Selective peptide bond hydrolysis of cysteine peptides in the presence of Ni(II) ions

Article abstract of DOI:10.1016/j.jinorgbio.2010.09.003

Recently, we described a sequence-specific R1-(Ser/Thr) peptide bond hydrolysis reaction in peptides of a general sequence R1-(Ser/Thr)-Xaa-His-Zaa- R, which occurs in the presence of Ni(II) ions [A. Kr??el, E. Kopera, A. M. Protas, A. Wys?ouch-Cieszyńska, J. Poznański, W. Bal, J. Am. Chem. Soc. 132 (2010) 3355-3366]. In this study we explored the possibility of substituting the Ser/Thr and the His residues, necessary for the reaction to occur according to the Ni(II)-assisted acyl shift reaction mechanism, with Cys residues. We tested this concept by synthesizing three homologous peptides: R1-Ser-Arg-Cys-Trp-R2, R1-Cys-Arg-His-Trp-R2, and R1-Cys-Arg-Cys-Trp-R2, and the R1-Ser-Arg-His-Trp-R2 peptide as comparator (R1 and R2 were CH3CO-Gly-Ala and Lys-Phe-Leu-NH2, respectively). We studied their hydrolysis in the presence of Ni(II) ions, under anaerobic conditions and in the presence of TCEP as a thiol group antioxidant. We measured hydrolysis rates using HPLC and identified products of reaction using electrospray mass spectrometry. Potentiometry and UV-vis spectroscopy were used to assess Ni(II) complexation. We demonstrated that Ni(II) is not compatible with the Cys substitution of the Ser/Thr acyl acceptor residue, but the substitution of the Ni(II) binding His residue with a Cys yields a peptide susceptible to Ni(II)-related hydrolysis. The relatively high activity of the R1-Ser-Arg-Cys-Trp-R2 peptide at pH 7.0 suggests that this peptide and its Cys-containing analogs might be useful in practical applications of Ni(II)-dependent peptide bond hydrolysis.

Full text of DOI:10.1016/j.jinorgbio.2010.09.003

Journal of Inorganic Biochemistry 105 (2011) 10–16
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Selective peptide bond hydrolysis of cysteine peptides in the presence of Ni(II) ions
Anna Maria Protas a, Arkadiusz Bonna a, Edyta Kopera a, Wojciech Bal a,b,
⁎
a
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland
b
Central Institute for Labour Protection – National Research Institute, Czerniakowska 16, 00-701 Warsaw, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 June 2010
Received in revised form 16 September 2010
Accepted 20 September 2010
Available online 29 September 2010
Recently, we described a sequence-specific R1-(Ser/Thr) peptide bond hydrolysis reaction in peptides of a
general sequence R1–(Ser/Thr)–Xaa–His–Zaa–R, which occurs in the presence of Ni(II) ions [A. Krężel, E.
Kopera, A. M. Protas, A. Wysłouch-Cieszyńska, J. Poznański, W. Bal, J. Am. Chem. Soc. 132 (2010) 3355–3366].
In this study we explored the possibility of substituting the Ser/Thr and the His residues, necessary for the
reaction to occur according to the Ni(II)-assisted acyl shift reaction mechanism, with Cys residues. We tested
this concept by synthesizing three homologous peptides: R1-Ser-Arg-Cys-Trp-R2, R1-Cys-Arg-His-Trp-R2,
and R1-Cys-Arg-Cys-Trp-R2, and the R1-Ser-Arg-His-Trp-R2 peptide as comparator (R1 and R2 were CH3CO-
Gly-Ala and Lys-Phe-Leu-NH2, respectively). We studied their hydrolysis in the presence of Ni(II) ions, under
anaerobic conditions and in the presence of TCEP as a thiol group antioxidant. We measured hydrolysis rates
using HPLC and identified products of reaction using electrospray mass spectrometry. Potentiometry and UV–
vis spectroscopy were used to assess Ni(II) complexation. We demonstrated that Ni(II) is not compatible with
the Cys substitution of the Ser/Thr acyl acceptor residue, but the substitution of the Ni(II) binding His residue
with a Cys yields a peptide susceptible to Ni(II)-related hydrolysis. The relatively high activity of the R1-Ser-
Arg-Cys-Trp-R2 peptide at pH 7.0 suggests that this peptide and its Cys-containing analogs might be useful in
practical applications of Ni(II)-dependent peptide bond hydrolysis.
Keywords:
Peptide bond hydrolysis
Ni(II) complexes
© 2010 Elsevier Inc. All rights reserved.
1. Introduction
Gly-Ala and Lys-Phe-Leu-NH2, respectively [14]. We indicated R1-
SRHW-R2 as the sequence most susceptible to hydrolysis in the
Nowadays, there is a growing demand for new methods of
sequence-specific cleavage of the peptide bond. Reagents for cleavage
of the peptide bond used currently in research and biotechnological
manufacturing (e.g. new peptide and protein drugs) include natural
or engineered proteolytic enzymes [1], self-cleaving intein sequences
[2], and chemical agents [3]. Because of specific disadvantages of those
methods, alternative agents for sequence-specific cleavage of pep-
tides are searched for. Many metal ions and metal complexes were
found to promote or catalyse the hydrolysis of peptide bonds in short
peptides [4–6]. Cu(II) ions and metal complexes were also found to
cleave the –Lys226–Thr227– peptide bond in the human IgG1 [7] and to
fragment bovine serum albumin (BSA) [8,9].
We found that some peptides undergo a selective peptide bond
hydrolysis in the presence of Ni(II) ions [10–13]. In a recent paper we
described our systematic studies of this reaction. Using a combina-
torial library of R1-(Ser/Thr)-Xaa-His-Zaa-R2 peptides, where Xaa
residues included seventeen common α-amino acids (except of Asp,
Glu, and Cys), Zaa residues included nineteen common α-amino acids
(except of Cys), and invariable terminal chains R1 and R2 were CH3CO-
presence of Ni(II) ions. Studying a series of corresponding R1-(Ser/
Thr)-Xaa-His-Zaa-R2 peptides synthesized individually we documen-
ted the molecular mechanism of this reaction. It proceeds in a low-
spin 4 N Ni(II) complex, containing the Ni(II) ion anchored by the His
imidazole nitrogen and three preceding amide nitrogens. The Ser/Thr
hydroxyl group acts as a nucleophile accepting the N–O acyl shift of
the oxygen atom of R1-Ser/Thr peptide bond. The resulting ester is
hydrolyzed spontaneously, completing the reaction [15]. A similar
mechanism of sequence-specific peptide bond cleavage is used by
Nature (e.g. Hedgehog protein, intein system) as the first step of the
protein splicing process [2,16]. In this case, however, a Cys thiol,
rather than a Ser/Thr hydroxyl is a preferred acyl group acceptor, and
the thioester is the intermediate hydrolysis product. We therefore
hypothesized that R1-Cys-Xaa-His-Zaa-R2 peptides could provide an
alternative target for Ni(II)-dependent peptide bond hydrolysis.
Cys thiol groups can anchor Ni(II) ions to peptides similarly to His
imidazoles [17]. Therefore, we also hypothesized that a R1-(Ser/Thr)-
Xaa-Cys-Zaa-R2 peptides might be susceptible to Ni(II)-dependent
peptide bond hydrolysis.
To test these hypotheses, we synthesized three novel homologous
peptides: R1-Ser-Arg-Cys-Trp-R2, R1-Cys-Arg-His-Trp-R2, and R1-Cys-
Arg-Cys-Trp-R2, and studied their hydrolysis in the presence of Ni(II)
ions, under anaerobic conditions and in the presence of TCEP as a thiol
group antioxidant. We measured hydrolysis rates using HPLC and
⁎
Corresponding author. Institute of Biochemistry and Biophysics, Polish Academy of
Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland. Tel.: +48 22 592 2346; fax: +48 22
659 4636.
E-mail address: wbal@ibb.waw.pl (W. Bal).
0162-0134/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.jinorgbio.2010.09.003

A.M. Protas et al. / Journal of Inorganic Biochemistry 105 (2011) 10–16
11
identified products of reaction using electrospray mass spectrometry.
Potentiometry and UV–vis spectroscopy were used to assess Ni(II)
complexation. The results of these studies are presented below.
analytical column (5 mm particle size, 4.6×250 mm), respectively.
The mobile buffer consisted of (A) 0.1% TFA in water and (B) 0.1% TFA
in 90% acetonitrile in water. The pure lyophilized peptides were
characterized on a Premier ESI-Q-TOF MS (Waters), exhibiting correct
molecular masses.
2. Experimental
2.1. Peptide synthesis
2.2. Potentiometry
Starting materials. All N-α-9-fluorenylmethyloxycarbonyl amino
acids were purchased from Novabiochem. Trifluoroacetic acid (TFA),
piperidine, N-hydroxybenzotriazole (HOBt), N-[(1H-benzotriazol-1-yl)
(dimethylamino)methylene]-N-methylmethanaminium tetrafluorobo-
rate N-oxide (TBTU), chloranil, 1,2-ethanedithiol (EDT), triisopropylsi-
lane (TIS) and trifluoroacetic acid (TFA) were purchased from Fluka AG.
Acetonitrile (HPLC grade) was purchased from POCh. Acetic anhydride
was purchased from Merck. Dimethylformamide (DMF) was obtained
from Roth. TentaGel S RAM resin was obtained from Rapp Polymere
GmbH.
The peptides, R1-Ser-Arg-Cys-Trp-R2, R1-Cys-Arg-His-Trp-R2, R1-
Cys-Arg-Cys-Trp-R2, and R1-Ser-Arg-His-Trp-R2 (R1 denotes CH3CO-
Gly-Ala, and R2 denotes Lys-Phe-Leu-NH2) were synthesized manu-
ally by the solid-phase method using the Fmoc/But procedure [18].
The side chain protecting groups of the amino acids were Boc for Lys
and Trp, Pbf for Arg and trityl for Cys and His. All peptides were
synthesized according to the following procedure: (i) 2×5 min
deprotection steps using 20% piperidine in DMF; (ii) the coupling
reactions carried out with the protected amino acid diluted in a DMF
in the presence of 1% Triton using TBTU as a coupling reagent treated
with DIPEA and HOBt (Fmoc-AA:TBTU:DIPEA:HOBt; 5:4,9:10:5) for
1 h. The completeness of each coupling reaction was monitored by the
chloranil test. The acetylation of the N-termini of peptides was
obtained using 10% acetic anhydride in DCM. The protected peptidyl
resins were treated with cleavage cocktail – a mixture of 94% TFA, 1%
TIS, 2.5% water and 2.5% EDT – for 2.5 h. The cleaved peptides were
precipitated with cold diethyl ether and lyophilized.
Potentiometric titrations of R1-SRCW-R2 were performed on a
MOLSPIN automatic titrator, using InLab 422 combined glass-Ag/AgCl
clectrodes (Mettler-Toledo), which were calibrated daily by nitric acid
titrations [19]. 0.1 M NaOH (carbon dioxide free) was used as titrant.
Sample volumes of 1.5–2 ml were used. The samples contained 1.2–
1.5 mM peptide, dissolved in 4 mM HNO3/96 mM KNO3. The Ni(II)
complexformation wasstudied using a 5–10% excess of the peptideover
Ni(II). The pH range for all potentiometric experiments was from 2.4 to
11.8. All experiments were performed under argon at 25 °C. The data
were analyzed using the SUPERQUAD and the HYPERQUAD software
[20,21]. Three to five titrations were included simultaneously into
calculations, separately for protonation and Ni(II) complexation.
2.3. UV–vis spectroscopy
The spectra were recorded at 25 °C on a Cary 50-Bio spectropho-
tometer (Varian) over the spectral range of 300–1100 nm, using a
1 cm cuvette. The samples contained 0.95 mM peptide, 0.95 mM
TCEP, and no or 0.90 mM Ni2+. The samples were titrated with NaOH
by careful manual additions of very small amounts of the concen-
trated base solution. Changes of pH were monitored with a combined
glass-Ag/AgCl electrode (InLab 422, Mettler-Toledo) over the range of
2.4–11.4 pH units.
2.4. HPLC measurements of hydrolysis rates
The samples, incubated in a TB-941U thermoblock (IWElectronic,
Warsaw, Poland) at 60.0 0.1 °C, contained 1 mM peptide, 1 mM
TCEP (Tris(2-carboxyethyl)phosphine) and 2.5 mM Ni(II) in a 20 mM
Tris buffer at pH 8.2 or in a 20 mM Hepes buffer at pH 7.0. The
The peptides were purified and analyzed by HPLC using an
Empower system (Waters) equipped with a Vydac semi-preparative
column (5 mm particle size, 10×250 mm) and an ACE 5 C18-300
Fig. 1. HPLC chromatograms of 1 mM R1-SRCW-R2 peptide incubated in 20 mM Tris buffer in the presence of 1 mM TCEP without/with 2.5 mM Ni(II) at 60 °C and pH 8.2. S — substrate;
P — product, IP — intermediate product, D — dimer, * — product of decomposition.

12
A.M. Protas et al. / Journal of Inorganic Biochemistry 105 (2011) 10–16
Fig. 2. HPLC chromatograms of 1 mM R1-CRHW-R2 peptide incubated in 20 mM Tris buffer in the presence of 1 mM TCEP without/with 2.5 mM Ni(II) at 60 °C and pH 8.2. S — substrate;
D — dimer.
hydrolysis of air-sensitive Cys-containing peptides was performed
under a low-oxygen atmosphere (0.5–2% O2 in N2, Coy anaerobic
chamber). In all cases, the 20 μL aliquots of incubated samples were
periodically removed from reaction mixtures (200 μL initially). 50 μL
of 5% (v/v) TFA in water was added to aliquots in order to stop the
hydrolysis reaction. These samples were stored at 4 °C before the
injection to an Empower HPLC system, equipped with an autosampler
and a dual wavelength detector, set to 220 nm and 280 nm. An
analytical C18 column (ACE, 250×4.6 mm) was used. The eluting
solvent A was 0.1% (v/v) TFA in water and solvent B was 0.1% (v/v)
TFA in 90% (v/v) acetonitrile. In a typical analysis 30 μL of the sample
acidified with TFA was diluted with 180 μL of water and placed in a
glass insert in the autosampler carrousel. 150 μL of such sample was
injected automatically into the HPLC system. The substrate and
hydrolysis products were separated in a linear gradient of 20–40% B in
20 min at the flow rate of 1 mL/min. The peaks were identified on a
Premier ESI-MS.
Pseudo-1st order kinetic constants were calculated using Microcal
Origin v. 8.0 Pro (OriginLab), by integrating HPLC peaks, summing up
final and intermediate products, and substrate monomers with
Fig. 3. HPLC chromatograms of 1 mM R1-CRCW-R2 peptide incubated in 20 mM Tris buffer in the presence of 2 mM TCEP without/with 2.5 mM Ni(II) at 60 °C and pH 8.2. S — substrate,
* — product of decomposition.

A.M. Protas et al. / Journal of Inorganic Biochemistry 105 (2011) 10–16
13
dimers, where appropriate and fitting the data, expressed as fractions
of the total to the exponential first-order rate equation.
3. Results
3.1. Screening of hydrolytic reactivity of Cys peptides
The conditions of the selective hydrolysis of the R1-SRCW-R2, R1-
CRHW-R2 and R1-CRCW-R2 peptides (one letter amino acid codes are
used here and below for brevity) were based on those applied
previously for the R1-SRHW-R2 peptide, used in this study as a
comparator [14,15]. The peptides (1 mM) were incubated at 60 °C in a
20 mM Tris buffer at pH 8.2, in the presence of TCEP thiol protector,
used at a 1:1 molar ratio per peptide Cys thiol and with Ni(II) ions
(2.5 mM). Preliminary experiments demonstrated that a higher
excess of TCEP caused peptide decomposition under these conditions.
HPLC was used to separate and quantitate reaction products, which
were identified using ESI-MS. Figs. 1–3 present chromatograms of
reaction mixtures containing R1-SRCW-R2, R1-CRHW-R2, and R1-
CRCW-R2, respectively, in the absence and presence of Ni(II) ions at
times 0 and 24 h. The chromatogram of R1-SRCW-R2 recorded after
3 h of incubation with Ni(II) is shown additionally in Fig. 1. These
results show clearly that the hydrolysis occurred only in the R1-
SRCW-R2 peptide incubated with Ni(II), and that only disulfide dimers
and other oxidation products were formed in two other cases.
Similarly to previous studies [14,15], the reaction with R1-SRCW-R2
proceeded in two major steps. This is indicated by the presence of the
intermediate product, whose molecular mass is identical with the
substrate, but the HPLC retention time is different. The formation of a
disulfide dimer was also observed for this peptide, but it was a minor
product, at a level of up to 15%. The N-terminal hydrolysis product, R1-
OH did not adsorb to the HPLC column and left the system within the
solvent front. It was therefore omitted from the analysis. Our previous
experience demonstrated that, due its very low absorbance at 220 nm
this omission did not affect the accuracy of quantitative analysis [14,15].
Fig. 4. A. The species distribution for the R1-SRCW-R2 peptide (0.95 mM) and Ni(II) ions
(0.9 mM) at 25 °C, with values of A449 (■) and A580 (○) overlaid. The dashed line
represents the sum of all Ni(II) complexes. B. The UV–vis spectra of R1-SRCW-R2
complexes (source data for A449 and A580 in Panel A). Numbers in the box denote pH
values.
3.2. Potentiometric and UV–vis study of Ni(II) complex formation by
R1-SRCW-R2
band at 345 nm, confirming the participation of the thiol group in the
coordination [17,22].
Following the screening results, we studied protonation and Ni(II)
complex formation of the R1-SRCW-R2 peptide. The constants
obtained by potentiometric titrations are presented in Table 1, and
Fig. 4A shows the complex species distribution, along with the
corresponding UV–vis spectra (Fig. 4B). At weakly acidic conditions
(pH 5–7) a minor NiHL complex was formed, presumably by the
monodentate coordination to the thiol residue. The pH profile of the
UV–vis spectra suggests that this species is low-spin. The next species,
a definitely low-spin NiH−2L, was formed above pH 7, as an effect of a
concerted deprotonation of three amide nitrogens. The final species,
NiH−3L, resulted from the deprotonation of amine nitrogen Lys side
chain at alkaline pH. The pKa of the low-spin complex formation was
calculated using the spectroscopic data (λmax at 449 nm and 580 nm).
We also recorded the UV–vis spectra of R1-SRCW-R2, R1-CRCW-R2
and their Ni(II) complexes at pH 8.2 (Fig. 5). The spectrum of the R1-
SRCW-R2 complex similar to that of the R1-CRHW-R2 complex is
characteristic of a low-spin complex with sulfur coordination, as
indicated by a broad charge transfer feature around 300 nm. This
conclusion was additionally confirmed by a slow decay of this band
Its value, 7.34 0.13 was very similar to the pKa for the NiH−2
L
species, 7.33 0.06, corresponding to the major contribution of
NiH−2L to the pH profile of low-spin complex formation (see
Fig. 4A and B). All these species exhibited a Ni(II)-S charge transfer
Table 1
Logarithmic protonation and Ni(II) complex formation constants for R1-SRCW-R2
peptide determined at 25 °C and I=0.1 M (KNO3).
log β
HL
H2L
8.77 0.11
16.29 0.14
NiHL
NiH−2
NiH−3
11.85 0.15
−10.15 0.04
−18.82 0.06
Fig. 5. The UV–vis spectra of 0.45 mM R1-CRHW-R2 (solid grey line) and R1-CRCW-R2
(dotted grey line) peptides and Ni(II) complexes of R1-CRHW-R2 (solid black line) and
R1-CRCW-R2 (dotted black line) peptides at pH 8.2.
L
L

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A.M. Protas et al. / Journal of Inorganic Biochemistry 105 (2011) 10–16
Table 2
A comparison of pseudo-1st order rate constants of R1-SRHW-R2 and R1-SRCW-R2
peptides at pH 7.0 and 8.2.
pH
7.0
8.2
R1-SRHW-R2
R1-SRCW-R2
Ratio
(7.1 1.1)×10−5
(3.9 0.3)×10−5
1.82
(46.4 2.5)×10−5
(11.0 0.8)×10−5
4.22
upon exposure to ambient air. The high-intensity broad features of the
spectrum of the R1-CRCW-R2 complex, due to a strong mixing of Ni(II)
and S electronic levels, are typical for multiple sulfur coordination,
and therefore indicate the involvement of both Cys residues in Ni(II)
binding [23]. This band also decayed slowly upon exposure to air.
Fig. 7. The changes in the concentration of the product of hydrolysis (SRCW-R2 peptide)
during the reaction at pH 8.2 (■ — experimental data, line — pseudo-1st order rate law
fit) and pH 7.0 (○ — experimental data, line — pseudo-1st order rate law fit) at 60 °C.
3.3. Detailed study of R1-SRCW-R2 hydrolysis
The above experiments indicated that the hydrolytically active
low-spin complex of R1-SRCW-R2 might have been formed at a lower
pH compared to that of R1-SRHW-R2 studied by us previously [15].
Therefore, we compared the hydrolysis rates of these two peptides at
a standard pH 8.2, in a 20 mM Tris buffer, and at a lower pH, 7.0, in a
20 mM Hepes buffer. For both, the reactions were performed at 60 °C,
under low-oxygen conditions, and in the presence of equimolar TCEP
(1 mM). Both peptides underwent hydrolysis. As expected, the
reaction at pH 7.0 was slower than at pH 8.2 (Table 2), but
qualitatively its course was similar at both pH values (see Fig. 6 for
examples of chromatograms for R1-SRCW-R2). The yield of the final
product for R1-SRCW-R2 was lower at pH 7.0 (60%) compared to pH
8.2 (85%) (Fig. 7), because of the more extensive formation of a
disulfide dimer enabled by longer reaction times, which likely caused
TCEP deactivation.
general terms in Scheme 1. The first step involves the formation of a
square-planar complex anchored at a His residue side chain, and
involving three preceding amide nitrogens. The last of them belongs
to a Ser/Thr residue, thus enabling an acyl shift, yielding an ester
intermediate product, which, in turn undergoes spontaneous
hydrolysis, completing the cleavage. As outlined in the Introduction,
we hypothesized that a Cys residue might substitute for a Ser/Thr
residue, to form a thioester, or for a His residue, providing an
alternative anchor for the Ni(II) ion. The first of these options did not
work. We observed no hydrolysis in R1-CRHW-R2 and R1-CRCW-R2
peptides. In the absence of Ni(II) these peptides were eventually
oxidized by traces of oxygen, but the presence of Ni(II) prevented
oxidation (Figs. 2 and 3). The UV–vis spectra (Fig. 5), together with
these facts, demonstrated that Ni(II) was bonded to these Cys
residues in the course of incubation, and this prevented both the
oxidation and the formation of the hydrolytically productive
complex, analogous to that presented in Scheme 1.
4. Discussion
The molecular mechanism of the Ni(II)-dependent sequence-
selective peptide bond hydrolysis in R1-(Ser/Thr)-Xaa-His-Zaa-R2
peptides consists of three essential steps [15]. It is depicted in
Fig. 6. HPLC chromatograms of 1 mM R1-SRCW-R2 peptide incubated in 20 mM Hepes buffer in the presence of 1 mM TCEP without/with 2.5 mM Ni(II)at 60 °C and pH 7.0. S — substrate;
P — product, IP — intermediate product, D — dimer, # — impurity.

A.M. Protas et al. / Journal of Inorganic Biochemistry 105 (2011) 10–16
15
Scheme 1. Major species present in the process of Ni(II)-dependent peptide bond hydrolysis [14,15]: the substrate (S), the intermediate product (IP), the final product (P).
On the other hand, R1-SRCW-R2, underwent hydrolysis according
to the mechanism outlined in Scheme 1. In this case the Cys residue
replaced the His residue, forming the initial square-planar complex, as
presented in Scheme 2. The further steps of the reaction proceeded
according to Scheme 1, as indicated by the presence of the
intermediate ester, and the pseudo-1st order reaction kinetics
(Figs. 6 and 7).
The R1-SRCW-R2 peptide reacted more slowly than R1-SRHW-R2
both under pH 8.2 and 7.0 (Table 2). However, at pH 7.0 the ratio of
the respective rates was much lower than at pH 8.2. At a given pH
value, the rate in His-containing peptides was a product of the
maximal rate and the relative concentration of the productive square-
planar complex. A much lower pH of formation of such complex with
the R1-SRCW-R2 peptide (7.33 vs. 8.52 [15]) was therefore most likely
responsible for this effect. One has to note that the R1-SRHW-R2
sequence was selected on the basis of a large scale peptide library
screening, and that the variable residues selected (R and W) are not
necessarily optimal for the Cys analog. Therefore, the susceptibility of
the R1-SRCW-R2 peptide to hydrolysis at pH 7.0 is very promising and
warrants further studies aimed at finding hydrolyzable sequences for
biotechnological applications.
5. Conclusion
The molecular mechanism of Ni(II)-dependent peptide bond
hydrolysis, described by us previously for histidine-containing
peptides [14,15] is akin to that of intein molecular rearrangement
[2]. Following this analogy, in this work we aimed at finding out
whether this analogy could be extended over cysteine-containing
peptides. The results presented above demonstrated that Ni(II),
serving largely as a conformation inducing factor in our system is
not compatible with the Cys thiol as acyl group acceptor. On the other
hand, we demonstrated that another Cys residue is a sufficiently
effective site for anchoring Ni(II) at the peptide in a hydrolytically
productive conformation. The relatively high activity of the Cys-
containing peptide at pH 7.0 is suggests that its analogs might be
useful in practical applications of Ni(II)-dependent peptide bond
hydrolysis.
Abbreviations
BSA
bovine serum albumin
dichloromethane
N,N-diisopropylethylamine, or Hünig's base
dimethylformamide
DCM
DIPEA
DMF
EDT
1,2-ethanedithiol
ESI-MS electrospray ionization mass spectrometry or electrospray
mass spectrometry
Hepes
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid or 2-
[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid
nitric acid
N-hydroxybenzotriazol
high-performance liquid chromatography or high-pressure
liquid chromatography
HNO3
HOBt
HPLC
KNO3
NaOH
TBTU
potassium nitrate
sodium hydroxide
N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-
methylmethanaminium tetrafluoroborate N-oxide
Tris(2-carboxyethyl)phosphine
Scheme 2. The substrate complex in the Ni(II)-dependent peptide bond hydrolysis of
the R1-SRCW-R2 peptide.
TCEP

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A.M. Protas et al. / Journal of Inorganic Biochemistry 105 (2011) 10–16
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Tris
trifluoroacetic acid
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This study was financed by the Polish National Center for Research
and Development, grant no. KB/138/13749/IT1-B/U/08.
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