Bond-specific dissociation following excitation energy transfer for distance constraint determination in the gas phase

Article abstract of DOI:10.1021/ja507215q

Herein, we report chemistry that enables excitation energy transfer (EET) to be accurately measured via action spectroscopy on gaseous ions in an ion trap. It is demonstrated that EET between tryptophan or tyrosine and a disulfide bond leads to excited state, homolytic fragmentation of the disulfide bond. This phenomenon exhibits a tight distance dependence, which is consistent with Dexter exchange transfer. The extent of fragmentation of the disulfide bond can be used to determine the distance between the chromophore and disulfide bond. The chemistry is well suited for the examination of protein structure in the gas phase because native amino acids can serve as the donor/acceptor moieties. Furthermore, both tyrosine and tryptophan exhibit unique action spectra, meaning that the identity of the donating chromophore can be easily determined in addition to the distance between donor/acceptor. Application of the method to the Trpcage miniprotein reveals distance constraints that are consistent with a native-like fold for the +2 charge state in the gas phase. This structure is stabilized by several salt bridges, which have also been observed to be important previously in proteins that retain native-like structures in the gas phase. The ability of this method to measure specific distance constraints, potentially at numerous positions if combined with site-directed mutagenesis, significantly enhances our ability to examine protein structure in the gas phase.

Full text of DOI:10.1021/ja507215q

Article
pubs.acs.org/JACS
Open Access on 09/01/2015
Bond-Specific Dissociation Following Excitation Energy Transfer for
Distance Constraint Determination in the Gas Phase
Nathan G. Hendricks,† Nichole M. Lareau,‡ Sarah M. Stow,‡ John A. McLean,*,‡ and Ryan R. Julian*,†
†Department of Chemistry, University of California, Riverside, California 92521, United States
‡Department of Chemistry, Vanderbilt Institute of Chemical Biology (VICB), and Vanderbilt Institute for Integrative Biosystems
Research and Education (VIIBRE), Vanderbilt University, Nashville, Tennessee 37235, United States
S
* Supporting Information
ABSTRACT: Herein, we report chemistry that enables
excitation energy transfer (EET) to be accurately measured
via action spectroscopy on gaseous ions in an ion trap. It is
demonstrated that EET between tryptophan or tyrosine and a
disulfide bond leads to excited state, homolytic fragmentation
of the disulfide bond. This phenomenon exhibits a tight
distance dependence, which is consistent with Dexter exchange
transfer. The extent of fragmentation of the disulfide bond can
be used to determine the distance between the chromophore
and disulfide bond. The chemistry is well suited for the
examination of protein structure in the gas phase because
native amino acids can serve as the donor/acceptor moieties.
Furthermore, both tyrosine and tryptophan exhibit unique
action spectra, meaning that the identity of the donating chromophore can be easily determined in addition to the distance
between donor/acceptor. Application of the method to the Trpcage miniprotein reveals distance constraints that are consistent
with a native-like fold for the +2 charge state in the gas phase. This structure is stabilized by several salt bridges, which have also
been observed to be important previously in proteins that retain native-like structures in the gas phase. The ability of this method
to measure specific distance constraints, potentially at numerous positions if combined with site-directed mutagenesis,
significantly enhances our ability to examine protein structure in the gas phase.
phase.9 Although ion mobility provides direct measurement of
the overall collision cross-section of a protein, no specific
information about substructural elements is revealed. It can be
difficult to confidently discriminate numerous structural
INTRODUCTION
■
Meaningful examination of protein structure in the gas phase is
complicated by several fundamental challenges. Perhaps most
obviously, the gas phase is a foreign environment where
possibilities with this type of global structural information.
proteins are not typically found. Indeed, intact transfer of
Recent experiments relying on radical based chemistry have
proteins into the gas phase proved to be a challenge for some
helped to provide more specific information about the distances
between particular residues. For example, radicals can easily be
generated at specific sites with atomic precision in proteins.
Subsequently, radical directed dissociation (which typically
time, and the eventual solutions to this problem were
recognized with Nobel prizes.1,2 Subsequent improvements in
speed and sensitivity have now made mass spectrometry (MS)
the method of choice for protein identification and
sequencing.3 For these same reasons, considerable effort is
takes place at different sites than where the initial radical was
created) can easily be monitored by mass spectrometry. In this
currently aimed at developing methods that can extract
information about protein structure in the gas phase.4 However,
our understanding of how proteins are transferred into the gas
fashion, distance constraints between the initial and final radical
positions can be estimated.5,7 Similarly, MS/MS experiments
and ion/molecule reactions can be used to probe the
phase and how this process impacts structure is both imperfect
probability of recombination of two radicals created at two
and rapidly evolving. Important factors to be considered
include the instrument/source conditions and relevant time
scales over which protein structure can be preserved. Although
separate, specific sites.10 Although these experiments provide
detailed substructural information, the difficulty with these
methods is that radical migration within a protein is not easily
modeled or a function of any simple chemical or physical
property.
it is clear that significant features of solution phase protein
structure can be retained in the gas phase in some cases,5,6
there are also examples where solution phase structure appears
to be lost.7,8
Ion mobility is the most commonly implemented exper-
imental technique for examining protein structure in the gas
Received: July 16, 2014
Published: September 1, 2014
© 2014 American Chemical Society
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An alternative way of probing specific distances (and thus
structural features) in proteins relies on excitation energy
transfer (EET).11 EET encompasses a number of mechanisms
molecular dynamics calculations, it is demonstrated that the
data is consistent with retention of the native structure for the
+2 charge state.
including Dexter energy transfer and more popularly, Forster
̈
resonance energy transfer (FRET). FRET is notable for the
transfer of energy at distances that can exceed 40 Å, making it a
valuable tool for examining protein conformation, enzymatic
activity, metabolic pathways, and many other aspects of biology
where distance probes are useful.12−15 Dexter energy transfer or
Dexter exchange transfer is also a distance-sensitive form of
EET that requires overlapping orbitals between chromophores
and results from the synchronous exchange of two electrons.16
Because of the requirement that molecular orbitals must
overlap, Dexter energy transfer occurs at much shorter
distances than those that are typically associated with FRET.
Additionally, the distance dependence for Dexter energy
transfer is exponential and is rarely observed to occur at
distances over 15 Å.11
EXPERIMENTAL SECTION
■
Peptides. The peptides Ac-RRWWCR-NH3, CGYGPKK-
KRKVGG, PHCKRM, VTCG, RGDC, EAGDDIVPCSMSYTWTGK,
CQDSETRTFY, and VCYDKSFPISHVR were purchased from
American Peptide Company. A series of nine polyalanine helical
peptides with sequences Ac-WAAAAAAAACK, Ac-AWAAAAAAACK,
Ac-AAWAAAAAACK, Ac-AAAWAAAAACK, Ac-AAAAWAAAACK,
Ac-AAAAAWAAACK, Ac-AAAAAAWAACK, Ac-AAAAAAAWACK,
and Ac-AAAAAAAAWCK were synthesized. Additionally, the
peptides, Ac-AAAAAAAAACK, VYCG, WWCG, and VWCG were
also synthesized. All peptides were synthesized according to standard
solid phase peptide synthesis procedures.33 The Trpcage variants C-
Trpcage, Trpcage-C, and Trpcage Y3F were also synthesized by solid
phase peptide synthesis.
Disulfide Formation. A 5 μL aliquot of 3 mM peptide solution
was combined with 1 μL of propylmercaptan and 10 μL of DMSO.
The mixture was heated in a water bath at 37 °C for 12 h and then
lyophilized to remove DMSO. Samples were resuspended in 200 μL
50:50:1 water:ACN:TFA. Disulfides between peptides were formed in
the same manner using 5 μL of a 3 mM solution of each peptide.
Synthesis of Indole-3-Methanethiol. Gramine, methyl iodide,
potassium thioacetate, and potassium carbonate were purchased from
Sigma-Aldrich. A total of 1.2 mmol of gramine was reacted with an
excess of methyl iodide (5 mmol) in 5 mL of 1:5 methanol:acetonitrile
and stirred for 3 h. The precipitate was collected by vacuum filtration
and rinsed with acetone. The resulting solid was then reacted with an
excess of potassium thioacetate (3.6 mmol) in a 5 mL 2:2:1 mixture of
tetrahydrofuran:acetonitrile:water. The mixture was stirred vigorously
for 4 h. A total of 2 mL of water and chloroform were added and the
organic layer was extracted and washed with water. The resulting
organic layer was dried by nitrogen. The product was redissolved in
methanol (4 mL) and an excess of potassium carbonate was added and
the mixture was stirred vigorously for 2 h. The final product identity
was confirmed by MS/MS. Disulfide modifications of indole-3-
methanethiol and 1H-indole-3-thiol (purchased from Sigma) were
performed in the same fashion as peptide modifications.
The implementation of traditional EET based methods in gas
phase experiments is difficult. Both the number of ions that can
be interrogated in gas phase experiments and optical access to
them make direct observation of emission difficult to achieve.
Nevertheless, a few groups have succeeded with the traditional
approach.17−26 Similar challenges exist for gas phase spectros-
copy experiments, which led to the development of action
spectroscopy.27 In these experiments fragmentation, which is
easily and sensitively measured by a mass spectrometer, is
monitored rather than absorption or emission. In theory, a
similar scheme should work for EET, where photon absorption
would occur at one chromophore, followed by energy transfer
and fragmentation at the other. There is a subtle (but very
important) difference between action EET and action spec-
troscopy, namely, for action EET to work, energy transfer must
lead to excitation of a dissociative excited state28,29 in the
acceptor.30 The reason for this requirement is simple. If
dissociation occurred following internal conversion of the
energy into vibrational excitation, then intramolecular vibra-
tional energy redistribution would lead to identical products
whether energy conversion occurred in the donor or the
acceptor. Therefore, no characteristic peaks would be generated
that would uniquely indicate energy transfer. On the other
hand, if excitation of the donor leads to specific fragmentation
of the acceptor prior to internal conversion, then energy
transfer is straightforwardly identified.
In solution, previous studies have found that disulfide
reduction by UV light is enhanced by the presence of
tryptophan.31 Additional studies have found that disulfides
participate in photochemical reactions in conjunction with
tryptophan.32 Accordingly, there is some evidence that
disulfides may participate with tryptophan in EET. However,
to the best of our knowledge, no such investigation has been
performed on the gas phase chemistry of this system.
In this work, we reveal an EET pair suitable for action
spectroscopy. It is demonstrated that excitation of tryptophan/
tyrosine leads to direct dissociation of proximal disulfide bonds.
Spectra for the tryptophan, disulfide, tryptophan/disulfide, and
tyrosine/disulfide pairs are reported in the range of 250−300
nm and confirm that energy transfer occurs, followed by excited
state fragmentation. The distance dependence of this process
leads to loss of energy transfer by ∼15 Å, which is consistent
with Dexter exchange transfer. Action spectra were acquired
and fit to reveal distance constraints for the gas phase structure
of the Trpcage in the +2 charge state. In combination with
Molecular Modeling. All calculations and molecular modeling
were performed in Maestro (Schrodinger Inc., Portland, Oregon)
̈
using the OPLS atomic force fields. To estimate disulfide-to-
tryptophan distances in peptides, the cysteine and tryptophan side
chains were rotated in sterically allowed conformations to find the
maximum and minimum distances between the disulfide S atoms and
tryptophan C4 and C6. MD runs were performed with no solvent at
270 K for 10 ns with 100 ps equilibration time and 1.5 ps time-step
intervals.
Simulated annealing calculations were performed on the Trpcage
variants up to temperatures of 700 or 1000 K. Each simulation
included 50 cycles, each cycle beginning at 300 K, incrementally
raising to 400, 500, and 700 K or 1000 K before lowering to 300 K and
then 200 K.
Mass Spectrometry Disulfide Photodissociation Experi-
ments. Peptide solutions were sprayed through an ESI source into
an LTQ modified with a 266 nm Nd:YAG laser (Continuum, Santa
Clara, CA) interfaced with the ion trap. Seven UV-photodissociation
experiments are performed by isolating the parent ion and exposing it
to a single laser pulse in the ion trap prior to scanning out. The yield of
disulfide cleavage is calculated by
⎛
⎞
production intensity
production + precursor intensity
PD yield =
× 100
⎜
⎝
⎟
⎠
For the vast majority of peptides examined, only a single product ion is
observed in significant yield.
A second setup utilized an Nd:YAG optical parametric oscillator
(OPO) laser (Opotek Inc., Carlsbad, CA) interfaced with an LTQ.
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Ions are exposed to a single laser pulse per scan. This setup was used
to obtain action spectra of ions in the range of 250−300 nm. Laser
power levels for each wavelength are recorded for normalization.
Action spectra are compiled by calculating the PD yield for each
wavelength and normalizing them according to differences in the laser
power. It should be noted that the laser power of the OPO is generally
one-fourth of that of the Nd:YAG at 266 nm laser, and comparing PD
yields obtained between the two instruments reflects this.
Ion Mobility−Mass Spectrometry Experiments. Peptides were
introduced by nanoelectrospray ionization (nESI) on a Waters Synapt
G2 HDMS in 50:50 H2O:ACN with 0.1% TFA. Tetraalkyl ammonium
salts served as ion mobility calibrants to convert drift times to nitrogen
collision cross section (CCS) values.34−36 The N2 CCS values for the
theoretically generated conformations were calculated using a modified
version of the trajectory method.34
repulsion, two peptide radicals generated by disulfide bond
cleavage can simply recombine38 to regenerate the disulfide
bond if the fragments are temporarily held together by
noncovalent interactions.
In order to potentially quantify the contribution of EET to
disulfide PD yield, it is desirable to avoid complications arising
from such noncovalent interactions. This can be accomplished
in a straightforward manner by replacing one of the peptides
with a small organic molecule that is not likely to form any
significant noncovalent bonds. Propyl mercaptan (PM) is a
small saturated hydrocarbon that cannot form hydrogen bonds
or participate in any other strong intermolecular interactions.
PM also readily forms disulfide bonds with peptides containing
free cysteine residues. Accordingly, cleaving disulfide bonds
formed with PM should give a more accurate reflection of the
underlying photochemistry.
RESULTS AND DISCUSSION
■
Photochemistry. It has been demonstrated previously that
disulfide bonds can be homolytically cleaved very specifically in
the gas phase by UV light.29,37 For example, if two peptides
connected by a disulfide bond are irradiated by 266 nm light,
only the disulfide bond will be broken and two radical peptides
will be produced. The efficiency of this process can vary
substantially as shown in Table 1, where several examples of PD
Table 2 shows the PD yields for various peptides connected
to PM via a disulfide bond. The PD yields are very high for
Table 2. PD yields for peptides tagged with PM
peptide bound to propyl mercaptan
PD yield
charge state
VWCG + PM
58%
53%
24%
20%
16%
7%
1+
1+
3+
2+
1+
2+
1+
1+
1+
1+
1+
1+
Ac-RRWWCR-NH3 + PM
CGYGPKKKRKVGG + PM
CGYGPKKKRKVGG + PM
CGYGPKKKRKVGG + PM
PHCKRM + PM
Table 1. PD Yields for Selected Peptide Pairs
PD
yield
charge
state
peptides
Ac-RRWWCR-NH3 + EAGDDIVPCSMSYTWTGA
CGYGPKKKRKVGG + VCYDKSFPISHVR
VTCG + Ac-RRWWCR-NH3
37%
24%
13%
13%
<1%
<1%
<1%
<1%
<1%
4+
6+
2+
5+
4+
3+
2+
1+
1+
PHCKRM + PM
9%
VYCG + PM
17%
7%
VTCG + PM
CGYGPKKKRKVGG + CQDSETRTFY
CGYGPKKKRKVGG + CQDSETRTFY
CGYGPKKKRKVGG + CQDSETRTFY
CGYGPKKKRKVGG + CQDSETRTFY
RGDC + VTCG
RGDC + PM
6%
AcA5WA3CK + PM
AcA9CK + PM
72%
8%
RGDC + PHCKRM
peptides which contain chromophores, as was also the case for
some peptides in Table 1. Additionally, the PD yields for
peptides modified with PM are higher than the PD yields
obtained from related peptide pairs. For example, Ac-
RRWWCR-NH3 has a yield of 53% when modified with PM,
whereas the yields obtained when attached to other peptides
are only 37% and 13% as seen in Table 1. Even peptides that
contain no chromophores (such as RGDC or VTCG) give PD
yields of 6% and 7%, respectively, whereas these peptides only
have a PD yield of less than 1% if bound to each other by a
disulfide bond. All of these results are consistent with the idea
that peptide pairs can form strong interfering noncovalent
interactions and that PM is not sticky and, thus, gives more
accurate PD yields.
The results in Table 2 reveal a strong correlation between the
presence of aromatic residues and high disulfide PD yield. In
order to investigate whether disulfide dissociation is truly due
to EET, we recorded the action spectra for tryptophan, a PM-
modified cysteine-containing peptide (AcA9CK-PM), and a
peptide which contains both cysteine and tryptophan
(AcA5WA3CK-PM). The results are shown in Figure 1. The
AcA9CK-PM spectrum is characterized by weak absorption and
featureless decay in the region from 250 to 300 nm. The
tryptophan spectrum shows broad absorption with a single
peak at 287 nm. This spectrum is consistent with previously
reported spectra for tryptophan in the gas phase.39 The peptide
that contains both moieties yields a spectrum that closely
resembles the tryptophan spectrum, although it is red-shifted by
yields for disulfide bond cleavage from precursor selected
peptide pairs are given. The PD yields range from less than 1%
to up to 37%. There are several potential factors that may
influence PD yields in these experiments. For example, the
peptide structure or charge state could potentially influence
photon absorption. Alternatively, the presence of noncovalent
interactions between the two peptides might prevent
observation of the individual peptide radicals even if the
disulfide bond were cleaved. In addition, it is also possible that
native chromophores could enhance dissociation for some
peptide pairs via EET.
Close examination of the results in Table 1 suggests that
some of these factors are likely influencing the observed PD
yields. For example, sequences containing chromophoric
residues (Trp, Tyr, and Phe) appear to undergo disulfide
cleavage more efficiently than peptides that do not. This
suggests that EET may occur from native chromophores to the
disulfide bond. Another notable trend is that higher charge
states tend to have the best PD yields for peptide pairs that are
observed in multiple charge states. This is evident in
CGYGPKKKRKVGG + CQDSETRTFY where only the
highest observable charge state has a PD yield greater than
1%, as shown in Table 1. Although the charge state per se may
not be important, it is likely for peptide pairs that charge
repulsion overcomes any noncovalent interactions that
otherwise inhibit dissociation. In the absence of Coulombic
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dissociation in the excited state. In contrast, more typical results
consisting of a variety of b and y ions are observed by CID.
Importantly, CID in an ion trap strongly favors fragmentation
via the lowest energy dissociation pathways. It is clear in Figure
2b that homolytic cleavage of the disulfide bond does not occur
via a low energy dissociation pathway and is not favored
following vibrational excitation. Taken together, the results in
Figures 1 and 2 indicate that the dominant cleavage of the
disulfide bond following photoactivation most likely occurs due
to excitation of a dissociative excited state. Importantly, this
type of chemistry should also exhibit a distance dependence
related to the underlying mode of EET.
In order to evaluate the distance dependence for Trp/PM
EET, we sought to measure the disulfide PD yield for peptides
where the distances between donor and acceptor could be
reasonably estimated. Previous work has indicated that peptides
based on an Ac-AxK (where x ≥ 7) motif tend to form alpha
helices in the gas phase.40,41 Building on this general motif, we
synthesized Ac-WA8CK, which has two of the alanine residues
at opposite ends of the helix replaced with PM modified
cysteine and tryptophan. The collision cross section for this
peptide as determined by ion mobility is consistent with an α
helical structure (the predicted cross section from molecular
modeling matches the experimental data within ∼3%,
Supporting Information Table S1). The estimated distance
between the donor and acceptor for this peptide is ∼14.5 2.5
Å. The large uncertainty in the distance comes from the
flexibility of the side chains, which are assumed to retain
significant rotational freedom. The PD yield for Ac-WA8CK is
fairly low at 24%. Nevertheless, the PD yield is still appreciably
greater than the 8% obtained for Ac-A9CK, which contains no
tryptophan. The results from Ac-A9CK serve as the baseline for
inherent non-EET assisted disulfide bond fragmentation in a
polyalanine based peptide. It is clear that even at a distance of
∼14.5 Å, some EET is occurring.
Figure 1. Action spectra for AcA5WA3CK-PM, tryptophan, and
AcA9CK-PM. Note: PD yields here reflect the lower power of the
OPO laser setup.
∼10 nm. This spectrum was obtained solely by monitoring
homolytic fragmentation of the disulfide bond, which is by far
the most dominant fragment observed at all wavelengths. The
resemblance of the AcA5WA3CK-PM spectrum to the spectrum
for tryptophan itself and the observed red-shift are both
consistent with fragmentation that occurs following EET.
The results in Figure 1 unambiguously establish that PD of
the disulfide bond is occurring after excitation of the
tryptophan chromophore. In order to determine whether this
fragmentation occurs due to EET to a dissociative excited state,
we compared the PD spectrum for AcA5WA3CK-PM to that
obtained by typical collision induced dissociation (CID) and
the results are shown in Figure 2. The PD spectrum confirms
that homolytic fragmentation of the disulfide bond yields by far
the most abundant product ion, which is consistent with direct
We also attempted to use the polyalanine scaffold to
determine the close contact EET efficiency; however, as the
distance between donor and acceptor is decreased by altering
donor/acceptor positions in the polyalanine chain, the
rotational freedom of the side chains becomes increasingly
problematic. Furthermore, it is not clear that peptides where
tryptophan replaces a central alanine are able to retain entirely
helical structures (i.e., cross sections from ion mobility
exceeded 3% difference relative to predicted values; see
Supporting Information Table S1). Therefore, we adopted an
alternative approach where we simply attached the donor and
acceptor to each other via short covalent bonds. Direct
attachment of the disulfide bond to the indole chromophore
is possible and represents the closest distance that the two
moieties can be brought together. By attachment of an amine
group on the other side of the disulfide (see structure in Figure
3a), a suitable ion can be generated for examination by ESI.
The PD spectrum for the protonated molecule is shown in
Figure 3a and it contains several interesting features. The
expected product at 77 Da results from homolytic cleavage of
the disulfide bond. The intensity of this peak is lower than
might be anticipated, given the unavoidable proximity of the
donor and acceptor. In addition, several unexpected fragments
are observed. For example, there is a peak at 148 Da that
corresponds to the radical cation of the indole chromophore.
This peak is most likely generated by dissociative electron
transfer from the indole ring to the disulfide bond. Indeed, the
action spectra for homolytic cleavage of the disulfide bond and
Figure 2. (a) PD and (b) CID spectra for AcA5WA3CK-PM.
* precursor.
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disulfide donor/acceptor pair. Insertion of an additional CH2
group in between the chromophores results in reduced PD
yield (88%), consistent with this idea.
Given the distance constraints that we have established up to
this point, it is possible to narrow down the mode by which
EET is likely occurring for this system. The observation that the
PD yield decays significantly within 15 Å suggests that FRET is
not responsible for most of the energy transfer. Instead, we
propose that the indole/disulfide EET occurs primarily via
Dexter exchange transfer (DET), which has an exponential
distance dependence and typically occurs within 15 Å
(Supporting Information Figure S2).
In proteins, Trp is not the only chromophore that may
contribute to the cleavage of disulfide bonds via EET. Tyrosine
also has significant absorption in the near UV, and would be
expected to facilitate EET mediated disulfide bond cleavage. To
explore this possibility further, we replaced tryptophan with
tyrosine to create the peptide Ac-A5YA3CK-PM, which can be
compared with the Trp analog from Figure 2. The results are
shown in Figure 4. The comparison reveals that tyrosine yields
Figure 3. (a) PD of the molecule shown to the far right. Structures
corresponding to the products of interest are also shown. (b) CID of
the same molecule. (c) PD of the same molecule in complex with 18-
crown-6.
electron transfer to the indole are distinct (see Supporting
Information Figure S1), suggesting formation via distinct
chemical processes. No similar signs of electron transfer
chemistry were detected in any experiments with peptides.
The remaining smaller peaks likely originate from internal
conversion of the photon energy to vibrational excitation as the
peaks at 108, 118, and 180 Da are also observed by CID
(Figure 3b). Other peaks in Figure 3a, such as 104 and 121 Da,
appear to be the products of rearrangements following a
combination of electronic and vibrational excitation.
The extremely close proximity of the protonated amine
appears to significantly influence the fragmentation behavior of
this small molecule. In order to explore this possibility further,
we solvated the ammonium group by noncovalent complex-
ation with 18-crown-6 ether (18C6), which forms three strong
hydrogen bonds with protonated primary amines.42 The PD
spectrum for the 18C6 complex is shown in Figure 3c. The
electron transfer product is no longer observed to any
significant extent, and most other dissociation channels are
suppressed as well. Homolytic cleavage of the disulfide bond is
enhanced significantly and the resulting PD yield is quite high
(∼93%). The distance between chromophore centers for the
donor and acceptor in this system is ∼4 Å, and the PD yield is
likely the maximum value that can be obtained for the indole/
Figure 4. Action spectra of Ac-A5WA3CK-PM as compared with Ac-
A5YA3CK-PM.
a distinct spectrum, which is similar to action spectra acquired
for tyrosine itself. The distinctive shoulder occurs at a unique
wavelength for both Trp and Tyr, which should allow for
contributions from the two chromophores to be distinguished.
Furthermore, the Tyr spectrum loses intensity at shorter
wavelengths more rapidly than Trp. The relative intensity of the
Tyr spectrum is also significantly smaller than that for Trp,
suggesting that Tyr will only make significant contributions to
PD yield at very short distances.
Trpcage. We now apply these tools to the examination of
protein structure. The Trpcage is a very small and fast folding
protein that was designed computationally.43 It is comprised of
only 20 amino acids with the sequence NLYIQWLKDG
GPSSGRPPPS. It contains one Tyr and one Trp. The native
fold traps the Trp side chain in the center of a hydrophobic
pocket created by a small α helix and a run of proline residues;
this pocket is the defining feature of the structure and the
source of the molecule’s name. A + − + salt bridge can form
between the Lys, Asp, and Arg side chains. This salt bridge has
been demonstrated to be important for the stability of the
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protein in solution.44,45 An additional salt bridge can form
between the N- and C-termini. The stability and importance of
these salt bridges in the gas phase has been questioned.46
Furthermore, previous work has demonstrated that Trpcage
can adopt non-native structures in the gas phase.18 This is not
surprising because the gas phase is a very different environment
than the aqueous solution for which the protein was
engineered. Nevertheless, other studies have suggested that
native-like Trpcage structures can be stable in the gas phase
under appropriate conditions.47,48
There are no cysteine residues in the standard Trpcage,
therefore, we added a cysteine to both the N-terminus and C-
terminus (called C-Trpcage and Trpcage-C, respectively).
NMR experiments reveal that the N and C-terminal residues
are fairly disordered, which suggests these additions are not in
sterically confined locations and are not anticipated to
significantly impact the structure or folding of the protein in
solution.
which is characteristic of EET from tyrosine and indicates very
close Tyr/disulfide proximity. At the same time, the shoulder at
∼287 nm is indicative of EET from Trp, suggesting that both
chromophores contribute to the spectrum. In order to explore
this idea further, a combination spectrum created by additively
combining the Tyr and Trp spectra is shown in Figure 5b. The
tyrosine spectrum from Ac-A5YA3CK-PM was combined in a
1:1 ratio with the spectrum from Ac-WA8CK-PM. This would
correspond to a situation where the tyrosine residue is close at
<6 Å, and the tryptophan residue is significantly farther away
(>10 Å). As can be seen in Figure 5b, the combination
spectrum is a reasonably good match to the experimental C-
Trpcage-PM 2+ spectrum. The primary differences are due to
less distinct absorption at ∼282 and ∼287 nm, which may be
caused by internal solvation of the chromophores. Solvation of
tyrosine has previously been demonstrated to yield less distinct
spectral features.49 Alternate ratios of 2:1 or 1:2 mixtures of the
same spectra yielded less satisfactory fits (see Supporting
Information Figure S3).
The remaining spectra in Figure 5a are significantly flatter
and consistent with minimal tyrosine character. To test this
hypothesis, a variant of Trpcage-C was synthesized with
phenylalanine substituted for tyrosine. Although phenylalanine
is also a potential chromophore, the observed absorption in the
250−300 nm range is so poor that we do not detect any
appreciable dissociation of phenylalanine containing peptides.
Accordingly, spectra for the Y3F mutant of the Trpcage should
reveal exclusively EET due to tryptophan. The results are
shown in Supporting Information Figure S4 overlaid with the
spectra collected from the tyrosine-containing version. Differ-
ences in the spectra are minimal which supports the idea that
tyrosine is too far from the C-terminal cysteine to contribute
substantially to EET.
Examination of the action spectra for each charge state,
which are shown in Figure 5, reveals several important
structural features. C-Trpcage-PM 2+ exhibits the largest PD
yield and the shape of the spectrum is noticeably different from
the spectra for other charge states. Importantly, the intensity for
this spectrum decreases more rapidly at shorter wavelengths,
The C-Trpcage-PM 2+ is the best candidate for more in
depth structural characterization because distance constraint
information is provided for both chromophores. Simulated
annealing was carried out on the C-Trpcage-PM 2+ starting
from the native structure. Two separate simulated annealing
approaches were used to generate low energy gas-phase
structures from the NMR structure: one reaching temperatures
of 1000 K and another reaching temperatures of 700 K. High
temperature simulated annealing is preferable to find the global
energy minimum because significant activation may be required
to overcome the energy barriers needed to reach such a
structure. Accordingly, the global energy minimum structure
generated from simulated annealing at 1000 K may not be the
structure that is actually observed. In fact, the structure that is
generated (Supporting Information Figure S5) bears no
resemblance to the solution phase structure and is also distinct
from those noted in previous studies of the Trpcage.47,48 In
contrast, the structure generated from a more modest 700 K
annealing resembles the solution-phase structure while being
significantly lower in energy than structures generated from
other conformational searches or simple energy minimization.
The lowest energy structure obtained from the 700 K
simulated annealing is shown in Figure 6 in comparison with
the native structure. Most of the structure is unperturbed, with
a few interesting exceptions. Notably, the N-terminus has a
different orientation that allows for extensive hydrogen bonding
between the N- and C- termini. The distances between the
chromophores and the disulfide bond in the structure in Figure
6 are completely consistent with the experimental data in
Figure 5. It should be noted that one charge is located at the N-
Figure 5. (a) Action spectra for the C-Trpcage 2+, Trpcage-C 2+ and
Trpcage-C 3+. (b) Comparison of the action spectrum of the C-
Trpcage (2+) and the combination spectrum from Ac-A5YA3CK-PM
and Ac-WA8CK-PM.
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Article
the native-like structure to the lowest energy gas phase
structure. For the +3 charge states, the results indicate a
more unfolded structure, which results in a slightly increased
Trp/disulfide interaction distance. This is consistent with
previous observations by ion mobility illustrating Coulomb
induced unfolding of proteins in higher charge states.52
CONCLUSIONS
■
We have developed an action EET system based on Trp/Tyr
and disulfide bonds suitable for experiments on ions in the gas
phase. Excitation of Trp/Tyr by UV light leads to EET and
excited state dissociation of disulfide S−S bonds in a distance
dependent fashion. Trp can facilitate homolytic fragmentation
of disulfide bonds out to ∼15 Å, whereas Tyr is only active at
very close proximity (<6 Å). These distance regimes suggest
that the majority of the EET for these chromophores occurs via
Dexter exchange transfer and that FRET is likely a minor
contributor. The combination of distance information with
initiating chromophore identity provides a powerful new
platform for investigating protein structure in the gas phase.
Furthermore, the ability to use native chromophores (i.e., Trp
and Tyr) in combination with disulfide bonds generated from
cysteine residues provides facile access to the powerful suite of
molecular biology tools that have been developed to
manipulate protein sequence. If a native sequence does not
contain probes in appropriate locations, standard site-directed
mutagenesis can be used to incorporate the desired residues.
This presents a considerable advantage over existing methods
of applying EET which require more disruptive addition of
larger synthetic chromophores. This method should lead to
enhancement of our understanding of protein structure in the
gas phase.
Figure 6. Comparison of the C-Trpcage-PM simulated annealing
structure (blue) superimposed with the Trpcage NMR structure
(orange).
terminus, which remains hydrogen bonded to the C-terminus,
stabilizing the native-like fold. The second charge is located at
arginine, which is in a + − + salt bridge with Asp and Lys.
Previous results have suggested that Trpcage does not retain
salt bridges in the gas phase, and that the +2 charge state may
not have a native-like structure.46 There are several possible
explanations for these apparent contradictions. The conditions
of our source may be gentler, facilitating the retention of the
native like structure. It is also possible that the salt bridge may
convert to simple hydrogen bond interactions via proton
transfer without significantly influencing the remainder of the
structure, though previous results with other proteins have
demonstrated salt bridges are frequently critical for the
retention of native structure in the gas phase.50,51 It is also
possible that the additional cysteine residue may stabilize the
native structure by enabling extra hydrogen bonding between
the termini. Comparison of our data with previously proposed
structures that differ substantially from the native fold does not
yield good agreement (i.e., the Tyr and Trp distances are not
consistent with our results). Taking all of the evidence into
consideration, we conclude that it is possible to preserve a
native like state for the Trpcage in the +2 charge state under
appropriately gentle source conditions.
Detailed evaluation of the remaining structures is more
difficult because the data only reveal a fairly long-range
interaction with Trp and absence of a close range interaction
with Tyr. The data for Trpcage-C-PM 2+ are consistent with
the native fold structure, although many other structures would
likely be suitable as well. Activation of the C-Trpcage 2+
structure by harsher source conditions or supplemental
collisional energy result in significantly lowered PD yields,
whereas native MS solvent conditions (1 mM NH4OAc) do not
significantly change the yield (Supporting Information Table
S2). These observations for C-Trpcage 2+ are consistent with
the expected behavior that would accompany a transition from
ASSOCIATED CONTENT
■
S
* Supporting Information
Additional spectra, ion mobility results, and structures for the
Trpcage. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Authors
john.a.mclean@vanderbilt.edu
ryan.julian@ucr.edu
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank Richard Hooley, Mike Pirrung, and Chris
Switzer for helpful advice on synthetic procedures. We thank
the NSF (CHE-0747481) and NIH (GM103531) for financial
support and Greg Beran, Arun Agarwal for stimulating
conversation. Financial support for J.A.M., N.M.L., and S.M.S.
was provided by the National Institutes of Health
(UH2TR000491) and the Vanderbilt Chemical Biology
Interface training grant (T32GM065086, N.M.L.), the
Vanderbilt University College of Arts and Science, the
Vanderbilt Institute of Chemical Biology, and the Vanderbilt
Institute for Integrative Biosystems Research and Education.
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