Cyanylated Cysteine: A Covalently Attached Vibrational Probe of Protein-Lipid Contacts.

Abstract

Cyanylated cysteine, or beta-thiocyanatoalanine, is an artificial amino acid that can be introduced into peptides and proteins by post-translational chemical modification of solvent-exposed cysteine side chains, and thus it can be used in any protein with a suitable expression and mutagenesis system. In this study, cyanylated cysteine is introduced at selected sites in two model peptides that have been shown to bind to membrane interfaces: a membrane-binding sequence of the human myelin basic protein and the antimicrobial peptide CM15. Far-UV circular dichroism indicates that the secondary structures of the bound peptides are not influenced by introduction of the artificial side chain. Infrared spectra of both systems in buffer and exposed to dodecylphosphocholine micelles indicate that the CN stretching absorption band of cyanylated cysteine can clearly distinguish between membrane burial and solvent exposure of the artificial side chain. Since infrared spectroscopy can be applied in a wide variety of lipid systems, and since cyanylated cysteine can be introduced into proteins of arbitrary size via mutagenesis and post-translational modification, this new probe could see wide use in characterizing the protein-lipid interactions of membrane proteins.

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Published on Web Date: February 12, 2010
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Cyanylated Cysteine: A Covalently Attached Vibrational
Probe of Protein-Lipid Contacts
Heather A. McMahon, Katherine N. Alfieri, Katherine A. A. Clark, and Casey H. Londergan*
Department of Chemistry, Haverford College, 370 Lancaster Avenue, Haverford, Pennsylvania 19041
ABSTRACT
acid that can be introduced into peptides and proteins by post-translational chemical
modificationofsolvent-exposedcysteinesidechains,andthusitcanbeusedinany
protein with a suitable expression and mutagenesis system. In this study, cyany-
lated cysteine is introduced at selected sites in two model peptides that have been
shown to bind to membrane interfaces: a membrane-binding sequence of the
human myelin basic protein and the antimicrobial peptide CM15. Far-UV circular
dichroism indicates that the secondary structures of the bound peptides are not
influenced by introduction of the artificial side chain. Infrared spectra of both
systems in buffer and exposed to dodecylphosphocholine micelles indicate that
the CN stretching absorption band of cyanylated cysteine can clearly distinguish
between membrane burial and solvent exposure of the artificial side chain. Since
infrared spectroscopy can be applied in a wide variety of lipid systems, and since
cyanylated cysteine can be introduced into proteins of arbitrary size via mutagen-
esis and post-translational modification, this new probe could see wide use in
characterizing the protein-lipid interactions of membrane proteins.
Cyanylated cysteine, or β-thiocyanatoalanine, is an artificial amino
SECTIONBiophysical Chemistry
T
ment, despitemanyrecentexperimentaladvancesinthe iso-
lation and structural characterization of membrane proteins.
A site-specific probe of such interactions with fast time
resolution could be used broadly to characterize theinterplay
between membrane proteins and their often heterogeneous
environments. X-ray crystallography has successfully assi-
gned the structure of many membrane proteins crystallized
with help from added surfactants and mesophase-based
crystallizationtechniques,1buttheseexcellenthigh-resolution
structures are static and limited to their artificial crystalline
environments. Techniques for characterizing membrane-
protein interfacial geometry and interactions include nuclear
magnetic resonance (NMR), electron paramagnetic reso-
nance site-directed spin labeling (EPR-SDSL), fluorescent
probes such as the naturallyoccurring tryptophan side chain,
and several recently popularized vibrational probes. New
model lipid and surfactant systems, as well as techniques
for orienting samples preferentially versus the applied field,
have led to many advances in membrane protein structure
determination via NMR;2a quite promising development is
the recent extension of solid-state NMR techniques to mem-
brane proteins bound to larger and slower-moving lipid
particles than the artificial micelles and bicelles needed
for conventional solution NMR experiments.3In particular,
for short membrane-bound peptides and proteins, aligned
bilayer samples spun at the magic angle have provided
he dynamic, atom-level interactions that occur bet-
weenmembrane-boundproteinsandtheirnativelipid
systemsaretypicallynotwell-characterizedbyexperi-
excellent structural resolution of antimicrobial peptide
sequences in contact with lipids of varying composition, with
solution exposure or burial depth left to either paramagnetic
relaxationmeasurementsorthrough-spacemagneticinterac-
tions with magnetically active lipid nuclei.4However, there
remains no single NMR experiment that is universally appli-
cable to membrane proteins, with each particular system
requiring a different sample preparation and usually multiple
instrumental approaches.
Natural lipid systems often form very large (100s of
nanometers or larger) objects with complicated and hetero-
geneous phase behavior: site-specific optical probes that
report on variations in either the peptide or lipid structure at
theirinterfacemightfindmoregeneraluseindeterminingthe
role that proteins play in modulating and organizing this
heterogeneous phase behavior. Tryptophan fluorescence5
has been used widely to note the extent of membrane burial
of single native Trp side chains. However, in membrane-
boundproteinswithmultipleornotryptophansintheirnative
sequence, the protein-membrane interface may be signi-
ficantlydisruptedbymutationsrequiredtoaddorremoveTrp
side chains or other artificial fluorophores. Since there are
oftennowavelength-dependentfluorescencespectralproper-
ties that vary with membrane burial, the main fluorescence
Received Date: January 6, 2010
Accepted Date: February 5, 2010

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quantitythatistypicallyusedtocalculatethedepthofburialis
the extent of quenching,6which must be modulated by
introducing or excluding third-party quenching agents in
either aqueous or lipid phases. EPR-SDSL is also able to
measure the extent of membrane burial of a covalently
attachedspinlabel,similarlybyitsinteractionwiththird-party
magnetically active species introduced artificially in either
phase.7The nanosecond-time scale sensitivity of EPR spin
labels is a widely used probe of site-specific protein folding
dynamics;8however, the dynamics of the lipid interface can
range over many other orders of magnitude, including the
faster picosecond time scale directly accessible to vibrational
spectroscopy.
Nitrile-containing artificial amino acids such as p-cyano-
phenylalanine (PheCN)9and 5-cyano-tryptophan10have
been shown to be quite sensitive probes of site-specific
protein-lipid contacts using infrared spectroscopy. However,
their use is restricted (with limited exceptions11) to syntheti-
cally tractable peptides containing suitable mutation sites for
the larger phenylalanine and tryptophan side chains. For
C?N-containing side chains, the CN stretching peak occurs
in a relatively isolated region of the IR spectrum (usually
between 2100 and 2300 cm-1), so a more universally in-
corporable amino acid containing this vibrational chromo-
phore might see wide use in membrane proteins.
β-Thiocyanato-alanine, or cyanylated cysteine (C*) is a
CtN-containing amino acid that can be introduced using
site-selective chemical modification at cysteine, thus it could
be more easily incorporated into larger and more diverse
proteinsthanthepreviouslymentionedartificialaminoacids.
The CN stretching band of aliphatic thiocyanate has a quali-
tatively similar, yet quantitatively quite different, response to
its environment12,13comparedto theCCN-containing probes
mentioned above. Although the -SCN group is apparently
able to accept very weak and nonspecific hydrogen bonds at
the N atom according to density functional theory (DFT)
calculations,14solvent-dependent studies have shown that
aliphatic SCN does not form well-defined 1:1 hydrogen-
bonded complexes with any functional groups that naturally
occurinproteinsorlipids,thusmakingC*arelativelypassive
probeofitsenvironment.ComparedtoPheCN,thefrequency
variation of C* in response to environmental changes is
less dramatic, but this is balanced by its smaller size and
its possible incorporation into a much wider variety of pro-
teins via post-translational modification rather than peptide
synthesis.
The incorporation of C* in proteins follows a similar
strategyusedinEPR-SDSL,namely,site-directedmutagenesis
ofasingle-cysteinemutanttoaproteinofinterestfollowedby
covalent chemical addition of the probe moiety at the free
cysteine thiol group.15However, in the context of membrane
proteins, cyanylated cysteine has two distinct advantages
over other covalently attached EPR or fluorescent probes:
its remarkablysmallsizeversus other probesand its abilityto
report membrane exposure of specific sites without its pre-
sence of third-party quenching or paramagnetic species. This
study was designed to demonstrate using two different
well-characterized systems that the CN stretching band of
C* is in fact sensitive to the formation of site-specific mem-
brane contacts in a diagnostic way.
To examine the sensitivity of C* to membrane-interface
binding, two model peptides with single-site mutations to C*
were chosen (see Table 1 for sequences). The first peptide is
aminoacids81-95ofhumanmyelinbasicprotein(MBP),16a
membrane-binding domain whose bound orientation has
been previously characterized by EPR-SDSL17and NMR18
studies and whose release from the membrane presents an
antigenictarget.MBP16isoneexampleofaproteinassociated
with disease pathogenesis that has important membrane-
bindingandmechanicalactivity.ThesecondpeptideisCM15,
whose bound orientation versus bacterially derived mem-
branes and detergent micelles has also been studied pre-
viously by EPR-SDSL and NMR techniques.19CM15, an
artificial hybrid of the N-termini of cecropin A and melittin,
is the shortest potent antimicrobial peptide sequence. It
disrupts and reorganizes membranes through an unknown
mechanism that begins with its binding to the membrane
surface.19Each of these two peptides is unstructured in
solution and helical when bound to lipids. In each peptide,
wehavechosentwocentrallylocatedresiduestoreplacewith
C*, one of which is known to be solvent-exposed, and one of
which isburied inthe lipidinterface. Forbestagreement with
thepreviousstudiesthatemployedNMRandEPRtechniques,
the lipid system used here is dodecylphosphocholine (DPC)
micelles. It is important to keep in mind, however, that future
use ofC*asa probe ofmembrane binding will not be limited
to micelles or other such “artificial” lipid systems. Indeed,
given its ability to avoid complexation with natural hydrogen
bond donors, it is expected that C* should be useful in
complex lipid systems containing hydrogen bond donors
such as cholesterol and sphingolipids without strong pertur-
bation of the lipid media by the probe and possible spectral
discrimination of phase segregation by the probe's IR spec-
trum.Thelocalstructureanddynamicsofmorecomplexlipid
media will be the subject of future work; the purpose of the
presentstudyistoevaluatethesensitivityofC*tomembrane
bindinginanotherwisewell-understoodcontext.Duetotheir
short length, the peptides studied here were constructed via
solid-phase peptide synthesis; but in a general sense, it is
important to note that the C* probe is not in any way
restricted to use in short, synthetic peptides.
The probe CN group was introduced into the CM15 and
MBPsingle-cysteinemutantsbystandardmodificationchem-
istry(seebelow)withnear-unityyieldsinallcases.Sincethese
are partially amphipathic peptides, their aqueous solubi-
lities are limited to ∼1 mM or less. Each of these peptides is
also strongly cationic at neutral pH, so substantial buffer
Table 1. Sequences of Model Peptides, Where C* = Cyanylated
Cysteine
CM15
CM15 A10C*
CM15 I8C*
MBP 81-95
MBP N89C*
MBP F87C*
Ac-KWKLFKKIGAVLKVL-NH2
Ac-KWKLFKKIGC*VLKVL-NH2
Ac-KWKLFKKC*GAVLKVL-NH2
Ac-NPVVHFFKNIVTPRT-NH2
Ac-NPVVHFFKC*IVTPRT-NH2
Ac-NPVVHFC*KNIVTPRT-NH2
C* solvent-exposed
C* buried
C* solvent-exposed
C* buried

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concentrations were used to prevent the concentrated pep-
tides from raising their own local pH outside the window in
which the probe group is stable (pH 6.0-8.0 according to
previous work as well as ours).
Far-UV circular dichroism (CD) was used to verify that
mutation and cyanylation did not perturb the helical second-
ary structure of the peptides' DPC-bound states. Figure 1
presents far-UV CD spectra in aqueous buffer solution and
in the presence of 100 mM DPC for all six peptides. For each
mutant peptide, substitution of C* led to no observable
changes in the global secondary structure of the DPC-bound
peptidesascomparedtotheC*-freepeptide.Interestingly,the
aqueousstructuresofthepeptidesdidchangetosomeextent,
indicatingthatthemutationtoC*didintroducestericchanges
that biased the unstructured ensemble toward a less random-
coil-likeaveragestructure.However,thehelicalpropensityofeach
peptide in its bound form is unaltered with the addition of C*.
Previous studies on the solvent dependence of aliphatic
thiocyanate's CN stretching band indicate that its frequency
depends on the dipolar character and hydrogen bonding
ability of the solvent.12The CN stretching band of methyl
thiocyanate in solvents relevant to this work (most of which
were not included in the original solvent study) is shown in
Figure2.Theprobevibrationexhibitsaredshiftof4-5cm-1
versusitsaqueouspositioninless-hydrogenbondingsolvents
with the exception of alkanes, which are not able to stabilize
the charge distribution of the non-H-bonded -SCN moiety.
The CN line width narrows considerably in tetrahydrofuran
(THF) and alkane solvents compared to other more dipolar
solvents, likely due to a smaller inhomogeneous frequency
distribution imposed by the dipoles of surrounding solvent
molecules. If THF is the best solvent model of the membrane
interface (largely alkane but with substantial dipole variations),
thentheexpectationisthataC*sidechain'sstretchingvibration
should red-shift by 4-5 cm-1when buried in the membrane
interface versus its frequency when solvent-exposed. The band
might also narrow due to dipolar and/or dynamical differences
betweenaqueousandlipidenvironments.Thebandwidthofthis
particular vibration was shown previously to be particularly
sensitive to picosecond time scale solvent dynamics.12
The CN stretching band of each cyanylated peptide was
recorded under the same aqueous and DPC-exposed condi-
tions as the CD spectra. Peptide concentrations were limited
by solubility for the aqueous samples and by maintaining a
suitably large lipid:peptide ratio for the DPC samples, and
since the CN stretching band of SCN is weaker than for CCN,
the approximate optical density of the CN band is approxi-
mately 100 μOD for each cyanylated sample. Figure 3 com-
pares the infrared spectrum in the CN stretching region for
each peptide in aqueous buffer versus DPC micelle solutions.
CN stretching bands for the MBP mutants (Figure 3a,b)
show that there is a distinct difference between the mem-
brane-buried versus the solvent-exposed C*. As shown in
Figure3a,therearenosignificantchangestothelinewidthor
central peak frequency between the solvent-exposed N89C*
probe in buffer and that in DPC micelles. However, the
membrane-buried F87C*, shown in Figure 2d, exhibits a
red shift of ∼4 cm-1(from 2163 to 2159 cm-1) as well as a
substantial narrowing in the presence of the lipid.
ForCM15,inFigure3c,thereisnosignificantchangeinthe
line width or central peak frequency between the solvent-
exposed A10C* probe in buffer versus DPC. However, the CN
stretchingbandofthemembrane-buriedI8C*proberedshifts
by ∼4 cm-1with DPC (Figure 3d) from 2163 to 2159 cm-1.
The observed CN stretching red shift of about 4 cm-1for
thepresumablymicelle-buriedprobegroupsisinquantitative
Figure 1. Far-UV CD spectra of all peptides in phosphate buffer
and DPC-bound conditions: (a) native-sequence MBP 81-95, (b)
MBPN89C*,(c)MBPF87C*,(d)unlabeledCM15,(e)CM15A10C*,
(f) CM15 I8C*. Note: since the MBP sequence does not contain a
good UV chromophore for concentration determination, scaled
raw ellipticities of samples are presented, and shape comparisons
are most useful.
Figure 2. Solvent dependence of the CN stretching infrared
absorption band of methyl thiocyanate in solvents relevant to
membrane binding studies.

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agreement with a THF-like environment when compared to
the spectra of MeSCN. A recent study of C* inserted into the
aqueous, water-soluble alanine peptides (AAAAK)5observed
a red shift of approximately 1.5 cm-1upon the formation of
helicalsecondarystructure.20However,thisredshiftwasfrom
2164.5 to 2163.0 cm-1and accompanied a decrease in the
temperature ofthe sample; a similar temperature-dependent
shiftwasalsoobservedforMeSCNoverthesametemperature
range. In the aqueous helical peptides, formation of helical
structure did correspond with a change in the observed CN
stretchingbandwidth.However,inthecaseofthemembrane-
bound peptides investigated here, the CN stretching band of
C* is observed well outside the frequency window of any CN
stretching band observed for water-exposed C*, thus we can
say that the red shift is unmistakably due to burial in a
nonaqueous and non-H-bonding environment.
In two different peptide systems with different neigh-
boring side chains and different physiological effects on
natural membranes, the C* probe is shown here to be
sensitive to lipid burial and reports the orientation of the
peptide at the membrane interface. The difference in line
width between the two buried side chains (Figure 3b,d)
suggests the possibility of depth and/or dynamic differ-
ences at the two peptide-micelle interfaces. This differ-
ence is interesting, given the contrasting natural roles of
these two peptide sequences: MBP serves to stabilize and
solidify the myelin sheath, at least partly due to lipid
interactions, and CM15's toxicity derives from its ability
to permeabilize bacterial membranes. Recent detailed
NMR experiments by Zangger et al.21suggest that CM15
isnearlycompletelyburiedinDPCmicelles;thisisnotquite
what is suggested by our results for the A10C* mutant of
CM15, in which the CN band of the C* probe is nearly
indistinguishable from that of A10C* in aqueous solution.
This may be due to fact that the CN stretching band is
sensitive simply to the presence of water in its local
environment, rather than to the artificially introduced
solution-phase paramagnetic species that are needed to
reveal solvent exposure in NMR experiments. Solid-state
NMR measurements of oriented samples that explicitly
take into account membrane-peptide interactions via
peptide-lipid nuclear Overhauser effects (NOEs) have
recently been able to quantify membrane burial depth
dependence in some antimicrobial peptides better than
paramagnetic exposure.22It is our hope that our approach
might in time also lead to clear quantitation of burial depth
in membrane-bound proteins of arbitrary size.
Future experiments will address each peptide in the
context of its natural lipid system, with variability in the
linewidthandfrequencyofburiedlabelsexpectedtoreport
on local membrane dynamics and speciation near the
peptides.ThepriorobservationthataliphaticSCNisweakly
sensitive to H-bonding groups presents the intriguing possi-
bilitythatburied,covalentlyattachedSCNgroupscouldbeused
to document the presence of H-bonding physiological bilayer
componentssuchassterolsnearmembraneproteins.Fornow,
these results indicate that the cyanylated cysteine side chain
shouldbeasensitiveandwidelyincorporablevibrationalprobe
of membrane binding in membrane proteins.
EXPERIMENTAL METHODS
Materials. Fmoc-labeled amino acids and all peptide syn-
thesis reagents were purchased from Advanced Chem Tech,
exceptforN,N-diisopropylethylamine(DIEA;Pharmco-AAPER)
and acetic anhydride (Aldrich). All peptide cleavage reagents
Figure 3. CN stretching absorption bands for cyanylated peptides in aqueous solution and DPC micelles: (a) MBP N89C*, (b) MBP F87C*,
(c) CM15 A10C*, (d) CM15 I8C*. Intensities are scaled to the peak intensity for comparison; all optical densities are between 200 and 400 μOD.

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were purchased from Aldrich, and high-performance liquid
chromatography (HPLC) solvents were purchased from
Pharmco-AAPER. 5,50-Dithiobis(2-nitrobenzoic acid) (DTNB)
was purchased from Acros and D,L-dithiothreitol (DTT) and
NaCN were purchased form Aldrich. Methyl thiocyanate was
purchasedfromAcros.Allpurchaseditemsabovewereusedas
received. DPC was purchased from Avanti Polar Lipids dis-
solved in chloroform. All aqueous samples were constituted
using doubly deionized, Milli-Q quality water.
PeptideSynthesisandAnalysis.Allpeptidesweresynthe-
sized on an Applied Biosystems API 443A synthesizer using
standard fmoc chemistry with an HBTU/HOBt activation cock-
tail. All branched amino acids were double-coupled. The PAL
resinwasusedtofurnishaC-terminalcarboxamideoncleavage;
treatmentoftheresinboundpeptideswithaceticanhydridewas
used for N-acetylation. Purity and identity of peptides was veri-
fied by reverse-phase HPLC using an analytical-scale Dynamax
C18 column and matrix-assisted laser desorption/ionization
mass spectrometry (MALDI-MS; performed at the Wistar Insti-
tute, Philadelphia, PA). Since all peptides were nearly (95% or
greater) pure following peptide cleavage according to analytical
HPLC and MALDI analysis, preparative-scale HPLC was not
performed.
CyanylationofPeptides.Lyophilizedcysteine-containing
peptide from cleavage was treated for 20 min in 0.01 M
HCl solution and relyophilized to remove residual TFA. The
peptide was weighed after lyophilization, dissolved in
50 mM phosphate buffer, pH 7.0-7.5, and treated with
100? DTT to ensure the free thiol. Residual DTT and any
other small impurities were separated from the peptide
using a 28 cm column of Sephadex G-10, and the reduced
peptide was lyophilized. The peptide was redissolved in
50 mM phosphate buffer (CM15 peptides) or 20 mM phos-
phate (MBP peptides), pH 6.5-7.0, and treated with 5?
DTNBdissolvedin200mMphosphatebuffer,pH6.5-7.0,to
form a mixed disulfide at cysteine. In limited cases, absor-
bance of the sample was monitored at 412 nm to observe
releaseofthethionitrobenzoateanionandquantifytheyield
of the mixed disulfide; yield for all cyanylated peptides was
near unity when determined. The DTNB-adduct sample
was then treated with 50? NaCN, and the cyanylated
peptide was isolated using the same 28 cm Sephadex G-10
columnequilibratedwith50mM (CM15peptides) or20mM
(MBP peptides) sodium phosphate buffer, pH 6.5-7.0. Pep-
tide-containing fractions were concentrated about 5? using
a Speed Vac centrifugal vacuum device, and the presence of
the CtN label was verified using infrared spectroscopy. The
final concentrated sample concentration was 1-2 mM pep-
tide in 100-200 mM phosphate buffer, pH 6.5-7.0.
Lipid-Containing Samples. DPC was dried from chloro-
form by evaporating the solvent under a steady stream of
nitrogen. Dynamic light scattering using a DynaPro (Wyatt
Technologies,SantaBarbara,CA)ina1.5mmquartzcuvette
(Helma) indicated a uniform micelle size distribution of
∼2 nm. To obtain uniform micelle size, samples were
extruded15timesusingamini-extruder(AvantiPolarLipids,
Alabaster, AL). Speed-vac'ed peptide samples from above
were mixed with the dried lipid to yield samples that were
approximately 2 mM in peptide and 100 mM in DPC in a
200 mM phosphate buffer.
Far-UV CD. CDspectrawerecollectedfrom185to260nm
using an Aviv model 410 spectropolarimeter. Peptide samples
at1-2mMpeptideconcentration(and100-200mMsodium
phosphatebuffer) wereanalyzedina0.1mmquartzdemoun-
table cell (Starna Cells).
Infrared Spectroscopy. Cyanylated peptide samples
(in buffer or 100 mM DPC) were placed between the
windows of a 22 μm CaF2BioCell (BioTools, Jupiter, FL)
placed inside a BioJack temperature-circulating jacket held
at25?C.Allspectrawerecollectedat2cm-1resolutionusing
a Bruker Optics Vertex 70 FTIR spectrometer with a photo-
voltaic HgCdTe detector. A spectrum of buffer solution or
100 mM DPC in buffer solution was subtracted from the
raw spectrum, and further baseline correction was accomp-
lishedbyfittingthebaseline(outsidetheregionfrom2145to
2185 cm-1) to a polynomial and subtracting the fit.
AUTHOR INFORMATION
Corresponding Author:
*To whom correspondence should be addressed. E-mail: clonderg@
haverford.edu.
ACKNOWLEDGMENT We gratefully acknowledge the groups of
Karin Akerfeldt and Robert Fairman at Haverford College for help
withpeptidesynthesisandcirculardichroism.C.H.L.acknowledges
the Camille and Henry Dreyfus Foundation for a New Faculty Start-
Up Award, the Research Corporation for a Cottrell College Science
Award, and the National Institute of General Medical Sciences for
AREA Grant R15GM088749. K.N.A. thanks the Howard Hughes
Medical Institute fora summer fellowship. This content is solely the
responsibility of the authors and does not necessarily represent the
official views of any of the funding agencies above.
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