaturing capability to Gdm+ is tetrapropylammonium (TPA+)(23).
However, a recent study by Dempsey et al. (24) found thatalthough
TPA+, like Gdm+, can denature a tryptophan (Trp) zip-per β-hairpin
(i.e., trpzip1) at a sufficiently high concentration(>1.0 M), it
stabilizes this β-hairpin at lower concentrations andalso the
α-helical conformation of an alanine-based peptide. Thisobservation
led them to conclude that a TPA+ ion, which has four
roughly flat faces, can interact favorably with planar aromatic
sidechains but is excluded from interacting with peptide backbone
dueto its large size (24). Although the finding that TPA+ can
denaturetrpzip1 is consistent with an earlier molecular dynamics
(MD)simulation (23), which revealed that TPA+ has a longer
residencetime on the indole ring of Trp than on other nonaromatic
sidechains in the peptide melittin, the stabilizing actions of TPA+
re-main to be explained (24). In addition, results obtained with
di-electric spectroscopy (25) and ultrafast infrared (IR)
spectroscopy(26) on a series of tetra-n-alkylammonium salts
indicated that ionsconsisting of long alkyl chains, such as TPA+,
can cause the motionof nearby water molecules to slow down, owing
to their largerhydrophobic surfaces and hence the so-called
hydrophobic hydra-tion. However, it is not clear how such hydration
property of TPA+
would contribute to its interaction with different protein
moieties.Although previous studies (23, 24) have provided many
insights
on the protein denaturation mechanisms of Gdm+ and TPA+
ions,their molecular actions have not been directly probed from
theperspective of the protein. In this regard, it would be quite
advan-tageous to devise an experimental approach that is capable of
re-vealing information on specific interactions of interest, such
asinteractions between Gdm+/TPA+ ions and a specific amino acidside
chain. To achieve this goal, herein we use multiple
spectroscopicmethods and two unnatural amino acids,
p-cyano-phenylalanine(PheCN) and 5-cyano-tryptophan (TrpCN), to
directly assesswhether protein aromatic side chains can
preferentially interactwith Gdm+ and TPA+ ions. Both the C≡N
stretching vibration(27, 28) and fluorescence quantum yield (29,
30) of PheCN andTrpCN have been shown to be sensitive to their
local environment.Thus, the premise of our study is that any
specific ion–side chain
Significance
Ever since the discovery by Franz Hofmeister in 1888 that a
seriesof salts (now commonly referred to as the Hofmeister series)
canhave different but consistent effects on the solubility and
hencestability of proteins, many studies have been devoted to
un-derstanding how such effects are achieved. However, examiningany
specific mode of ion–protein interactions has proven to
bedifficult. Herein, we demonstrate, using guanidinium and
tetra-propylammonium as examples, that it is possible to use
multiplespectroscopic methods and unnatural amino acids that
havedistinct, environment-sensitive fluorescence and infrared
prop-erties to explicitly assess how these ions interact with a
specifictype of amino acid side chains, thus yielding microscopic
insightsinto their protein-denaturing mechanisms.
Author contributions: B.D. and F.G. designed research; B.D.,
D.M., and J.C. performedresearch; B.D. analyzed data; and B.D.,
D.M., and F.G. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.1To whom correspondence
should be addressed. Email: [email protected].
This article contains supporting information online at
www.pnas.org/lookup/suppl/doi:10.1073/pnas.1618071114/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1618071114 PNAS | January 31,
2017 | vol. 114 | no. 5 | 1003–1008
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interaction will manifest itself as a change in the local
hydrationand electrostatic environment of the spectroscopic probe
used,which can be assessed by either infrared (IR) or
fluorescencespectroscopy. Several previous studies (27, 31–33) have
demon-strated that 2D IR spectroscopy is ideally suited to study
suchchanges because it is capable of reporting the
frequency–fre-quency correlation function of the IR vibration in
question andhence the dynamics of motions (arising from both
hydration andprotein conformational heterogeneities) that
contribute to theoverall bandwidth of the IR spectrum. Our results
show thatcontrary to the conventional expectation, Gdm+ does not
specif-ically stack on aromatic side chains. On the other hand,
consistentwith molecular dynamics simulations (23), TPA+ exhibits a
strongaffinity toward aromatic side chains, and at ∼1.3 M, TPA+
ag-gregates to form clusters that provide an interfacial region,
which,similar to that found in lipid membranes and micelles (34),
canhelp anchor Trp side chains and also promote α-helix formation
inalanine-based peptides, due to dehydration and hence a
lowerdielectric environment.
Results and DiscussionTo access whether Gdm+ and TPA+ can
specifically interact withthe two aforementioned aromatic unnatural
amino acid sidechains (i.e., PheCN and TrpCN) in a polypeptide
environment, wefirst examined the dynamics of the C≡N stretching
vibration oftwo peptides, Gly-PheCN-Gly and Gly-TrpCN-Gly
(hereafter re-ferred to as GFCNG and GWCNG, respectively), in the
presenceand absence of Gdm+ and TPA+ ions using Fourier
transforminfrared (FTIR) and 2D IR spectroscopies. We chose
thesepeptide sequences to ensure the full exposure of the
respectivearomatic side chains to solvent and to avoid any side
chain–sidechain interactions. The latter could complicate
interpretation ofthe spectroscopic results. We then further
verified our finding onTrpCN–TPA
+ interactions using a longer peptide with the fol-lowing
sequence: SWCNTAENGKATWCNK (hereafter referredto as 2WCNP). In
addition, we also used an alanine-based pep-tide (sequence:
GKAAAAKWCNAAAWCNKAAAAKG) and anantimicrobial peptide (sequence:
INWKGIAAMAKKLL), bothof which can be induced to form α-helix by
TFE, to test the helix-inducing ability of TPA+ ions. Wherever
applicable, we also car-ried out fluorescence and circular
dichroism (CD) measurementsto substantiate the IR results.
Linear and 2D IR Studies. As shown (Fig. 1 and SI Appendix,
TableS1 and Fig. S1), the FTIR spectra of GFCNG and GWCNGobtained
under different solvent conditions indicate that bothGdm+ and TPA+
(at high concentrations) affect the C≡Nstretching vibrations of
PheCN and TrpCN in these peptides. Spe-cifically, in 6.0 M GdmCl
solution the C≡N stretching bandsof both peptides show a small but
measurable red-shift (i.e.,∼1.6 cm−1 for GFCNG and ∼1.3 cm−1 for
GWCNG) in comparisonwith their respective peak frequencies in pure
water. In addition,the width of these bands shows a slight increase
(
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For an oscillator that is capable of forming hydrogen bonds
withwater, such as the CN group in the current case, the
exponentialcomponent in Eq. 1 is typically attributed to the
dynamics of hy-drogen bonds formed between the oscillator and water
molecules(27, 31–33). As shown (SI Appendix, Table S1), for both
GFCNGand GWCNG, the major effect of 6.0 M GdmCl is manifested by
amodest increase in the offset B value, which, according to
previousstudies (31), could be attributed to the increase in
viscosity. Thus,this finding is consistent with the linear IR
results and also theabovementioned notion that GdmCl does not
increase in any sig-nificant manner the inhomogeneity of the
environment sampled byPheCN and TrpCN in these peptides. On the
contrary, the effect of2.0 M TPACl on the spectral diffusion
dynamics of the C≡Nstretching vibration of both peptides is much
more pronounced (SIAppendix, Table S1), with a substantial increase
in the B value inboth cases. This result indicates that at a
concentration of 2.0 M,TPA+ ions can effectively slow down certain
dynamic motions inboth peptides that contribute to the spectral
diffusion dynamics ofthe C≡N stretching vibration and hence its IR
bandwidth. Thus,this finding corroborates the conclusion reached
above that TPA+
ions specifically accumulate around aromatic side chains,
likelythrough the stacking interactions suggested by Mason et al.
(23).A closer comparison of the spectral diffusion dynamics of
the
C≡N probes in GFCNG and GWCNG obtained in 2.0 M solution ofTPACl
further indicates that TPA+ ions affect the hydration statusof
PheCN and TrpCN differently (Fig. 3 and SI Appendix, Fig. S3). Itis
apparent that for GFCNG, interacting with TPA
+ ions does notresult in any significant change in the
hydrogen-bonding compo-nent of the spectral diffusion dynamics,
whereas for GWCNG, in-teractions between TrpCN and TPA
+ ions essentially eliminate thispicosecond dynamic component.
In other words, these results showthat interaction with TPA+ ions
causes the TrpCN side chain to bemore dehydrated than that of
PheCN, a picture that is consistentwith the MD simulation of Mason
et al. (23), which suggested thatTPA+ ions interact more strongly
with the indole ring of Trp thanwith other hydrophobic groups in
the antimicrobial peptidemelittin. Despite this stronger
interaction, however, based on the
2D IR results alone we cannot rule out the possibility that
thereare water molecules surrounding the TrpCN side chain in
thepresence of 2.0 M TPA+. Nonetheless, these water molecules,
ifany, should be less mobile than bulk water and hence do notcause
the C≡N stretching frequency to fluctuate on the timescaleof our
experiment, as observed in other exmples (32, 39).
Fluorescence Studies. To help better understand the results
obtainedwith linear and nonlinear IR spectroscopic methods, we also
per-formed fluorescence measurements. It has been shown that
thefluorescence quantum yields of PheCN and TrpCN are sensitive
tointeractions with water molecules (29, 30), with a relation
thathydration increases/decreases the fluorescence intensity of
PheCN/TrpCN. Thus, both unnatural amino acids can in principle be
usedas fluorescence probes to assess how Gdm+ and TPA+ affect
thelocal hydration status of PheCN and TrpCN in GFCNG and
GWCNG.However, we only carried out fluorescence experiment on
GWCNGbecause Cl− is known to significantly quench the fluorescence
ofPheCN (29). As shown (Fig. 4, Inset), the TrpCN fluorescence
in-tensity of GWCNG in 6.0 M GdmCl solution is slightly lower
thanthat in pure H2O. However, addition of 2.0 M TPACl leads to
asignificant increase in the fluorescence intensity of TrpCN
(∼9.7times) in comparison with that obtained in pure H2O. Taken
to-gether, these results indicate that TPA+ ions (at 2.0 M), but
notGdm+ ions (at 6.0 M), show preferential interactions with
TrpCN(and hence Trp), resulting in either partial or complete
dehydrationof its side chain, as concluded from the IR
experiments.
Concentration-Dependent Studies. To gain further insight into
theinteraction between TPA+ and aromatic side chains, we carried
outsimilar linear and nonlinear IR studies on GFCNG and GWCNG
insolutions of different molar concentrations of TPACl ([TPACl]).As
expected, for both peptides, increasing [TPACl] leads to a
de-crease/increase in the frequency/bandwidth of the C≡N
stretchingvibration (SI Appendix, Table S3 and Figs. S4–S6), which
is ac-companied by an increase in the static component in the
respectiveC≡N spectral diffusion dynamics (SI Appendix, Table S3).
In ad-dition, although the hydrogen-bonding dynamics component
ofGFCNG does not show a significant dependence on [TPACl], thesame
component of GWCNG becomes practically undetectablewithin our
experimental uncertainty when [TPACl] is increased toabout 1.5 M
(SI Appendix, Figs. S7–S10). As indicated (SI Appendix,
Fig. 2. Representative 2D IR spectra of GWCNG in the C≡N
stretching fre-quency region obtained under different solvent
conditions and at differentwaiting times, as indicated.
0.0
0.2
0.4
0.6
0.0 1.0 2.0 3.0 4.0 5.0
Waiting Time, T (ps)
CLS
-1
H2O 6.0 M GdmCl 2.0 M TPAClH2
Fig. 3. CLS−1 versus T plots of the C≡N stretching vibration of
GWCNG obtainedunder different solvent conditions, as indicated.
Ding et al. PNAS | January 31, 2017 | vol. 114 | no. 5 |
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Fig. S11), a further fluorescence titration experiment reveals
thatthe fluorescence intensity of TrpCN in GWCNG exhibits an
unusualdependence on [TPACl]—it increases linearly as a function
of[TPACl] with one slope in the range of 0–1.3 M but a steeper
slopebetween 1.3 and 2.0 M (Fig. 4). This indicates that there is a
distincttransitional event occurring at about 1.3 M. A dielectric
spectros-copy study by Buchner et al. (25) on tetrapropylammonium
bro-mide (TPABr) aqueous solutions showed that the
dispersionamplitude of the water trapped in the hydration shell of
TPA+ ionsincreases with [TPABr] and reaches a maximum at 1.3 M,
which,based on neutron scattering experiments (40), was attributed
toTPA+ aggregation taking place at concentrations above this
criticalvalue. Similarly, the study of Bakker and coworkers (26) on
thedynamics of water surrounding tetra-n-alkylammonium ions
withlong alkyl chains also suggested ion aggregation. Thus, taking
thoseprevious findings into consideration, our IR and fluorescence
re-sults provide direct evidence that aggregated and
nonaggregatedTPA+ ions have different effects on the hydration
dynamics of theside chain of TrpCN and, perhaps more importantly,
that aggre-gated TPA+ ions are more effective in dehydrating TrpCN
(andhence Trp). The latter is reminiscent of the phenomenon that
Trpprefers the interfacial region of lipid membranes (41) and
associ-ation with this region leads to dehydration of its indole
moiety (42).Thus, this result suggests that above the critical
concentration of1.3 M, TPA+ ions aggregate to form clusters that
exhibit a distinctwater-hydrophobic interface. Furthermore,
considering the findingof Dempsey et al. (24) that TPA+ shows a
denaturing effect towardtrpzip1 only when its concentration is
higher than 1 M, we believethat the aggregated and nonaggregated
TPA+ ions have differenteffects on protein hydrophobic interactions
and that the protein-denaturing ability of TPA+ arises from its
aggregated clusters.
Effect on a Longer Peptide. To further validate the findings
obtainedwith GWCNG, we used the same approach to study a longer
pep-tide, 2WCNP. As shown (SI Appendix, Table S4 and Fig. S12),
thelinear IR spectroscopic properties of the C≡N stretching
vibrationof 2WCNP obtained in 6.0 M GdmCl and 2.0 M TPACl
solutionsare almost identical to those of GWCNG obtained under the
samesolvent conditions. However, the C≡N stretching vibrational
band
of 2WCNP in pure water is broader than (by ∼1.1 cm−1) and
red-shifted from (by ∼0.8 cm−1) that of GWCNG. This suggests that
incomparison with GWCNG, the longer 2WCNP peptide can sample
alarger conformational space, as expected. In fact, the 2D
IRspectra of 2WCNP in pure water (SI Appendix, Fig. S13) show
thatthere are two resolvable spectral features within the profile
of thelinear C≡N stretching band, at ∼2,225 (strong) and ∼2,217
cm−1(weak), indicative of two distinct population ensembles. As
shown(SI Appendix, Fig. S14), the CD spectrum of 2WCNP in pure
waterconsists of not only a broad, negative-going band at 198
nmexpected for a disordered peptide but also a weak CD band at∼245
nm. Previously, we have shown that in a well-folded
peptidestructure, a pair of nearby TrpCN residues can produce a
strong CDcouplet at this wavelength due to excitonic coupling (43).
Thus, theCD spectrum of 2WCNP is not only consistent with the 2D
IRresults but also suggests that the minor population ensemble
cor-responds to a more compact conformation wherein the two
aro-matic side chains are in close proximity. In 6.0 M GdmCl
solutionthis excitonic CD band only becomes weaker, which supports
thenotion that Gdm+ ions do not specifically and strongly interact
withthe side chain of TrpCN. One the other hand, this excitonic
CDband is not detectable in 2.0 M TPA solution, suggesting,
onceagain, preferential binding of TPA+ to TrpCN side chains.
Whatis more, the CD spectrum of 2WCNP in 2.0 M TPACl solution
iscomposed of two negative-going peaks, centered at ∼205 and∼225
nm, suggesting that TPA+ ions promote formation of(partial)
α-helical structures. This finding is consistent with thestudy of
Dempsey et al. (24), which showed that 2 M TPAClpromotes α-helix
formation in an alanine-based peptide.As shown (Fig. 5), the
spectral diffusion dynamics of the C≡N
stretching vibration of 2WCNP in pure water exhibits a similar
hy-drogen-bonding kinetic phase comparing to that of GWCNG;however,
the corresponding static component is much larger. Thisis well
expected for a longer peptide consisting of different sidechains
because a greater portion of the conformational
dynamicscontributing to the linear IR bandwidth will occur on a
timescalethat is too slow to be explicitly time resolved. As
similarly observedfor GWCNG, addition of 2.0 M TPACl to 2WCNP
solution essen-tially abolishes any observable hydrogen-bonding
dynamics andalso substantially increases the static component in
the spectraldiffusion dynamics (Fig. 5). Thus, these results
provide confirma-tion that TPA+ ions have a strong affinity toward
Trp and theunderlying Trp–TPA+ interactions lead to dehydration of
the in-dole ring. This notion is further substantiated by the fact
that theTrpCN fluorescence intensity of 2WCNP in 2.0 M TPACl
solution is∼10 times larger than that in pure water (SI Appendix,
Fig. S15).Addition of 6.0 M GdmCl results in a decrease in the
static com-ponent of the spectral diffusion dynamics of the C≡N
stretchingvibration of 2WCNP (Fig. 5). This result has two
implications: (i) itsupports the notion that Gdm+ ions do not
strongly interact withprotein aromatic side chains and (ii) it
shows that as observed in aprevious study (31), Gdm+ reduces the
distribution of peptideconformational states that interconvert on a
timescale too slow tobe captured by 2D IR measurements.
Implication on the Protein Denaturizing Mechanisms of TPA+
andGdm+. Because of its enormous importance in chemistry and
bi-ology, the effect of ions on the activity and structural
integrity ofproteins has been the subject of extensive studies (5).
For a specificion, its mechanism of action is often assessed by
examining itsability to alter the hydrogen-bonding structure and
dynamics ofwater, its affinity toward specific amino acid side
chains, and itsinteraction with protein backbone units. For Gdm+
and TPA+, ithas been suggested that they denature proteins by
directly inter-acting with various protein components (7, 11,
18–24). By takingadvantage of two amino acid-based spectroscopic
probes andmultiple spectroscopic techniques, herein we assess
whether theseions can alter the hydration and conformational
dynamics of
0.0
0.4
0.8
1.2
0.0 0.7 1.4 2.1
0.0
0.4
0.8
1.2
325 425 525
[TPACl] (M)
Nor
mal
ized
Inte
nsity
Wavelength (nm)
Nor
mal
ized
Inte
nsity H2O GdmCl TPAClH2
Fig. 4. Normalized TrpCN fluorescence intensity (integrated
area) of GWCNGas a function of TPACl concentration. The lines
represent linear regressionsof the data obtained below [TPACl] =
1.2 M and above [TPACl] = 1.4 M.These two lines cross at a TPACl
concentration of ∼1.3 M. Shown in the insetare the TrpCN
fluorescence spectra of GWCNG obtained under different sol-vent
conditions, as indicated.
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aromatic side chains in a peptide environment. Taken together,
ourresults show that Gdm+ does not strongly bind to aromatic
sidechains and hence is more likely to denature proteins through
in-teractions with the backbone. Of course, as a salt, GdmCl can
alsoscreen electrostatic interactions that stabilize a protein’s
nativestate. On the other hand, TPA+ does interact favorably with
aro-matic side chains, especially the indole ring of Trp.
Furthermore,TPA+ exhibits a behavior common to detergents or
surfactants (44)as it undergoes a distinct transition when the
concentration reachesa critical value (∼1.3 M) to form aggregates
or clusters. Althoughthe current study cannot reveal the structural
characteristics ofsuch TPA+ clusters, we believe that their mode of
interaction withproteins is, at least to some extent, similar to
that of ionic surfac-tants. This is because similar to the
surfactant SDS, which denaturesglobular proteins by forming
micelles and produces a denatured-state rich in α-helices (44),
TPA+ clusters are able to disrupt thenative hydrophobic
interactions among aromatic side chains in aβ-hairpin, causing it
to unfold (24), and at the same time, areable to promote α-helical
structure formation in an alanine-based peptide (24). Despite these
similarities, clusters formedby TPA+ ions are expected to be
different from SDS micelles inmany aspects. This is because the
polar group of TPA+, unlikethat of SDS or other surfactant
molecules, is situated at thecenter of the molecular ion and hence
renders it impossible toyield a membrane-like environment as SDS
does. Indeed, ourCD results (SI Appendix, Figs. S16 and S17) show
that althoughTPA+ clusters can induce α-helix formation in an
alanine-basedpeptide, as observed by Dempsey et al. (24), they do
not increasethe α-helicity of a Trp3/TrpCN mutant of the
antimicrobial peptidemastoparan-X that is known to form an
amphipathic α-helix uponbinding to the interfacial region of lipid
micelles or membranes (30).These results suggest that unlike
membrane-mimic micelles formedby surfactants, a single TPA+ cluster
is unable to provide a con-tinuous water-hydrophobic interface
large enough to host an entireα-helix. Instead, the helix-promoting
ability of TPA+ clusters islikely achieved by interacting with
aromatic and hydrophobic sidechains in the peptide, which brings
the corresponding backboneunit(s) to a relatively low dielectric
environment and hence increasesthe possibility of local, backbone
hydrogen bond formation (45). Thispicture is consistent with the
fact that the α-helix–promoting abilityof TPA+ clusters depends on
the peptide sequence. For an alanine-rich α-helix, most side chains
are not only identical but also quiteuniformly and symmetrically
distributed along the helical axis. This
uniform and symmetrical distribution of TPA+-interacting
hydro-phobic side chains allows multiple TPA+ clusters to be
accumulatedaround the peptide, which, collectively, yields a
channel-like envi-ronment favorable for α-helix folding. However,
for a peptide withmany different side chains, although simultaneous
interactions withmultiple TPA+ clusters are still feasible, there
is no guarantee thatthese clusters are adjacent to each other, thus
reducing the possibilityof forming a continuous membrane-like
environment across theentire peptide and hence α-helix formation.
Finally, it is worth notingthat unlike clusters formed by
protein-protecting cosolvents (46) thatare excluded from the
protein surface, the excluded-volume orcrowding effect of TPA+
clusters is more localized and, as a result,will not increase the
protein stability through this entropic cause.
ConclusionsThere are numerous examples in the literature
documenting andinvestigating the effect of ions on the physical
and/or chemicalproperties of proteins and, among which, studies on
protein-denaturing ions, such as Gdm+, are especially prevalent due
to theirimportant utility in protein science. Despite previous
efforts, how-ever, we still lack a molecular-level understanding of
the protein-denaturing mechanism of many ions, especially organic
ions. This isbecause such ions can, in principle, engage in
specific interactionswith protein side chains, an aspect that is
difficult to assess experi-mentally. Herein, we use two unnatural
amino acids, PheCN andTrpCN, both of which have been shown to be
useful as site-specificIR and fluorescence probes of protein local
environment, andmultiple spectroscopic techniques to examine
whether Gdm+ andTPA+ specifically interact with aromatic side
chains in a peptideenvironment. Our 2D IR results show no evidence
of this interactionfor Gdm+ because it does not change the spectral
diffusion dynamicsof the site-specific IR probes in any significant
manner at the con-centration of 6.0 M. Thus, as suggested by other
studies (7, 9, 11,47), Gdm+ most likely denatures proteins by
binding to theirbackbone units or charged side chains. On the other
hand, TPA+
does exert a strong effect on the spectral and dynamic
properties ofthose probes and, at 2.0 M, causes TrpCN to be
dehydrated.Moreover, our fluorescence data show that TPA+ undergoes
atransition in ionic aggregation state at ∼1.3 M, akin to the
micelli-zation behavior of surfactants. Taken together, these
findings sug-gest a protein-denaturing action, similar to that
found for ionicmicelles, wherein TPA+ clusters disrupt native
hydrophobic inter-actions by accumulating around hydrophobic side
chains. In addi-tion, as observed for micelles and membranes, the
TPA+ clustersappear to have an interfacial region that promotes
α-helix formationin certain peptides and also provides an anchoring
position for theside chain of TrpCN (and hence Trp). Furthermore,
considering theimportance and complexity of the ion–protein
interaction problem,we believe that the current study demonstrates
an approach of usingsite-specific vibrational and fluorescent
probes in different proteinmoieties to help tease out relevant
molecular information aboutspecific interactions between the ion
and side chain of interest.Currently, we are using different
unnatural amino acid-based IRprobes and 2D IR spectroscopy to
investigate whether Gdm+ ionsspecifically interact with other types
of amino acid side chain.
Materials and MethodsAll peptides used in the current study were
synthesized using standard9-fluorenylmethoxy-carbonyl (Fmoc)
methods. FTIR spectra were collected ona Thermo Nicolet 6700 FTIR
spectrometer. Fluorescence spectra were col-lected on a Jobin Yvon
Horiba Fluorolog 3.10 spectrofluorometer. CD spectrawere measured
on an Aviv 62A DS spectropolarimeter (Aviv Associates). The2D IR
spectra were collected on a home-built 2D IR apparatus in a
boxcargeometry with heterodyne detection. The details of the sample
preparationand all spectroscopic measurements are given in SI
Appendix.
ACKNOWLEDGMENTS. We gratefully acknowledge financial support
fromthe National Institutes of Health (Grant P41-GM104605).
0.0
0.3
0.6
0.9
0.0 1.0 2.0 3.0 4.0 5.0
Waiting Time, T (ps)
CLS
-1
H2O 6.0 M GdmCl 2.0 M TPACl
T (ps)
Fig. 5. CLS−1 versus T plots of the C≡N stretching vibration of
2WCNPobtained under different solvent conditions, as indicated.
Ding et al. PNAS | January 31, 2017 | vol. 114 | no. 5 |
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