Direct visualization of disulfide bonds through diselenide proxies using77Se NMR spectroscopy

Article abstract of DOI:10.1002/anie.200905206

Chemical Equation Presentation Se-ing is believing: Many proteins are cross-braced by disulfide bonds that frequently play key roles in protein structure, folding, and function. Unfortunately, the methods available for assignment of disulfide-bond connect

Full text of DOI:10.1002/anie.200905206

Communications
DOI: 10.1002/anie.200905206
Protein Structures
Direct Visualization of Disulfide Bonds through Diselenide Proxies
Using 77Se NMR Spectroscopy**
Mehdi Mobli, Aline Dantas de Araffljo, Lynette K. Lambert, Gregory K. Pierens,
Monique J. Windley, Graham M. Nicholson, Paul F. Alewood, and Glenn F. King*
Disulfide bridges are a common posttranslational modifica-
tion in proteins that result from the formation of a covalent
bond between the thiol groups of two cysteine residues.
Disulfide bridges are important for directing and stabilizing
the three-dimensional (3D) structure of proteins[1] and they
are often functionally critical.[2,3] Incorrect pairing of disulfide
bonds typically leads to a nonnative 3D fold accompanied by
a loss of protein function. The importance of this structural
motif is highlighted by the fact that 24% of the circa 53000
protein structures deposited in the Protein Data Bank (PDB)
contain at least one disulfide bond.
Disulfide bonds are highly prevalent in three important
classes of secreted proteins: peptide hormones, growth
factors, and peptide toxins excreted in animal venoms.[4]
NMR spectroscopy is an ideal technique for determining
the 3D structure of these small proteins; of the 4285 structures
available in the PDB for proteins smaller than 8 kDa, 71%
were determined using NMR methods. However, this raises a
conundrum—NMR spectroscopy is a poor method for
establishing disulfide-bond connectivities owing to the
absence of a sulfur isotope with favorable NMR properties.
In some cases, dipolar interactions between the b-methylene
protons of covalently linked cysteine residues can be used to
infer disulfide bridges,[5] but these connectivities are often
ambiguous when multiple disulfide bridges are present.[6,7]
Thus, disulfide bonds often constitute a “blind spot” in NMR
structural analyses.
is a genetically encoded amino acid residue that is identical to
cysteine except for the replacement of sulfur with sele-
nium.[8,9] The human selenoproteome comprises 25 seleno-
proteins.[10] Sec residues can be introduced into proteins by
chemical synthesis[11–14] or recombinant expression,[15,16] and
replacement of disulfide bonds with diselenide bonds has
been demonstrated previously not to alter protein structure or
function.[13,14,17]
With a natural abundance of 7.6%, 77Se is an NMR-active
s = 1/2 nucleus that provides a route for direct determination
of disulfide bridge connectivities using NMR scalar couplings.
We demonstrate this approach using a 37-residue spider toxin
(k-ACTX-Hv1c) that contains four disulfide bonds, including
a rare and functionally critical vicinal disulfide bridge
between the adjacent cysteine residues Cys13 and Cys14.[2]
Since recombinant expression would lead to replacement of
all Cys residues with Sec, for the sake of simplicity we used
solid-phase peptide synthesis to produce a toxin in which only
Cys13 and Cys14 were replaced with Sec residues (see the
Supporting Information).
We determined the 3D solution structure of the
[Sec13,Sec14] toxin using homonuclear NMR methods (see
the Supporting Information) and found it to be equivalent to
that of the native toxin; the two structures can be super-
imposed over the well-ordered region (residues 1–34) with a
backbone root-mean-square deviation (rmsd) of 0.91 ꢀ
(Figure 1a). k-ACTX-Hv1c is a high-affinity blocker of
calcium-activated potassium (KCa) channels in insects, and
the vicinal disulfide bond is a key component of the channel
binding site.[2,18,19] Figure 1b shows that the [Sec13,Sec14]
variant is equipotent with the native toxin in blocking KCa
currents in cockroach neurons (see the Supporting Informa-
tion). Thus, replacing the functionally critical vicinal disulfide
bridge in k-ACTX-Hv1c with a vicinal diselenide bond does
not affect either the folding or function of the protein.
Figure 2 shows that the diselenide connectivities in the
[Sec13,Sec14] toxin (which serve as a proxy for the disulfide
connectivities) can be unequivocally determined from scalar
couplings present in a 2D 1H–77Se heteronuclear multiple
bond correlation (HMBC) experiment. The two intense cross-
peaks in Figure 2b result from large intraresidue two-bond
couplings (ca. 35 Hz) between each pair of Sec methylene
protons and the 77Se nucleus of the same residue. The two less
intense but clearly visible cross-peaks arise from interresidue
three-bond couplings across the diselenide bond between
methylene protons and 77Se. This latter coupling, although
small (ca. 2 Hz), allows unequivocal assignment of the
diselenide bond connectivity.
One possible solution to this “disulfide blind spot” would
be to replace the NMR-inactive 32S nucleus with a nucleus of
similar chemical, but more favorable magnetic, properties.
Such a replacement occurs naturally, albeit infrequently.
Selenocysteine (Sec), often referred to as the 21st amino acid,
[*] Dr. M. Mobli, Dr. A. D. de Araffljo, Prof. P. F. Alewood, Prof. G. F. King
Institute for Molecular Bioscience, The University of Queensland
St. Lucia QLD 4072 (Australia)
Fax: (+61)7-3346-2101
E-mail: glenn.king@imb.uq.edu.au
Homepage: http://www.imb.uq.edu.au
Dr. L. K. Lambert, Dr. G. K. Pierens
Centre for Magnetic Resonance, The University of Queensland
St. Lucia QLD 4072 (Australia)
M. J. Windley, Prof. G. M. Nicholson
Department of Medical and Molecular Biosciences
University of Technology, Sydney (Australia)
[**] This work was supported by Discovery Grants DP0774245 and
DP0878450 from the Australian Research Council and the
Queensland Smart State Research Facilities Fund.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200905206.
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Angewandte
Chemie
Figure 2. Direct determination of disulfide-bond connectivities using
77Se NMR spectroscopy. a) 3D structure of the [Sec13,Sec14] analogue
of k-ACTX-Hv1c.[2] The three disulfide bonds and single diselenide
bond are shown as green and red tubes, respectively. A magnified
version of the vicinal diselenide bridge is shown on the right with the
NMR-active nuclei labeled. b) 1H–77Se HMBC, c) 1H–77Se HMQC, and
d) 77Se–77Se COSY spectra of the [Sec13, Sec14] analogue acquired at
500 MHz. The spectrum shown in (b) was acquired using a natural-
abundance sample, whereas the spectra shown in (c) and (d) were
acquired using a sample containing 77Se-enriched Sec residues. See
the Supporting Information for experimental details.
Figure 1. Structure and function of a [Sec13,Sec14] analogue of the
spider toxin k-ACTX-Hv1c. a) Stereoview of the 3D structure of the
[Sec13,Sec14] analogue determined in the current study (green back-
bone with orange disulfide/diselenide bonds) superimposed on the
previously determined structure of the native toxin (blue backbone
with red disulfide bonds; PDB file 1L0). The lowest-energy structure in
each ensemble was used for the overlay. The vicinal disulfide/disele-
nide bond is highlighted with an asterisk. b) Concentration-depend-
ence of the blocking of KCa currents in cockroach dorsal unpaired
median (DUM) neurons by native k-ACTX-Hv1c (green circle) and the
[Sec13,Sec14] analogue (blue circle). The calculated IC50 values were
3.5Æ1.2 nm and 1.5Æ0.3 nm, respectively. See the Supporting Infor-
mation for experimental details.
disulfide bridges in much the same way that the study of
hydrogen bonds in proteins has been stimulated by the
development of methods for their direct observation using
NMR spectroscopy.[22,23]
Received: September 17, 2009
Published online: November 4, 2009
Additional experiments applicable to larger proteins
become possible if the cysteine residues are replaced with
Sec enriched with 77Se. This involves incorporation of 77Se-
enriched metallic selenium into the appropriate selenocys-
teine framework for subsequent use in peptide synthesis or
recombinant expression. We synthesized a version of k-
ACTX-Hv1c in which Cys13 and Cys14 were replaced with
Sec residues enriched with 98% 77Se (see the Supporting
Information). This allowed direct observation of diselenide
bonds through one-bond 77Se–77Se scalar couplings in a two-
dimensional 77Se–77Se COSY experiment (Figure 2d). The
connected Sec residues were then assigned by a simple 2D
heteronuclear multiple quantum coherence (HMQC) experi-
ment that allowed each 77Se nucleus to be correlated with the
b-methylene protons of the same Sec residue (Figure 2c).
This NMR approach is the first method to allow direct
observation of disulfide bridges in native proteins in solution.
Although the methodology described here is ideally
suited to synthetic peptides, it is equally applicable to larger
proteins produced using recombinant methods.[20] Sec incor-
poration can be achieved by growing cysteine auxotrophs of
E. coli[15,16] or yeast[21] in media enriched with Sec. We
anticipate that this new approach will stimulate the study of
Keywords: disulfide bonds · NMR spectroscopy · peptides ·
.
selenium · solid-phase synthesis
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