Selective incorporation of nitrile-based infrared probes into proteins via cysteine alkylation

Article abstract of DOI:10.1021/bi101711a

The nitrile stretching vibration is increasingly used as a sensitive infrared probe of local protein environments. However, site-specific incorporation of a nitrile moiety into proteins is difficult. Here we show that various aromatic nitriles can be easily incorporated into peptides and proteins via either thiol alkylation or arylation reaction.

Full text of DOI:10.1021/bi101711a

10354 Biochemistry 2010, 49, 10354–10356
DOI: 10.1021/bi101711a
Selective Incorporation of Nitrile-Based Infrared Probes into Proteins via
Cysteine Alkylation†
Hyunil Jo,§, Robert M. Culik,§, Ivan V. Korendovych,§ William F. DeGrado,*,§,‡ and Feng Gai*,‡
§
‡Department of Chemistry and Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia,
Pennsylvania 19104, United States. These authors contributed equally to this work.
Received October 23, 2010; Revised Manuscript Received November 12, 2010
ABSTRACT: The nitrile stretching vibration is increasingly
used as a sensitive infrared probe of local protein environ-
ments. However, site-specific incorporation of a nitrile
moiety into proteins is difficult. Here we show that various
aromatic nitriles can be easily incorporated into peptides
and proteins via either thiol alkylation or arylation reaction.
tives shows an approximately 3 cm-1 shift to a higher energy as
compared to that of the aryl derivatives). Because any interac-
tions that decrease or increase the electron density of the CtN
bond will result in an increase or decrease, respectively, in the
CtN stretching vibrational frequency of nitriles (33), these
results can be understood qualitatively in the context of the effect
of an activating substituent on the cyanobenzyl ring (i.e., sulfur
vs methylene and para vs ortho position with respect to the nitrile
group). Furthermore, in comparison with those obtained in
water, the CtN stretching bands of these probes in tetrahydro-
furan (THF) show a 7-8 cm-1 shift toward a lower frequency
and also a concomitant decrease in the bandwidth by a factor of
approximately 2 (Table 1), demonstrating the potential utility of
these aromatic nitriles as local environmental probes.
Moreover, it is interesting to note that in aqueous solution the
CtN stretching bandwidths of 2 and 4 are noticeably larger than
those of 1 and 3. This finding is consistent with the study of
Waegele et al. (34), which showed that the CtN stretching vibra-
tion of an aromatic nitrile can be influenced by direct interactions
between the nitrile group and solvent molecules and also indi-
rectly by the solvation status of the aromatic ring. In other words,
the larger bandwidth of 2 and 4 arises most likely from their
asymmetric molecular shape (with respect to the nitrile group),
which leads to a more heterogeneous solvation of the respective
aromatic ring and hence a broader vibrational transition.
Considering the fact that the synthesis of 1 and 2 involves
conditions much milder [e.g., the reaction solution contains only
a small amount of organic solvent, and similar conditions have
been used in chemical modification of proteins (35)] than those
used in the synthesis of 3 and 4 and that the extinction coefficient
of the CtN stretching vibration of 1 and 3 is about one order
of magnitude larger than that of 2 and 4 (Figure S1 of the
Supporting Information), only probe 1 is used in the subsequent
proof-of-principle tests involving peptides and proteins.
First, the method of cysteine alkylation is applied to two
cysteine mutants of mastoparan-X (MpX), W3C and A8C. These
mutants are chosen because upon association with calmodulin
(CaM) the side chains of Trp3 and Ala8 of MpX are known to
situate inside the peptide-protein binding groove and, as a result,
become less solvent-exposed (36). It is found that both peptides
are efficiently labeled by p-cyanobenzyl bromide with >80%
yield (determined by LC-MS) under the conditions specified in
Scheme 1 (the corresponding nitrile-containing peptides are
hereafter termed W3C-CN and A8C-CN). The FTIR spectra
of W3C-CN (Figure 1) and A8C-CN (Figure S2 of the Support-
ing Information) also support the site-specific incorporation of
probe 1 into these peptides, as their CtN stretching bands in
The CtN (nitrile) stretching vibration has recently emerged
as a valuable infrared (IR) probe of the conformation and local
environment of biological molecules (1-26) because of its sen-
sitivity to various factors (13, 14, 17), such as local electric field
and hydrogen bonding interactions (27). For example, it has been
used to probe insertion of a peptide into membranes (6), protein-
ligand interactions (25), and the dehydration status of an anti-
microbial peptide encapsulated in reverse micelles (8). For chem-
ically synthesizable peptides, site-specific incorporation of a nitrile
group can be readily achieved via the use of nitrile-derivatized
non-natural amino acids, such as cyanoalanine (AlaCN) and
p-cyanophenylalanine (PheCN). For proteins that cannot be
chemically synthesized, however, selective incorporation of a
nitrile moiety is rather difficult. Currently, only the chemical
method developed by Boxer and co-workers is available, which
directly converts a cysteine thiol into a thiocyanate (7). Addi-
tionally, it has been shown that PheCN can be incorporated into
proteins by using an orthogonal tRNA-synthetase pair (28, 29),
but the techniques involved are time-intensive and available to
only a handful of laboratories worldwide. Thus, it would be quite
helpful to develop an alternative and easier method for selective
incorporation of different nitrile moieties into proteins. Here, we
show that cysteine alkylation and arylation reactions under mild
conditions can be used for such a purpose.
We tested the feasibility of the proposed method on four
cyanobenzyl derivatives (Scheme 1), based on the consideration
that the oscillator strength and stark tuning rate of aromatic
nitriles are normally larger than those of alkyl nitriles (3, 4).
As shown (Scheme 1), these model probes can be quite easily
attached to the cysteine side chain via either thiol alkylation or
arylation (30-32). Similar to that of PheCN (3), the CtN
stretching frequency of these nitrile derivatives in water is found
to be in the range of 2233-2241 cm-1 (Table 1 and Figure S1 of
the Supporting Information), with the exact value depending
on the molecular structure (e.g., the band of the benzylic deriva-
†We thank the National Institutes of Health (GM-065978 and
GM008275) and the National Science Foundation (DMR05-20020)
for funding.
*To whom correspondence should be addressed. Telephone: (215)
573-6256. Fax: (215) 573-2112. E-mail: wdegrado@mail.med.upenn.
edu (W.F.D.) or gai@sas.upenn.edu (F.G.).
r
pubs.acs.org/Biochemistry
Published on Web 11/15/2010
2010 American Chemical Society

Rapid Report
Biochemistry, Vol. 49, No. 49, 2010 10355
Scheme 1
Table 1: Band Positions (ν), Full Widths at Half-Maximum (Δν), and
Estimated Molar Extinction Coefficients (ε) of the CtN Stretching Vibra-
tion of Various Probes in H2O and THF
F
IGURE 2: CtNstretchingbandofthenitrile-labeledhumancalmod-
ulin-like protein [in 50 mM HEPES buffer (pH 7.4)] in the absence
(O) and presence (4) of Ca2þ (30 mM). The protein concentrations
were approximately 1-2 mM (blue) and 200 μM (red), respectively.
The solid lines are respective fits of these data to a Lorentzian
function, and the thin dashed line indicates the peak position of the
nitrile band of probe 1 in water.
1
2
3
4
ν (cm-1, H2O)
Δν (cm-1, H2O)
ν (cm-1, THF)
Δν (cm-1, THF)
2236.6
11.8
2240.3
14.4
2233.7
11.2
2237.9
16.4
2228.5
7.4
2232.3
7.8
2226.8
6.1
2229.6
7.1
the reaction product confirms the successful incorporation of
probe 1 into the protein of interest, and the corresponding yield
was estimated to be >50%. More importantly, the far-UV CD
spectrum of the nitrile-labeled CALM3 is almost identical to that
of the parent protein (Figure S3 of the Supporting Information),
indicating that the labeling reaction does not change the struc-
tural integrity of the protein in question. The latter notion is
further corroborated by the dependence of the bandwidth of the
CtN stretching vibration on Ca2þ. As indicated (Figure 2), upon
addition of Ca2þ, the bandwidth of the IR transition is decreased
from ∼19 to ∼11 cm-1, a phenomenon expected to occur as Ca2þ
is known to rigidify the calcium-binding domain (37, 38) where
the labeled cysteine is located. In addition, the peak position of
this nitrile band (ν =2236.0 cm-1) does not change upon addi-
tion of Ca2þ, which is consistent with the fact that the labeled
cysteine residue is exposed to solvent in CaM structures deter-
mined in the absence and presence of Ca2þ (38).
ε (M-1 cm-1
)
210 ( 60
22 ( 10
240 ( 60
24 ( 10
In conclusion, we have demonstrated a post-translational
method allowing site-specific incorporation of nitrile-based IR
probes into peptides and proteins via cysteine alkylation or aryla-
tion. Because this method involves relatively routine and mild
reaction conditions, we expect that it will find wide application in
biophysical studies of proteins.
F
IGURE 1: CtN stretching bands of W3C-CN obtained in the pre-
sence (O) and absence (4) of CaM [50 mM HEPES buffer (pH 7.4)
and 30 mM CaCl2]. In the former case, the concentrations of W3C-
CN and CaM were estimated to be 1-2 mM. Lines are respective
fits of these data to a Lorentzian function with the following param-
eters: for W3C-CN, ν =2236.5 cm-1 and Δν =10.7 cm-1, and for
SUPPORTING INFORMATION AVAILABLE
Materials, methods, and FTIR spectra. This material is
available free of charge via the Internet at http://pubs.acs.org.
the W3C-CN-CaM complex, ν = 2231.2 cm-1 and Δν = 11.7 cm-1
.
aqueous solution are centered at ∼2236.5 cm-1 but shift to lower
wavenumbers upon binding to CaM, as expected. In addition, the
nitrile bandwidth of the free peptide is found to be slightly
narrower than that of the peptide-CaM complex, likely because
both bound and unbound peptides exist in the complex solution.
A similar finding was also observed in a previous study (3).
Second, we tested the applicability of the cysteine alkylation
reaction to proteins by applying it to human calmodulin-like
protein CALM3, which contains a unique cysteine residue (Sup-
porting Information). As shown (Figure 2), the IR spectrum of
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