Dynamic interchanging native states of lymphotactin examined by SNAPP-MS

Article abstract of DOI:10.1007/s13361-010-0042-3

The human chemokine lymphotactin (Ltn) is a remarkable protein that interconverts between two unrelated native state structures in the condensed phase. It is possible to shift the equilibrium toward either conformation with selected sequence substitutions

Full text of DOI:10.1007/s13361-010-0042-3

B The Author(s) 2011. This article is published with open access at Springerlink.com
J. Am. Soc. Mass Spectrom. (2011) 22:399Y407
DOI: 10.1007/s13361-010-0042-3
RESEARCH ARTICLE
Dynamic Interchanging Native States
of Lymphotactin Examined by SNAPP-MS
Qingyu Sun,1 Robert C. Tyler,2 Brian F. Volkman,2 Ryan R. Julian1
1Department of Chemistry, University of California, Riverside, CA 92521, USA
2Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA
Abstract
The human chemokine lymphotactin (Ltn) is a remarkable protein that interconverts between two
unrelated native state structures in the condensed phase. It is possible to shift the equilibrium
toward either conformation with selected sequence substitutions. Previous results have shown
that a disulfide-stabilized variant preferentially adopts the canonical chemokine fold (Ltn10),
while a single amino acid change (W55D) favors the novel Ltn40 dimeric structure. Selective
noncovalent adduct protein probing (SNAPP) is a recently developed method for examining
solution phase protein structure. Herein, it is demonstrated that SNAPP can easily recognize
and distinguish between the Ltn10 and Ltn40 states of lymphotactin in aqueous solution. The
effects of organic denaturants, acid, and disulfide bond reduction and blocking were also
examined using SNAPP for the CC3, W55D, and wild type proteins. Only disulfide reduction was
shown to significantly perturb the protein, and resulted in considerably decreased adduct
formation consistent with loss of tertiary/secondary structure. Cold denaturation experiments
demonstrated that wild-type Ltn is the most temperature sensitive of the three proteins.
Examination of the higher charge states in all experiments, which are presumed to represent
transition state structures between Ltn-10 and Ltn-40, reveals increased 18C6 attachment
relative to the more folded structures. This observation is consistent with increased competitive
intramolecular hydrogen bonding, which may guide the transition. Experiments examining the
gas phase structures revealed that all three proteins can be structurally distinguished in the gas
phase. In addition, the gas phase experiments enabled identification of preferred adduct binding
sites.
Key words: h/d exchange, Protein structure, Covalent labeling
Scheme 1 [2]. One structure represents the typical chemo-
Introduction
kine fold, a single alpha helix adjacent to three beta-sheet
strands, and is referred to as the Ltn10 structure. The other
structure is known as Ltn40 and is characterized by
dimerization of two proteins, with both adopting four β-
sheet strands oriented in a symmetrical head to tail fashion.
Although both structures share β-sheet motifs, the composi-
tion of the beta sheets is quite different between the two
structural forms. Similarly, the hydrophobic cores and other
tertiary and quaternary structural characteristics are essen-
tially entirely dissimilar. Under typical biological conditions,
both forms of the wild type protein exist in equilibrium and
ymphotactin is an unusual protein in many ways. It is a
L
human chemokine that can adopt two natively folded
states, which are both functionally relevant in vivo [1]. The
two native state structures are very dissimilar as shown in
Electronic supplementary material The online version of this article
(doi:10.1007/s13361-010-0042-3) contains supplementary material, which
is available to authorized users.
Correspondence to: Ryan R. Julian; e-mail: ryan.julian@ucr.edu
Received: 15 September 2010
Revised: 15 November 2010
Accepted: 15 November 2010
Published online: 15 January 2011

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Q. Sun, et al.: Lymphotactin by SNAPP-MS
Scheme 1. The two structures of lymphotactin
interconvert on a timescale of seconds [2]. In the Volkman
laboratory, two mutants have been developed that adopt only
one of the two structural motifs [3]. The CC3 mutant
contains an additional disulfide bond that favors the typical
Ltn10 structure. Disruption of the Ltn10 hydrophobic core in
the W55D mutant pushes the structural equilibrium strongly
towards the Ltn40 structure. These mutants can be utilized to
examine aspects of each structure independently.
conformation. Changes to the protein structure will typically
lead to changes in the SNAPP distribution; therefore the
strength of SNAPP is in measuring structural differences for
either static or dynamic systems.
SNAPP is closely related to other mass spectrometry
(MS) based protein structure determination methods such as
charge state distribution analysis [8–10], hydrogen/deute-
rium (H/D) exchange [9–11], and covalent labeling [12], but
also differs in several important ways. H/D exchange
monitors protein structure as a function of the exchange of
amide hydrogens. The overall number, locations, and rates
of exchanges can be used to interrogate structure [13].
Various covalent labeling methods can also be used to probe
structure, although in this case reactions are ideally restricted
to a single modification to avoid perturbation of the target
structure [14]. The primary difference between SNAPP and
both of these methods is that the reporting chemistry in
SNAPP is highly reversible (if present at all) in solution and
information is only encoded during the later stages of ESI
when the protein is transitioning into the gas phase. This
enables SNAPP to probe highly dynamic systems where
structural changes may be occurring rapidly in solution, and
on the same timescale as H/D exchange or covalent labeling.
If, for example, a protein adopted an unfolded and folded
state under given conditions, the SNAPP distributions for
both would be determined independently during ESI and
observable in different charge states (due to the different
sizes) as long as the structural transition took longer than a
few milliseconds (the time required to desolvate the protein).
SNAPP is therefore an appropriate method to interrogate the
structural features of lymphotactin, which undergoes struc-
tural changes on a significantly longer timescale [2].
Selective noncovalent adduct protein probing (SNAPP) is
a recently developed method for examining protein structure
in solution [4]. This method relies on specific noncovalent
interactions to probe protein structure. Experiments are
conducted by introducing a reagent which binds weakly in
solution, such as 18-crown-6 ether (18C6), which becomes
strongly attached to the protein in the gas phase. 18C6
attaches to lysine in the gas phase due to three specific
hydrogen bonds between the protonated side-chain amine
and alternating oxygen atoms in the crown ether. The
binding energy for this attachment is ~54 kcal/mol [5]. Less
specific, although fairly strong noncovalent (42 kcal/mol)
associations between 18C6 and the protonated side chain of
arginine can form as well. SNAPP experiments are con-
ducted by subjecting an aqueous solution containing protein
and 18C6 to electrospray ionization (ESI). Source conditions
which favor both complete desolvation and retention of
noncovalent adducts must be employed. During the process
of ESI, binding interactions between 18C6 and protonated
side chains transition from weak to strong as the protein is
rapidly desolvated. Previous experiments have determined
that the local chemical environment surrounding each lysine
residue strongly influences the probability for binding 18C6,
with proximate salt bridge or hydrogen bonding interactions
interfering the most [6]. Structural information about the
protein is therefore obtained because the local chemical
environment surrounding each residue is a function of
protein structure [4,7]. In the end, a distribution of peaks
(known as a SNAPP distribution) representing the number of
18C6s attached to the protein is observed in the mass
spectrometer and is characteristic of a particular protein
Herein it is demonstrated that the Ltn10 and Ltn40
structures yield easily distinguishable SNAPP distributions.
The wild type protein yields results that are most similar to
the W55D variant. The addition of acid or organic
denaturants does not have a large impact on the SNAPP
distributions for the wild type, CC3, or W55D proteins.
Reduction and blocking of the disulfide bonds affects the

Q. Sun, et al.: Lymphotactin by SNAPP-MS
401
structures more dramatically and yields shifts in the SNAPP
distributions that are consistent with significant unfolding.
Data obtained from NMR experiments supports this con-
clusion. The effects of cold temperature on all three proteins
are explored. Finally, a combination of SNAPP and radical
chemistry is utilized to compare the gas phase structures of
the wild type, CC3, and W55D proteins.
1.24 μmol for CC3) to block all free thiol groups. The
blocked proteins were purified by a protein trap (Michrom
Bioresources, Inc. Auburn, CA, USA), lyophilized and
stored at –20 °C for subsequent use.
SNAPP Experiments
CC3, wild type Ltn, and W55D stock solutions were diluted
to 7 μM in H2O, respectively. The final concentration of
18C6 in SNAPP solutions was 84 μM. Mass spectra were
obtained using an LTQ linear ion trap mass spectrometer
(Thermo Fisher Scientific, San Jose, CA, USA) equipped
with a standard ESI-source. Protein samples mixed with
18C6 were directly infused into LTQ mass spectrometer.
The electrospray parameters, such as spray voltage, sheath
gas flow rates, capillary voltage, temperature, etc. were
optimized and were similar to parameters described pre-
viously for SNAPP experiments [4]. Once optimized, all the
parameters were maintained for all SNAPP experiments
presented herein. The following are the optimized source
parameters for all SNAPP experiments: spray voltage
4.8 kV, sheath gas flow rates 11, tube lens 160 V, capillary
voltage 44 V and capillary temperature 275 °C.
Experimental
Materials
Recombinant human lymphotactin proteins used in SNAPP
study were expressed and purified as previously described
[15]. Lymphotactin mutants CC3 and W55D were prepared
by site-directed mutagenesis using the Stratagene Quick-
Change kit following established protocols [3]. Purified
proteins were lyophilized and stored at –20 °C for
subsequent study. 18-Crown-6 ether used for SNAPP studies
was purchased from Alfa Aesar (Ward Hill, MA, USA).
Dithiothreitol (DTT) and iodoacetamide (IAA) were pur-
chased from Sigma Aldrich (St. Louis, MO, USA). H2O
used for SNAPP experiments was purified to 18.2MΩ
resistivity using Millipore Direct-Q (Billerica, MA, USA).
All the solvents were obtained from Fisher Scientific
(Fairlawn, NJ) and used as received. 2-(hydroxymethylio-
dobenzoylester)-18C6 (IBA-18C6), employed in the radical
directed dissociation experiments, was prepared as follows:
0.50 mmol DCC in 5.0 mL dioxane was added to a 50 mL
round bottom flask containing 0.50 mmol of 4-iodobenzoic
acid and 0.50 mmol 2-hydroxymethyl-18-crown-6 ether. A
catalytic amount of DMAP (~10 mg) was added. After a
12 h reaction period, a crystalline hair-like precipitate was
observed. The precipitate was removed by filtration. The
filtrate was then passed through celite and evaporated over
nitrogen. The product was recovered as a white solid.
Radical Directed Dissociation of Ltn Proteins
Five μM lymphotactin protein was mixed with 15 μM IBA-
18C6 in 50/50 H2O/MeOH solution and directly infused into
the LTQ linear ion trap mass spectrometer. The posterior
plate of the LTQ is modified with a quartz window to transit
fourth harmonic (266 nm) laser pulses from a flash lamp
pumped Nd/YAG laser (Continuum, Santa Clara, CA,
USA). The laser pulses are synchronized to the end of the
isolation step of a typical MS2 experiment by a TTL trigger
signal from the mass spectrometer to the laser via a digital
delay generator (Berkeley Nucleonics, San Rafael, CA,
USA). The isolation window width of MS2-MS4 experi-
ments was set to 5 Da.
Results and Discussion
The stackplot in Figure 1a shows the mass spectra for
lymphotactin alone acquired in water (front), water/methanol
(middle), and water/methanol/acetic acid (back). These
solvent systems are increasingly denaturing for most
proteins and will typically yield substantial shifts in the
charge state distribution towards higher charge states [8,16].
The increase in charge state is typically rationalized by the
notion that denatured proteins are less compact and can
therefore accommodate additional charges without increas-
ing Coulombic repulsion. Interestingly, the typical shift
towards higher charge states is not observed for lympho-
tactin. In fact, the charge state distributions barely change at
all. Therefore, charge state distributions alone yield virtually
no information about the structure of lymphotactin. As
detailed in the Introduction, it is known that lymphotactin
S–S Bond Reduction and Cysteine Protection
of Ltn Proteins
To reduce disulfide bonds in the lymphotactin proteins, 60
μL solution containing 10 nmol protein and excess amount
of DTT (10 mM) was incubated at 54 °C for 45 min. Free
cysteine and excess DTT were treated with a stoichiometric
amount of IAA (1.22 μmol for wild type Ltn and W55D,

402
Q. Sun, et al.: Lymphotactin by SNAPP-MS
Figure 1. (a) ESI-MS spectra for wild type lymphotactin acquired in water, 50/50 water/methanol, and 49/49/1 water/methanol/
acetic acid from front to back, respectively. SNAPP distributions for wild type lymphotactin in various charge states are shown
in (b) +8, (c) +9, (d) +10, and (e) +12
has two highly dissimilar native structures, which are
under equilibrium. It is likely that transitioning from one
structure to the other requires sampling of an unfolded
state due to the large disparity between the two
conformations. The data in Figure 1a is consistent with
the notion that lymphotactin accesses an unfolded state
even in an aqueous solution, and therefore the addition
of methanol and acid does not significantly shift the
charge state distribution because unfolded structures are
already being sampled. From these data alone, it is clear
that lymphotactin is an unusual protein.
In Figure 1b–d, several representative SNAPP distribu-
tions for lymphotactin in the same series of solvents are
shown. These distributions have been extracted from the raw
mass spectra and illustrate the number of 18C6 adducts
which attach to lymphotactin, separated by charge state. The
addition of 18C6 reveals another layer of structural
information, allowing identical charge states to be compared
with each other. The SNAPP distributions for the +8 through
+10 charge states are fairly similar to each other and are also
fairly constant in the various solvents. These distributions
most likely represent the more folded conformations of the
protein. In the +11 and +12 charge states, the SNAPP
distributions begin to shift towards an increasing number of
adducts (as indicated by the small relative abundance of the
non-adduct peak in the +12 charge state). The average
number of 18C6 adducts goes from 1.3 for the +8 charge
state to 1.6 for the +12 charge state. Some differences
between the various solvent systems also become notable.
Interestingly, the number of crown adducts increases with
the addition of methanol and then decreases again with the
addition of acid. For systems where structural changes are
not dominant, it is known that the addition of acid leads to a
decrease in crown binding due to competitive binding by the
acid [4]. This confirms that acid does not significantly
influence the structure of lymphotactin. Given that the
protein is highly basic (6 Lys, 8 Arg), with few acidic
residues, this result is perhaps not surprising.

Q. Sun, et al.: Lymphotactin by SNAPP-MS
403
attachment in W55D is less than that observed in CC3.
Therefore, the differential response of SNAPP to these two
proteins is clearly due to structural differences. Salt bridges
are known to significantly interfere with 18C6 binding [4].
Both the Ltn10 and Ltn40 structures contain two salt bridges
(Arg61-Asp64 and Arg35-Glu31 for Ltn10, Arg9-Asp50
and Glu31-Lys25 (intermolecular) for Ltn40). However,
lysine is the preferred target for 18C6, and the transition for
Lys25 from being freely available in Ltn10 to being
incorporated into a salt bridge in Ltn40 likely accounts for
some of the reduced 18C6 binding to the W55D mutant.
Although the wild type protein can adopt either the Ltn10 or
Ltn40 structures, it is clear from the data in Figure 2 that the
wild type Ltn SNAPP distributions are most similar to those
from the W55D mutant. The similarity likely results from a
combination of two factors. (1) It is known that wild type Ltn
favors the Ltn40 structure at low ionic strength [17]. (2) The
W55D mutant is able to freely access unfolded states which are
expected to be present in wild type Ltn, but the CC3 mutant is
significantly constrained by an additional disulfide bond,
which likely interferes with adopting similar unfolded states.
Despite similarity to W55D, wild type Ltn does present some
intermediate behavior as well (for example in the 8-2 and 12-1
distributions where the wild type Ltn peaks are more similar to
the CC3 values), suggesting that the equilibrium is not entirely
shifted to the Ltn40 structure. It is worth noting that the dimeric
Scheme 2. Sequences for each lymphotactin variant
In Figure 2, the SNAPP distributions obtained in water
for wild type Ltn, Ltn10 (CC3), and Ltn40 (W55D) are
shown. There is a clear and reproducible difference between
the SNAPP distributions for the CC3 and W55D mutants.
Less 18C6 attachment to the W55D protein is observed in
every case. SNAPP-MS is therefore easily able to distin-
guish the two structural variants of lymphotactin from each
other when examined separately. As shown in Scheme 2,
these two mutants vary in sequence by only six amino acids
(W55D also contains three additional N-terminal residues,
none of them are targets for 18C6, nor do they alter the
Ltn40 conformation). Interestingly, one of the mutations,
Ala in CC3 for Arg in W55D, creates an additional target
residue for 18C6 binding. Despite this change, 18C6
Figure 2. SNAPP distributions for three variants of lymphotactin sampled from water. The CC3 and W55D mutants clearly
produce distinguishable distributions

404
Q. Sun, et al.: Lymphotactin by SNAPP-MS
association for Ltn40 known to dominate in solution is not
observed in the gas phase. Presumably, this is due to loss of the
hydrophobic effect as the lymphotactin dimer is desolvated,
which is primarily driving the association in solution. Never-
theless, it is clear from the data in Figure 2 that the structural
information encoded by attachment of 18C6 is locked in prior
to the loss of the dimer during the transition to the gas phase.
Characterization of the transition between the two forms
of lymphotactin is an active area of interest [18]. The ability
of SNAPP to examine and potentially separate out highly
dynamic structural features is an important advantage which
should facilitate characterization of the transition structures.
As described above, the higher charge states most likely
represent structural states, which are at least partially
unfolded and therefore capable of transitioning between the
two structural forms. One possibility for transition is that the
unfolded protein becomes entirely disordered, and subse-
quently refolds into either native state with probabilities
determined by the nature of the solution. To investigate this
theory, we obtained SNAPP distributions for proteins where
the disulfide bonds had been reduced and blocked. Results
from NMR experiments indicate that elimination of the
disulfide bonds leads to highly disordered states (see
Supporting Information). In Figure 3 the average number
of 18C6 adducts for blocked and disulfide bound protein are
plotted as a function of charge state. Direct comparison
reveals reduced 18C6 complexation following disulfide
reduction and capping for each protein.
These results can be rationalized by examining potential
competitive intramolecular binding sites. Lymphotactin has
few acidic residues, suggesting that the primary competition for
lysine or arginine side chain binding should originate from
hydrogen bonding with the backbone or side chains. When the
protein is in the folded state, many backbone hydrogen binding
sites will be unavailable due to involvement in secondary
structure. For example, in an α helix, all of the internal amides
are hydrogen bound to each other. It has been demonstrated
previously that loss of secondary structure to random coil
structure can lead to significantly reduced crown attachment
due to competitive hydrogen bonding with the backbone [4]. In
the case of lymphotactin, the reduction in crown binding is
consistent with adoption of a highly disordered state where
disruption of backbone hydrogen bonding leads to competitive
inhibition of 18C6 binding. Comparison of the higher charge
states for the blocked and unblocked structures (i.e., +11 and
+12 in Figure 3) reveals significantly different behavior,
suggesting that the transition structures are likely not entirely
disordered. Similarly, the average binding numbers for
unblocked protein are consistent with results given above and
suggest that the wild type Ltn and W55D proteins access
similar transition state structures, while CC3 does not
(Figure 3d). For the blocked proteins, all three have virtually
Figure 3. The average number of 18C6 adducts is shown as a function of charge state for (a) CC3, (b) wild type Ltn, and (c)
W55D. The protected (disulfide reduced and capped) proteins are shown in dotted lines. (d) All proteins are shown together to
demonstrate relative binding. The key is identical to (a)–(c)

Q. Sun, et al.: Lymphotactin by SNAPP-MS
405
identical average 18C6 binding in the +11 and +12 charge
states (Figure 3d), suggesting absence of any structural differ-
ences for the unfolded, disordered states.
results suggest that out of the three variants examined, wild
type Ltn is most susceptible to temperature denaturation. The
drop in number of 18C6 adducts at lower temperatures is
similar to the trend observed in Figure 3 and suggests cold
denaturation is responsible for the shift in the SNAPP
distribution, rather than some other type of structural shift
(for example skewing the equilibrium in favor of the CC3
structure, which would lead to an increase in 18C6 attach-
ment). It should be remembered that wild type Ltn is the only
form of the protein, which is capable of simultaneously
interchanging between the Ltn-10 and Ltn-40 structures. It is
likely that this ability necessitates that any particular folded
state be in a rather shallow well of stability, making
susceptibility to denaturation more likely.
Another interesting variable, which can produce structural
change and is known to influence lymphotactin, is temperature.
To examine temperature effects, we simply gathered data on
identical solutions at room temperature and with the addition of
an ice pack to the syringe containing the protein solution. The
temperature of the syringe is ~0 °C, although the solution will
warm slightly passing through the line to the electrospray tip.
Results were acquired after 20 min equilibration time and are
shown in Figure 4. Both the CC3 and W55D distributions do
not undergo any compelling shifts in 18C6 adduction after
application of the ice pack. Interestingly, wild type Ltn does
exhibit a decrease in 18C6 binding at lower temperature. It is
therefore clear that temperature induced structural shifts can be
observed by SNAPP. Conversely, merely dropping the temper-
ature does not necessarily influence SNAPP distributions since
the CC3 and W55D distributions remain unchanged. The
The SNAPP experiments reported up to this point were
designed to investigate the solution phase structure of
lymphotactin, even though ultimate detection took place in
the gas phase. It is also possible that information about
solution phase structures may be obtained by actually
Figure 4. SNAPP distributions acquired in water at room temperature and after the addition of an ice pack. Only the wild type
Ltn distribution shifts to any significant extent

406
Q. Sun, et al.: Lymphotactin by SNAPP-MS
examining molecules in the gas phase [19,20]. This would
be possible in at least two scenarios; (1) if the solution phase
structure was retained in the gas phase, direct analysis would
apply, or (2) if differences in the solution phase structures
led to differences in the gas phase structures, it would be
possible to monitor changes in structure. The second
scenario would of course only reveal differences in structure
rather than information about the structures themselves. It is
beyond the scope of the present experiments to attempt to
determine if the solution and gas phase structures for
lymphotactin are the same; however, we did examine the
gas phase structures of the wild type Ltn, CC3, and W55D
proteins to determine if they were distinguishable from each
other. This was accomplished by using IBA-18C6, which is
an 18C6 based molecule with a highly efficient radical
precursor attached. Following photoactivation with 266 nm
light, a radical is generated by dissociation of the carbon–
iodine bond. This radical is subsequently transferred to the
protein. As we have demonstrated previously, radical
migration and consequent dissociation in gaseous protein
ions is largely determined by the overall protein structure
[21]. Therefore, differences in protein structure would be
expected to yield differences in dissociation. Interestingly
with respect to SNAPP, this experiment also potentially
offers information about preferred binding sites for 18C6.
The results from radical directed dissociation of all three
proteins in the +7 charge state are shown in Figure 5. It is clear
that all three dissociation spectra are distinct, although the wild
type Ltn and W55D spectra share similarities. For CC3, there is
an abundant cleavage between N67 and T68, as shown in
Figure 5a. In addition, there is modest dissociation observed
along the remainder of the c-terminal portion of the protein.
Not surprisingly, there is no dissociation observed between the
portions of the protein which are linked by disulfide bonds.
This does not mean that there is no dissociation occurring in the
region, as such dissociation could not be observed unless two
backbone bonds were broken. There is also a very modest
amount of dissociation on the N-terminal side of the disulfide
linked portion, suggesting that indeed some segments of the
protein within the disulfide bonds are likely accessible to the
radical. The wild type Ltn in Figure 5b yields a significantly
different distribution. Dissociation at several residues in close
proximity to Lys76 is abundant, suggesting that this may be a
favorable site for attachment of IBA-18C6. In comparison with
CC3, several peaks towards the C-terminal portion of the protein
are missing. W55D yields results similar to those for wild type
Ltn, but even less dissociation is observed. Importantly, even
though there is an M to V substitution in the region where
dissociation is observed for the W55D mutant (see Scheme 2),
the mutation does not appear to affect dissociation, which is
actually observed at that location in all three proteins.
Figure 5. Dissociation points are shown as a function of
sequence for experiments probing the gas phase structures
of the +7 charge state for (a) CC3, (b) wild type Ltn, and (c)
W55D (the numbering of residues for W55D has been shifted
to be consistent with the other proteins, for actual numbering
see Scheme 2). Differences in dissociation indicate differ-
ences in structure as described in the text
the radical. This suggests that Lys76 is a preferred binding
site for 18C6. Due to the cyclic nature of all three proteins,
which all contain one or more disulfide bonds, it cannot be
determined whether there are other equally preferred binding
sites. In any case, the differences between the dissociation
spectra clearly indicate that the gas phase structures for the
proteins are dissimilar. Since the sequences of these proteins
are all very similar, the differences most likely result from
either retention or memory of the solution phase structures.
For both the Ltn10 and Ltn40 structures, portions of the
C-terminal tail of the protein are largely disordered as
determined by NMR (see italicized residues in Scheme 2).
These sections are therefore absent in the structures shown
Investigation of higher charge states, where tertiary
structure is lost in the gas phase due to Coulombic repulsion,
reveals that most of the dissociation is centered near Lys76.
In the absence of tertiary structure, dissociation is expected
to be most abundant in the vicinity of the starting location of

Q. Sun, et al.: Lymphotactin by SNAPP-MS
407
in Scheme 1, but they are still present and can contribute to
the results which are obtained in SNAPP experiments. These
regions contain several charged residues, which are potential
18C6 targets and the results from RDD suggest that at least
one lysine in this region is a favorable binding site.
Fortunately, SNAPP is capable of extracting structural
information even for proteins which are completely disor-
dered [22]. In the case of lymphotactin, some of the
differences between the observed SNAPP results and those
that would be predicted by the known structures may be
attributable to structural shifts in these disordered regions.
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Conclusions
The conformationally distinct Ltn10 and Ltn40 native states of
lymphotactin can clearly be distinguished and structurally
characterized by SNAPP-MS. The results also suggest that the
distribution of structures occupied by lymphotactin is not
significantly influenced by the addition of methanol or acid. In
contrast, reduction and blocking of the disulfide bonds produce
a denatured protein. Cold temperature experiments suggest that
out of three sequence variants examined, the wild type protein is
the most susceptible to denaturation. With regards to the
structural transition between the two native states of lympho-
tactin, the data reveal that slightly increased 18C6 binding is
observed for the transition structures relative to folded con-
formations. This observation is consistent with a small increase
in intramolecular hydrogen bonding interactions that enhances
the availability of lysine or arginine for 18C6 binding. These
intra-chain interactions may coordinate the transition between
Ltn10 and Ltn40. In the gas phase, the sequence variants of
lymphotactin also adopt distinct structures, as revealed by
radical directed dissociation. It is unclear whether the differences
result from retention of solution phase structure in the gas phase
or some other source. Overall, the results indicate that SNAPP is
a useful method for probing structure in proteins that undergo
slow global conformational transitions.
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(2002)
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folds. J. Chem. Phys. 131, 245105–254106 (2009)
Acknowledgments
The authors wish to thank the NIH for funding this research
(for BFV, grant R01 AI063325, and for RRJ, grant R01
GM084106).
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