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Divalent Metal-Ion Complexes with Dipeptide Ligands Having Phe
and His Side-Chain Anchors: Effects of Sequence, Metal Ion, and
Anchor
Robert C. Dunbar,*,† Giel Berden,‡ Jonathan K. Martens,‡ and Jos Oomens*,‡,§
†
Chemistry Department, Case Western Reserve University, Cleveland, Ohio 44106, United States
Radboud University, Institute for Molecules and Materials, FELIX Laboratory, Toernooiveld 7c, 6525ED Nijmegen, The
Netherlands
§
University of Amsterdam, Science Park 904, 1098XH Amsterdam, The Netherlands
‡
S Supporting Information
*
ABSTRACT: Conformational preferences have been surveyed
for divalent metal cation complexes with the dipeptide ligands
AlaPhe, PheAla, GlyHis, and HisGly. Density functional theory
results for a full set of complexes are presented, and previous
experimental infrared spectra, supplemented by a number of
newly recorded spectra obtained with infrared multiple photon
dissociation spectroscopy, provide experimental verification of
the preferred conformations in most cases. The overall
structural features of these complexes are shown, and attention
is given to comparisons involving peptide sequence, nature of the metal ion, and nature of the side-chain anchor. A regular
progression is observed as a function of binding strength, whereby the weakly binding metal ions (Ba2+ to Ca2+) transition from
carboxylate zwitterion (ZW) binding to charge-solvated (CS) binding, while the stronger binding metal ions (Ca2+ to Mg2+ to
Ni2+) transition from CS binding to metal-ion-backbone binding (Iminol) by direct metal−nitrogen bonds to the deprotonated
amide nitrogens. Two new sequence-dependent reversals are found between ZW and CS binding modes, such that Ba2+ and Ca2+
prefer ZW binding in the GlyHis case but prefer CS binding in the HisGly case. The overall binding strength for a given metal
ion is not strongly dependent on the sequence, but the histidine peptides are significantly more strongly bound (by 50−100
kJ mol−1) than the phenylalanine peptides.
■
Chart 1. Three Binding Motifs for HisGly with Metal Ionsa
INTRODUCTION
The side chains of histidine and phenylalanine (or, similarly,
tyrosine) often play an important role in the binding of metal
ions by peptides and proteins. The characterization of metal-ion
complexes in the gas phase can contribute useful perspectives
on such interactions, and the recent emergence of capabilities
for the spectroscopic characterization of small peptide
complexes in the gas phase has opened new possibilities
along these lines.1 Here we have set out to study and compare
the interactions across a representative series of metal ions with
simple peptide pairs, HisGly versus GlyHis and AlaPhe versus
PheAla. (The Gly and Ala residues are so similar in their
contributions to metal-ion binding by these peptides that our
choice of which one to use in each case has been governed by
questions of experimental expediency.)
Three alternative binding motifs have been found to describe
the ground-state conformations of the metal ion−peptide
complexes that have been observed or predicted in the gas
phase for peptides not having complications due to active side
chains (Chart 1). For the weakly binding metal ions, the
normal choice is chelation by the amide carbonyl oxygens,
known as charge-solvation (CS) binding.2−5 Also, by analogy
with the complexes of simple amino acids, there is the possible
© 2015 American Chemical Society
a
Green lines are metal-ion interactions with Lewis-basic chelation
sites; red dashed lines are probable hydrogen-bonding interactions.
formation and binding to the carboxylate zwitterion (ZW),
although this seems to be less favorable in general for peptides
than for simple amino acid complexes. (See refs 6−13 for a few
examples of the extensive study of this competition in the gas
Received: July 1, 2015
Revised: August 31, 2015
Published: September 1, 2015
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condensed phase17,19 is reflected when comparing these two
different side chains in the corresponding gas-phase complexation.
Characteristics of dipeptides having both Phe3,31−33 and
His30 residues have been reported in previous infraredspectroscopic studies using the free electron laser for infrared
experiments (FELIX) laser and infrared multiple photon
dissociation (IRMPD) spectroscopy. In addition to quantum
chemical calculations, we have recorded a number of IRMPD
spectra which will be presented here to provide new insights on
the questions outlined above. New spectra of metal-ion
complexes of AlaPhe and PheAla in the hydrogen-stretching
region in the vicinity of 3500 cm−1 resolve some important
ambiguities left open by the previous mid-IR spectra (1000−
1800 cm−1). New spectra of complexes of GlyHis in the mid-IR
region provide comparisons with the previous series of spectra
of the HisGly complexes.30
phase by various approaches.) Finally, for stronger-binding
metal ions, deprotonation of amide nitrogens (by tautomerization) becomes favorable, leading to chelation by direct metal−
nitrogen bonds in the “Iminol” (Im) binding motif.14 The CS
motif is characteristic of alkali metal ions and Ca2+ ions,
whereas the Im motif is characteristic of transition metal ions
like Co2+, Ni2+, Cu2+, Zn2+, Pd2+, and Cd2+. Consistently, Mg2+
and Mn2+ have been found to lie near the transition from CS to
Im. References 3, 14, and 15 show some examples of our
group’s study of the competition between CS and Im. Peptideand protein-related condensed-phase analogues of the CS motif
are common, for example, in calcium transporter proteins like
calmodulin.16 Solution-phase examples of the binding to
deprotonated amide nitrogens (analogous to the gas-phase
Im binding pattern) have been known since the early days of
peptide organometallic chemistry.17−22
The imidazole side chain of histidine often provides an
anchor ligand site for this binding mode, which gives impetus to
the present study of histidine-containing dipeptide complexes.
Our future plans for studying larger ligands will include
comparisons with the frequently studied protein domain
sometimes referred to as the ATCUN (amino terminal Cu(II)
and Ni(II)) structural pattern,18,19,23,24 which is characteristically anchored by His in third position. A few other widely
noted examples of the nitrogen backbone binding motif include
the binding of metal ions in oxytocin24,25 and the binding of
Cu(II) in a similar pattern in the prion protein.26,27 Finally, in
analogy to the gas-phase ZW pattern, it is not unusual to find
carboxylate chelation in the condensed phase, especially with
Asp and Glu side chains involved in the binding of metal ions in
proteins.28
In condensed-phase peptides, deprotonation of the backbone
amide nitrogen can be accompanied by removal of the proton
into the solvent environment (forming net anionic species, as
can be modeled by proton-loss processes in gas-phase ion
formation29) or onto remote locations in a larger structure
(forming zwitterionic species). In small isolated systems like the
present examples, however, attachment of the metal cation to
an amide nitrogen (without changing the net charge) must
accommodate the proton locally, and our work has focused on
the iminol tautomerization as the mechanism to achieve this
result.3,14,15,30
Several themes of interest are taken up in the work described
here. The overall theme is elucidating the factors that
determine the preferred binding mode for a given complex.
Within this framework we will address four specific questions:
(1) Considering the choice between ZW and CS, these
modes are competitive for the weak-binding metals, and we will
be looking at the transition between these two binding modes
as we go from Ba2+ to Ca2+ complexes.
(2) Considering the choice between CS and Im, it is found
that the weakest-binding metal Ba2+ never prefers Im, while
strongest-binding metals like Ni2+ and Cu2+ often prefer Im.
Here, our attention will be on the transitional region covering
the intermediate metals Ca2+ and Mg2+.
(3) As we refine the characterization of the foregoing two
transitional regions, we will address the effect of reversal of the
position of the side chain (C-terminal versus N-terminal).
(4) Finally, we will be concerned with how the observations
regarding these questions are similar or different for the
phenylalanine dipeptides versus the histidine peptides. It is of
interest to see whether the exceptional status of histidine as an
anchor for deprotonated nitrogen binding of peptides in the
■
METHODS
Structure Notation. A descriptive structure notation is
used (for example, Im [NONR]). The binding type is indicated
(charge solvated (CS), iminol (Im), or carboxylate zwitterion
(ZW)). Following in brackets is the set of metal-bound
chelation points. Deprotonated amide Nitrogens are given first
(N), then carbonyl oxygens (O), then N-terminal amino
nitrogen (N), and finally ring (R) (either imidazole nitrogen or
π-complexed phenyl). At the levels of computation used, a
proton which participates in a hydrogen bond between O and
N frequently yields two distinct potential energy minima, which
will be distinguished when necessary by an appended _OH or
_NH symbol indicating the shorter bond. Sometimes a prime
will be used, as in [OO′], to indicate monodentate
coordination by one carboxyl or carboxylate oxygen along
with complexation by the amide oxygen, distinguished from
[OO] which will indicate bidentate complexation by both
oxygens of the carboxylate.
Computational Methods. All calculations were carried out
using the Gaussian09 quantum chemical package.34 The default
computational level was B3LYP/6-31+G(d,p). For Ba2+
complexes, the SDD basis set with a relativistic effective core
potential was used on the metal ion, with the normal 631+G(d,p) (or 6-311++G(d,p)) basis functions on the H, C, O,
and N atoms. Previous density functional theory (DFT) studies
cited from our group and others have commonly used this or
similar relativistic effective core potentials on large atoms like
Ba. The principal reason for this is the known importance of
such effects for such large atoms, and the SDD pseudopotential
is a straightforward and proven way to take them into account.
A secondary advantage is to reduce the size of the calculation
without affecting accuracy compared with a simple all-electron
approach. For complexes of all other metals, all-electron
calculations were done using the chosen 6-31+G(d,p) or 6311++G(d,p) basis. The two different basis sets usually agreed
within a few kJ mol−1 and seldom disagreed by as much as 5
kJ mol−1, allowing us to consider the smaller basis set to be
large enough for reliable energy comparisons (to the extent of
the validity of the B3LYP functional) within the present set of
complexes. In citing relative energy values, the larger-basis-set
values have been reported, when available and without
comment. Some small effects are well-known to give minor
adjustments to binding energies, including basis set superposition error, thermal corrections from 0 K, and vibrational
zero-point energies. It was assumed that these effects would be
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Binding energies were calculated by a straightforward
subtraction of the energy of the separate metal ion plus neutral
ligand from the energy of the most stable conformation found
for the complex. The conformation of the neutral ligand was
the one with lowest energy coming from modest Amber
structure searches combined with DFT refinement. We would
not guarantee to have located the true most stable
conformations of the neutral ligands, and given the modest
computational level and the noninclusion of vibrational and
basis set superposition corrections, these binding energy values
should be taken as useful numbers for comparison, but not as
the most precise values that can be computed by current
methods.
IRMPD Experiments. IR spectra of the gaseous metal-ion
complexes were recorded employing a Fourier transform ion
cyclotron resonance mass spectrometer (FT-ICR MS) coupled
to the FELIX laser and to a benchtop optical parametric
oscillator/amplifier (OPO/OPA) system, as has been detailed
elsewhere.32,40−42 Metal-ion peptide complexes were generated
by electrospray ionization (ESI, Waters Z-Spray) from a
solution containing the peptide and metal salt in acetonitrile/
H2O (∼4:1). Target ions were trapped and mass-selected in the
FT-ICR cell and were irradiated with the wavelength-tunable
infrared light from FELIX (in the range of 1000−1850 cm−1)
or the OPO/OPA system (3000−3800 cm−1). When the sum
of all dissociation channels ratioed to the total ion count as a
function of laser frequency was plotted, an infrared action
spectrum was generated and interpreted as a surrogate IR
spectrum of the complex. DFT-computed linear IR spectra of
candidate ion structures were compared with the observed
spectra, with the calculated relative energetics providing
additional guidance, to assign conformational and tautomeric
isomers.
sufficiently small and similar for all systems so that they can be
ignored. In other reported studies of weakly bound neutral−
neutral complexes there has been concern about the adequacy
of the B3LYP functional to calculate the contribution of
dispersion to binding,35 but for ion−neutral complexes, like the
present systems, the binding is so strong compared with
dispersion forces that such concerns were assumed to be
insignificant.
For comparison of computed DFT spectra with observed
IRMPD spectra, the computed frequencies in the 1000−1900
cm−1 range were scaled by a factor of 0.975 (or 0.98 for the
larger basis set), which our experience suggests to be
appropriate at these levels of theory. In the 3000−3800 cm−1
range, vibrations have been scaled by 0.955, consistent with
previously reported scaling factors for this spectral range using
similar levels of theory.36−40 Computed spectra were
convoluted with a 20 cm−1 fwhm Gaussian line shape function
for comparison to experimental IR spectra.
A comprehensive search of configuration space was not
practicable, particularly for the transition metals, at a level of
computation sufficiently high to give reliable energies. The
conformations to be studied were developed by a combination
of molecular mechanics searching of Mg2+ or Ca2+ structures
using HyperChem and the Amber force field, along with
manual inclusion of structures known from prior studies to be
chemically reasonable. Searching was done for CS structures
and carboxylate zwitterions. Iminol searching by molecular
mechanics was not attempted because the metal−nitrogen
bonds were not well-parametrized in the force fields used.
Iminol structures are generally highly constrained in such small
systems, so manual assignment of likely structures was
considered quite satisfactory. Promising conformations were
further refined at the B3LYP/3-21G level prior to final selection
and calculation with a more adequate basis set.
For most complexes, the calculated vibrational spectra were
insensitive to the basis set choice. More than 20 comparisons
were made between spectra calculated with both the 631+g(d,p) and 6-311++g(d,p) basis sets, and the agreement
was almost always very good, with peak-position deviations of
as much as 10 cm−1 being rare. Relative peak heights were not
always as reproducible between basis sets, with occasional
deviations of the order of a factor of 2 or more. This lack of
consistency of peak heights was not considered to be a problem
because IRMPD relative peak heights are commonly accepted
as being somewhat uncertain, with little emphasis being placed
on interpretations based on precise peak heights. Consistent
with previous work,30 most AlaPhe and PheAla spectrum plots
are presented at the small-basis level below, and the GlyHis
calculations at the large-basis level. However, the small basis
calculation of Ni2+FA [NONR] gave energy and spectrum
results that were clearly unreasonable and inconsistent with
other systems. Taking this as a possible indication that this basis
set was too small for reliable all-electron calculations for Ni2+
complexes, it was considered more reliable for this metal ion to
present both energies and spectra using the large-basis
calculations, as will be seen in Figure 8 below.
For the nickel complexes, the triplet spin state was found to
be substantially more stable than the corresponding singlet in
most cases where singlet state trials were made. In our ongoing
studies of Ni(II) iminol complexes of peptides larger than the
present subjects, some singlet ground states have been
identified, but singlet spin is apparently not preferable for the
dipeptides studied here.
■
RESULTS AND DISCUSSION
Comprehensive computational results will be surveyed in order
to achieve a full predicted description of the conformational
trends for each of these ligands. The new spectroscopic results
will be described for both sets of ligands. Following that, in a
final section, conclusions will be drawn about the questions
posed above.
Thermochemistry. Histidine Dipeptides. Figure 1 displays
the metal-ion dependence of the calculated relative energies of
the principal structures for each of the two peptide isomers.
The metals are ordered along the horizontal axis in order of
increasing binding energy (see the discussion of binding
energies below and, for example, ref 15). The energies of the
various conformations are also noted along with structure
diagrams in the Supporting Information (Table S1). In large
measure, the plots for the two isomers are similar. As expected,
ZW and CS conformations dominate for weak-binding metals
and CS for intermediate binding; Im becomes dominant for
strong-binders. One observable CS-binding effect of the sidechain position is that in the HG case, folding to give fourcoordinate CS [OONR] is relatively favorable, while for the
GH case it is more favorable to open up to give threecoordinate CS [OOR].
Carboxylate Zwitterions. The lowest-energy zwitterion-type
complexes were found to relocate the carboxyl proton to the
imidazole nitrogen in preference to the amino nitrogen. This is
as expected because primary amines are less basic than
imidazoles,43 and in addition histidine itself is preferentially
protonated on the imidazole44 (although the energy difference
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possible for Ba2+, but is closer to being competitive for Ca2+.
For both of the present ligand isomers, iminol binding appears
to be somewhat unfavorable at Ca2+, but not by amounts that
are decisively beyond computational uncertainty or slightly
disequilibrated ion populations. For Mg2+ and stronger metal
ions, iminol binding is favored over CS. At this level of theory
Ca2+ is predicted to favor the zwitterion complex for GH, and
the CS complex for HG.
In almost all cases where iminol binding is favored, it is the
iminol conformation Im [NONR] with the imidazole bound to
the metal that is favored. The Im [NON] conformation, with
the imidazole remote from the metal and strongly hydrogenbonded to the iminol OH, becomes gradually more favorable as
the metal ion binding becomes stronger (compare Im [NONR]
and Im [NON]) but is indicated as the ground state only for
Cu2+ with GH. In the Im [NON] conformation, the hydrogenbonding proton lies quite strongly on the side of the imidazole
nitrogen, effectively creating a zwitterion form of the ligand.
Moving the proton back to the oxygen side of this
unsymmetrical hydrogen bond to form the iminol conformation is calculated to cost about 50 kJ mol−1.
Phenylalanine Dipeptides. Figure 2 shows the computed
relative energies of the low-lying conformations of the PheAla
Figure 1. Calculated energies of HisGly and GlyHis complexes. (See
Table S1 for structure drawings.) For each metal−ligand combination,
the energies are given relative to the most stable conformation for the
given peptide−metal combination.
is not large). There is a marked difference in zwitterion
favorability depending on the side-chain position. For HG
binding to the weak Ba2+ ion, the two-coordinate zwitterion
ZW [OO] is preferred, with the imidazole ring remote from the
metal ion and hydrogen-bonded to the amide oxygen. For
calcium, this binding mode is sharply higher in energy, making
the HG zwitterion highly unfavorable for calcium and more
strongly binding metals. For GH, a three-coordinate zwitterion,
ZW [OOO], becomes possible. This zwitterion is much more
favorable (by 50 kJ mol−1) than the corresponding HG
zwitterion, and is predicted to be the ground state for both
Ba2+ and Ca2+ complexes of GH. It is apparently highly
stabilized by a favorable salt-bridge Coulomb interaction,
analogous to the salt-bridge structures of simple amino acid
complexes (as for Ba2+His (ref 13)). However, the energy of
GH ZW [OOO] rises sharply for Mg and stronger binding
metals, and zwitterion conformations are not competitive with
other binding modes for these metal ions.
CS Binding. Looking at CS [OOR] and CS [OONR], we see
that the plots for these two structures are reasonably parallel
over the span of metal ions. This suggests that even for the
smallest metal ions, there is no added steric crowding resisting
bringing the additional amino nitrogen anchor into coordination with the metal. For HG compared with GH, the play of
steric factors makes it consistently more favorable (by about 20
or 30 kJ mol−1) to fold the amino nitrogen into coordination
with the metal (CS [OONR] rather than CS [OOR]).
Iminol Binding. The competition between CS and Iminol
binding reverses at Ca2+. Iminol binding is nowhere near
Figure 2. Calculated energies of PheAla and AlaPhe complexes with
M2+. (See Table S2 for structure drawings.) For each metal−ligand
combination, the energies are given relative to the most stable
conformation for the given metal−ligand combination.
and AlaPhe complexes. Structures and complete energy results
are displayed in the Table S2. Features similar to the histidine
dipeptides include the following: (i) ZW is competitive only for
the weakest-binding metal ions, and is much more stable
(relatively) for C−terminal AlaPhe than for N-terminal PheAla.
(ii) The transition from CS to Im preference occurs between
Ca and Mg. (iii) Four-coordinate Im [NONR] is more
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favorable than the three-coordinate conformations Im [NOR]
or Im [NON]. In fact, Im [NON] is very unfavorable in these
cases because no strong hydrogen bond is available to the
iminol OH to stabilize the free aromatic ring. However, at least
for the PheAla case, Im [NOR] is not unreasonably high in
energy and cannot be discounted as a possible ground state for
the strong-binding metals.
Spectroscopy. Spectra and Structures of Histidine
Dipeptides. Mid-IR spectra have been obtained for the Ba2+
and Ca2+ complexes with GlyHis and are shown in Figures 3
Figure 4. IRMPD spectrum of Ca2+GlyHis, along with computed
spectra (large basis) of possible structures of low relative energy. The
most likely interpretation is a mixture of comparable amounts of CS
[OOR] and ZW [OOO]. The spectrum of Ca2+HisGly (ref 30) is also
shown for comparison (light gray spectrum), showing a prominent
peak just below 1700 cm−1 that was interpreted as indicating a
substantial fraction of CS structures in that population.
Spectra and Structures of Phenylalanine Dipeptides. A fair
amount of progress has already been described in previous
work on the spectroscopic analysis of complexes of phenylalanine-containing small peptides, including the metal(II)
complexes of interest here. However, some questions have
remained open where convincing agreement was not achieved
between the observed results and computational predictions, or
because the spectra, restricted for the most part to the mid-IR,
were not sufficiently informative.
The previously discussed structure preferences correlating
the dipeptide ligand series FA, AF, and FF as a function of sidechain placement3,14,15,32,33 are revisited here with the help of
the new spectroscopic results. The recent OPO laser
capabilities at the FELIX laboratory have opened new
possibilities in the 3000−4000 cm−1 wavelength range, and
useful new insights have emerged. OPO spectra of the PheAla
complexes are displayed below, or in the Ba2+ case, in the
Supporting Information.
Spectroscopic evidence is particularly interesting for the Ca2+
and Mg2+ complexes because this is the region of ligand binding
strength which encompasses the crossover from CS to Iminol
structures for the PheAla and AlaPhe ligands. The Ca2+AlaPhe
spectrum in the OPO region (Figure 5) very clearly rules out
the Im [NONR] possibility, which would show its presence by
a pattern of two similar-sized OH stretching bands in this
wavelength region, due to the COOH and iminol OH stretch
modes. This complex is hence confidently assigned as CS.
Looking at the reverse residue sequence (Ca2+PheAla), it was
previously noted3 that the mid-IR spectrum did not give an
unambiguous assignment for that complex. However, the new
OPO spectrum (Figure 6) definitively rules out a predominant
contribution from Im [NONR] by the same argument as for
Ca2+AlaPhe, although the smaller features in the spectrum
suggest a possible admixture of a small fraction of Iminol in a
predominantly CS population. This spectroscopic evidence
firmly confirms Ca2+ as preferring CS binding for both PheAla
and AlaPhe.
Figure 3. IRMPD spectrum of Ba2+GlyHis (red spectrum), along with
computed spectra (large basis) of the possible structures with low
relative energies. The spectrum is a good match to a nearly pure
population of ZW [OOO]. The spectrum of Ba2+HisGly (ref 30) is
also shown for comparison (light gray spectrum), featuring a
prominent peak near 1700 cm−1 that is interpreted as a C-terminal
CO stretch vibration, suggesting a substantial fraction of CS
structures in that population.
and 4, respectively, along with the calculated spectra of the
energetically most likely structures composing the populations.
The question to address is the competition between ZW and
CS for these two weak-binding metal ions. The Ba2+GH
spectrum fits well to the prediction for pure ZW, in agreement
with the calculation showing that other structure types are
considerably higher in energy. This is a reversal from the
previously reported Ba2+HG complex, which was assigned as
pure CS.30 The spectrum of this latter Ba2+HG complex is also
shown in the figure, and it can be seen that the presence or
absence of the very strong peak near 1700 cm−1 (C-terminal
CO stretch, diagnostic for CS structures) highlights the
contrast between these two isomers.
In contrast, the Ca2+GH spectrum (Figure 4) shows a strong
peak near 1700 cm−1 which must correspond to CS. The
prominent feature at 1620 cm−1 also strongly suggests the
presence of ZW, and we assign this population as consisting of
comparable abundances of CS and ZW, which is not
unreasonable in view of the fairly small difference in the
calculated energies of these two conformers: 0 kJ mol−1 for
ZW[OOO] and 8 kJ mol−1 for CS[OOR]. This assignment also
offers a contrast, although less extreme than in the Ba2+ case,
between the GH and HG isomers: In the Ca2+HG case, whose
spectrum is also displayed in Figure 4, there is much less
indication of a ZW component in the population than in the
GH case.
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Figure 5. IRMPD spectrum of Ca2+AlaPhe. The population is
attributed to CS, with no significant contribution of either Im
[NONR] or ZW [OOR].
Figure 7. IRMPD spectrum of Mg2+PheAla. The double feature at
3500−3600 cm−1 strongly confirms a population consisting of Im
[NONR].
Progressing from Mg2+ to the stronger metal Ni2+ with
PheAla, we would confidently expect, like the Mg2+ case, to see
the same characteristic double-peak structure between 3500
and 3600 cm−1 corresponding to an Im [NONR] population,
so it was a surprise to observe only a single sharp peak in the
Ni2+PheAla OPO spectrum (Figure 8). The calculations suggest
an explanation for this anomaly, showing that the Im [NONR]
conformation may not be best for Ni2+, and that the amino
nitrogen can decouple from the metal with little or no energetic
cost, forming a hydrogen bond to the iminol OH to give the Im
[NOR_OH] conformation. The predicted Im [NOR_OH]
spectrum of the Ni2+PheAla complex matches well to the
Figure 6. IRMPD spectrum of Ca2+PheAla. A predominant CS
population is indicated.
Mg2+ provides a striking contrast. For the Mg2+PheAla
complex, the CS and Iminol conformations are virtually the
same in calculated energy. In Figure 7, the OPO region shows
the characteristic pattern of two peaks, with the Iminol OH
stretch peak at 3570 cm−1 clearly indicating a major
contribution of Im [NONR]. A simultaneous contribution
from the CS structure is not ruled out by the spectrum in this
OPO region, but in the mid-IR region the apparent absence in
Figure 7 of the characteristic Amide II peak expected near
1500−1550 cm−1 for the CS structure (as is clearly evident in
Figure 6) provides at least some evidence against a major
contribution from CS [OOR]. In the Mg2+AlaPhe case, the CS
[OOR] and Im [NONR] conformations are calculated as
nearly equal in energy (with a small advantage for the Iminol).
No spectrum is yet available, so an experimental confirmation is
not yet possible for the preference in this case. Taking the
computed result for Mg2+AlaPhe as an indication of a mixed
CS/Im population, we can tentatively conclude that going from
Ca2+ to Mg2+ for this ligand pair gives at least a partial switch
from CS to Iminol.
Figure 8. IRMPD spectrum of Ni2+PheAla. The single peak in the
3500 cm−1 region rules out the (lowest-energy) Im [NONR] structure
and strongly indicates a preference for the tridentate Im [NOR_OH]
coordination, with simultaneous H2N...HO hydrogen bonding between
the iminol OH and the N-terminal NH2.
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reversals between ZW and CS have previously been reported
for Ba2+ with Phe-containing dipeptides33 (as is noted in Figure
9), and Li+and Na+ with Arg-containing dipeptides.45 The side
chain in the N-terminal position is evidently better able to wrap
around and solvate the metal ion because it is always the Nterminal isomer that favors CS.
Given the computational predictions of a switch from CS to
Im binding between Ca2+ and Mg2+ for both AlaPhe and
GlyHis complexes, it would be very desirable to have
spectroscopic proof that these Mg2+ complexes do indeed
favor Im complexation. Unfortunately, it has not been possible
to prepare either of these complexes in the instruments we have
used in this study.
A mild contrast is observed for Ba2+ and Ca2+ between the
present dipeptides versus the simple amino acids that were
recently summarized in refs 41 and 1. For the simple amino
acids, these metal ions often favor zwitterionic binding, whereas
for the dipeptides, growing experience suggests that ZW
binding is the exception rather than the rule. Figure 9 illustrates
that the choice between zwitterion and charge-solvated is
closely dependent on the accidental details of steric constraints
and electrostatic stabilization for each individual ligand, but the
majority of systems prefer CS to ZW.
Table 1 shows computed electronic binding energies for the
set of complexes studied here. Of interest is the observation
observed spectrum in both the OPO and the mid-IR regions.
Such an opening up to go from a four-coordinate Mg2+
complex to a three-coordinate Ni2+ complex might be
rationalized by the slightly smaller size and greater steric
crowding of the Ni2+ ion. No experimental evidence is yet
available for the Ni2+AlaPhe complex, although the calculations
indicate a strong preference for an iminol conformation.
Dipeptide Trends Compared. Summarized in Figure 9 is
a comprehensive picture of dipeptide conformational prefer-
Table 1. Binding Energies of the Complexes in Their Most
Stable Conformations (kJ mol−1)
Figure 9. Predictions and observations of favored conformations.
Black bars are carboxylate zwitterions (ZW), red bars charge-solvated
complexes (CS), and blue bars iminol complexes (Im). The most
stable conformation (the one with lowest calculated energy) for each
metal−ligand pair is assigned a stability of zero and has the tallest bar,
and the less stable conformation types are displayed with negative
energies relative to this one. Conformations which are disfavored by
more than 20 kJ mol−1 are plotted at −20 kJ mol−1. Solid bars
designate complexes which have been observed and confirmed by the
IRMPD spectra. Hollow bars show relative energies for conformations
which have only been predicted from the calculations but not yet
directly observed. Shaded bars ([Ca FA Im] and [Mg FA CS]) are
less-stable conformations with significant calculated abundances whose
presence is consistent with, but not actually proven by, the
experimental spectra.
that the histidine dipeptides bind more strongly, by about 50−
100 kJ mol−1, than the corresponding phenylalanine complexes,
regardless of whether the binding mode is ZW, CS, or Im.
There is, however, no obviously strong difference in binding
energy between the N-terminal and C-terminal side-chain
isomers.
ences determined for the two side-chain-dominated ligand pairs
in the present study. The general behavior correlates with the
binding strength of the metal ion, in the sense that the assigned
preferences of binding motifs make the transitions from
zwitterion to charge-solvated to iminol as we go from
weakest-binding (Ba2+) to strongest (Ni2+). Ca2+ is the most
characteristically transitional metal ion in that it shows
meaningful competitions both for ZW versus CS, and also for
CS versus Iminol. Ba2+ shows transitional character between
ZW and CS, while Mg2+ shows transitional character between
CS and Im. Ni2+ always has a strong preference for Im.
Out of the present set of complexes, two new examples have
emerged where the amino acid sequence governs a direct
reversal, spectroscopically verified, of the character of complexation. These are the cases of Ba2+ and Ca2+ complexing to the
histidine-containing dipeptides. The complexes with the Nterminal histidine ligand (HisGly) are predominantly or fully
charge-solvated, while the complexes with the C-terminal
histidine ligand (GlyHis) are predominantly or fully zwitterionic. Such spectroscopically verified sequence-dependent
CONCLUSIONS
A full picture, with nearly comprehensive confirmation by
experiment, has been laid out for the conformational
preferences as summarized in Figure 9. Out of all the 12
metal−peptide pairs having spectroscopic data, only in the
Ba2+AlaPhe case does the observed structure (ZW) not match
the structure with lowest computed energy (CS) at the present
level of theory. The comprehensive agreement between the
experimental observations and the computational predictions of
the lowest-energy conformations can be considered as support
for the assumption that the process of electrospraying and
trapping the ions results in reasonable equilibration to the most
stable gas-phase distribution of conformations.
The new information from IRMPD spectra using the OPO
laser in the hydrogen stretching region was found to
complement the previous mid-IR spectra, making possible
quite detailed discrimination of different conformations. For
instance, the series of complexes of PheAla has been shown to
progress from pure CS [OOR] for Ba2+, to CS [OOR] with
some admixture of Im [NONR] for Ca2+, to pure Im [NONR]
for Mg2+, to the three-coordinate Im [NOR_OH] for Ni2+.
PheAla
AlaPhe
HisGly
GlyHis
Ba
Ca
Mg
Ni
537
538
601
628
676
673
756
745
1002
992
1122
1109
1403
1388
1530
1516
■
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DOI: 10.1021/acs.jpca.5b06315
J. Phys. Chem. A 2015, 119, 9901−9909
Article
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In most cases the preferred conformational type is the same
for the N-terminal and the C-terminal position of the side
chain. However, two CS/ZW reversals have been newly
characterized (as seen in Figure 9), for the Ba2+ and Ca2+
complexes of the histidine dipeptides, where the preferred
conformation switches between ZW and CS depending on
sequence. In CS/ZW reversals previously reported33,45as well as
the present examples, an N-terminal position of the side chain
is more favorable for CS binding than a C-terminal position of
the side chain. No obvious reversals were seen for the preferred
conformational type in CS versus Im cases as a function of
sequence. The phenylalanine peptides generally appeared to
favor CS somewhat more over Im as compared with the
histidine peptides.
Metal-ion binding is significantly stronger for the histidine
peptides than for the phenylalanine peptides, regardless of the
sequence and regardless of the nature of the preferred
conformation. This preference has to reflect a systematically
stronger interaction of the metal ions with the imidazole lonepair anchor point in histidine compared with the cation-π
anchor point of the aromatic phenyl side chain. Such a
preference goes along with the apparently privileged status of
histidine as an anchor for metal ions embedded in proteins.17,19
■
ASSOCIATED CONTENT
S Supporting Information
*
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jpca.5b06315.
Complete citation for ref 34; structures and energies of
metal-ion complexes of HisGly, GlyHis, PheAla, and
AlaPhe; and spectrum of Ba2+PheAla (PDF)
■
AUTHOR INFORMATION
Corresponding Authors
*E-mail: [email protected].
*E-mail: [email protected].
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
R.C.D. acknowledges support from the National Science
Foundation, Grant PIRE-0730072, and expresses gratitude to
the FELIX Laboratory for its continuing welcome. The FELIX
staff, and particularly Dr. Lex van der Meer and Dr. Britta
Redlich, are gratefully acknowledged for their assistance.
Financial support for this project was provided by NWO
Chemical Sciences under VICI Project 724.011.002. The
authors also thank NWO Physical Sciences (EW) and the
SARA Supercomputer Center for providing the computational
resources (Grants MP-264-14 and SH-260-14).
■
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