Site-specific conversion of cysteine thiols into thiocyanate creates an IR probe for electric fields in proteins

Article abstract of DOI:10.1021/ja0650403

The nitrile stretching mode of the thiocyanate moiety is a nearly ideal probe for measuring the local electric field arising from the organized environment of the interior of a protein. Nitriles were introduced into three proteins: ribonuclease S (RNase S

Full text of DOI:10.1021/ja0650403

Published on Web 09/27/2006
Site-Specific Conversion of Cysteine Thiols into Thiocyanate Creates an IR
Probe for Electric Fields in Proteins
Aaron T. Fafarman, Lauren J. Webb, Jessica I. Chuang, and Steven G. Boxer*
Department of Chemistry, Stanford UniVersity, Stanford, California 94305-5080
Received July 14, 2006; E-mail: sboxer@stanford.edu
In this paper, we develop a straightforward and general method
to introduce the thiocyanate nitrile stretch as a site-specific electric
field probe for proteins. Electrostatics affect nearly all aspects of
protein function, but general, site-specific probes for these fields
are not yet available. The nitrile stretch is a particularly attractive
probe because the frequency is in a relatively uncluttered region
of the IR spectrum (νjC≡N ∼ 2100-2240 cm-1) and is typically
quite intense (ꢀ ∼ 50-1000 M-1 cm-1), and νjC≡N is sensitive to
electric fields, that is, it has a relatively large Stark tuning rate
[∼0.4-1.1 cm-1/(MV/cm)].1 In some cases, it is possible to deliver
the nitrile probe on a substrate or inhibitor,2 and it may prove
possible to introduce nitrile-containing amino acids, such as 4-CN-
Phe, site-specifically into proteins by semi-synthesis or nonsense
suppression,3 but both methods often yield smaller quantities of
modified protein than typically used for biophysical studies and
are not readily compatible with diverse expression systems or multi-
Figure 1. IR absorption (top, left axis) and VSE (red, bottom, right axis)
subunit protein assemblies. Since the introduction of cysteine
residues is used widely as a site-specific labeling strategy (e.g.,
for spin4 or fluorescent labels), we exploit a well-known chemical
modification of cysteine residues to form thiocyanates as a general
method for introducing a very small IR-based probe of protein
electric fields.
spectra for 11 mM RNase S with homocysteine introduced at position 13
and labeled with CN. The VSE spectrum is the field-on minus field-off
difference spectrum obtained at 77 K in a 50/50 (v/v) glycerol/water glass.
The spectrum is scaled to a path length of 1 cm, a concentration of 1 M
and a field of 1 MV/cm.
Scheme 1. Cysteine Cyanylation
The strategy outlined in Scheme 1 for converting cysteine thiols
into thiocyanates5,6 is routinely employed as the first step in the
selective cleavage of peptide bonds at cysteine residues.7 Briefly,
the protein in buffer at pH 7 is reacted with 5,5′-dithiobis(2-
nitrobenzoic acid) (DTNB or Ellman’s reagent8) to form the mixed
protein-thionitrobenzoic acid disulfide (PS-TNB), followed by
displacement by cyanide (CN-), to form the protein-thiocyanate
(PS-CN).9 The electronic absorption of 2-nitro-5-thiobenzoate
(TNB) anion byproduct is conveniently monitored at 412 nm (ꢀ412
) 13 600 M-1 cm-1)8 to follow the course of reaction. We observed
that the PS-CN products in the examples that follow were stable
when stored at 4 °C over 4 days at pH 7,10 consistent with previous
reports.11
We have chosen three very different systems to demonstrate the
versatility and scope of this method: modification of S-peptide
bound to the ribonuclease S-protein (RNase S),12 human aldose
reductase (hALR2), which has multiple cysteine residues and for
which a number of CN-containing inhibitors related to diabetes
control are available,2,13 and the bacterial photosynthetic reaction
center (RC), which is a multi-subunit, integral membrane protein
containing many prosthetic groups.
homocysteine substituted at methionine 13, and the unique thiol of
the peptide was labeled according to Scheme 1 (see Supporting
Information for details) and combined with S-protein to form the
labeled RNase S complex. Formation of the complex was observed
by the shift of νjC≡N from 2161.2 cm-1 (fwhm ) 11.4 cm-1, ꢀ )
120 M-1 cm-1) for free, labeled S-peptide in buffer solution to
2155.4 cm-1 (fwhm ) 8.0 cm-1, ꢀ ) 130 M-1 cm-1) for the
complex. By analyzing the VSE spectrum, the Stark tuning
rate14,15,19 was determined to be 0.7 cm-1/(MV/cm), comparable
to the value observed in simpler model compounds in organic
glasses.1 Both ꢀ and the Stark tuning rate are quite large,
demonstrating the utility of thiocyanate as a probe.
The thiocyanate electric field probe was introduced into reactive
cysteine residues of the globular protein hALR2. hALR2 has seven
cysteines, none of which participates in disulfide bonds.20 Cysteine
298, which lies in the active site of the enzyme,20,21 has been shown
to be particularly reactive toward cysteine-thiol-modifying re-
agents.21,22 When reacted with 1.1 equiv of DTNB for 10 min, 1
equiv of TNB was released, and after displacement by cyanide,
the LC-MS determined that the mass was 26 Da higher than that
of the unmodified protein. The FTIR spectrum of the labeled protein
(Figure 2, red) exhibited a narrow peak at 2159.4 cm-1 with
The sensitivity of a vibrational frequency to an electric field is
calibrated by vibrational Stark effect (VSE) spectroscopy.14-16
Thiocyanate was introduced into RNase S and the VSE spectrum
recorded (Figure 1). RNase S is a noncovalent complex between
residues 1-20 (S-peptide) and 21-124 (S-protein) of bovine
ribonuclease A12 and is an extensively studied vehicle for the
introduction of non-natural biophysical probes into proteins.17,18 The
S-peptide was prepared by solid-phase peptide synthesis with
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C O M M U N I C A T I O N S
Figure 3. Room temperature IR spectra for the a Rb. capsulatus
photosynthetic reaction center mutant with a unique cysteine [I(L150)C/
C(L92/98/108/246/247)A] labeled with -S12CN (black) or -S13CN (red).
Spectra are for samples of approximately equal concentration.
Figure 2. Room temperature IR absorption spectra for hALR2 in a 1:1
complex with the CN-containing inhibitor shown (2232 cm-1) and bearing
one (red) or multiple (about 2-3 cysteines labeled, see text, black)
thiocyanate labels (2160 cm-1 region). Both spectra are scaled to the
absorption of the nitrile group in the inhibitor.
Acknowledgment. We thank Ian Suydam for his material and
intellectual input, and Professor Alberto Podjarny (Strasbourg) for
providing the gene for hALR2. L.J.W. is supported by an NRSA
postdoctoral fellowship 1F32GM076833-01, and J.I.C. by a Hertz
Graduate Fellowship. This work was supported in part by grants
from the NIH and the NSF Chemistry Division.
approximately equal intensity, but very different peak position, to
a nitrile on a bound inhibitor which makes a 1:1 complex with the
protein (see Supporting Information and Figure 2). It is possible to
modify additional labile cysteines by driving the reaction with the
addition of 10 mM DTNB over 4 h, after which we measured
between 2.2 and 2.8 total equivalents of TNB released.23 The
integrated area in the 2160 cm-1 region of multiply labeled hALR2
is between 2 and 3 times that of the singly labeled protein
(normalized to the inhibitor as an internal standard). Multiply
labeled hALR2 shows peaks at 2151.3 and 2161.3 cm-1, the latter
presumably the sum of contributions from the first labeled peak
centered at 2159.4 cm-1 with another shifted to slightly higher
energy. Importantly, this higher energy vibration contributing to
the 2161.3 cm-1 peak and the peak at 2151.3 cm-1 both report
environments in the enzyme that are distinct from that of the most
reactive site. Detailed assignments based on removal of individual
cysteines will be reported elsewhere.
In a final demonstration of the utility of this method, we
employed Scheme 1 to label the RC of Rb. capsulatus. Native
cysteines were removed, and a single cysteine replaced Ile(L150),
a moderately buried residue located on a short helix on the RC
periplasmic surface. Following successive reactions with DTNB
and cyanide for 8-16 h each, the overall labeling efficiency was
estimated at 60% (see Supporting Information). As shown in Figure
3, narrow peaks were observed at 2159 cm-1 for -S12CN and 2110
cm-1 for -S13CN with approximately equal intensities for identi-
cally treated RC samples, consistent with homogeneous, site-specific
labeling. The isotope shift (νjC≡N ) 49 cm-1) is as expected; this
simple isotope labeling strategy is especially useful for confirming
that small peaks are real in the presence of large backgrounds or
baseline offsets as often occurs with aqueous protein samples.
We have demonstrated the versatility of a simple reaction scheme
to introduce very small, stable nitrile electric field probes into
proteins with three very diverse systems. For each system, work is
underway to measure electric fields and changes that occur during
function, and these will be reported in full publications.
Supporting Information Available: Experimental procedures and
product characterization. This material is available free of charge via
the Internet at http://pubs.acs.org.
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