A simple protocol to study blue copper proteins by NMRIoannis Gelis1, Nikolaos Katsaros1, Claudio Luchinat2,3, Mario Piccioli2,4and Luisa Poggi2,4 1 NCSR Demokritos, Institute of Physica
Trang 1A simple protocol to study blue copper proteins by NMR
Ioannis Gelis1, Nikolaos Katsaros1, Claudio Luchinat2,3, Mario Piccioli2,4and Luisa Poggi2,4
1 NCSR Demokritos, Institute of Physical Chemistry, Agia Paraskevi Attikis, Greece; 2 Magnetic Resonance Center,
3 Department of Agricultural Biotechnology and 4 Department of Chemistry, University of Florence, Italy
In the case of oxidized plastocyanin from Synechocystis sp
PCC6803, an NMR approach based on classical two and
three dimensional experiments for sequential assignment
leaves unobserved 14 out of 98 amino acids A protocol
which simply makes use of tailored versions of 2D HSQC
and 3D CBCA(CO)NH and CBCANH leads to the
identi-fication of nine of the above 14 residues The proposed
protocol differs from previous aproaches in that it does not
involve the use of unconventional experiments designed
specifically for paramagnetic systems, and does not exploit
the occurrence of a corresponding diamagnetic species in
chemical exchange with the blue copper form This protocol
is expected to extend the popularity of NMR in the struc-tural studies of copper (II) proteins, allowing researchers to increase the amount of information available via NMR on the neighborhood of a paramagnetic center without requi-ring a specific expertise in the field The resulting 3D spectra are standard spectra that can be handled by any standard software for protein NMR data analysis
Keywords: blue copper proteins; NMR spectroscopy; struc-tural biology; paramagnetic proteins; plastocyanin
There is a strong interest in the structural biology
commu-nity for the study of copper trafficking and copper
homeostasis [1–11] This involves the understanding of the
role of metal ions in protein folding and misfolding related
diseases [12–20], as well as the understanding at the atomic
level of protein–protein interactions in electron-transfer
processes [21–29] Within this framework the search for
methodological advancements in NMR spectroscopy
tail-ored to the structural characterization of copper(II) proteins
may play a significant role
In paramagnetic metalloproteins, NMR signals of
pro-tons close to the metal ions are broadened, sometimes
beyond detection, by the presence of the paramagnetic
center [30,31] The extent of paramagnetic induced line
broadening depends on the electronic relaxation times of the
metal center [32–35] Tetragonal Cu(II), found in Type II
centers, has long electronic relaxation times [36] which make
the NMR lines of residues belonging to the coordination
sphere broad beyond detection [37,38] When Cu(II) adopts
a trigonal geometry, such as that provided by two histidines
and one cysteine residue in Type I centers, or blue copper
centers, the electronic relaxation times are about one order
of magnitude shorter Hence, signals belonging to Cu(II)
first coordination sphere, although severely broadened,
become observable [39] Because of the axial symmetry of
the g-tensor in Type I copper centers [40], the pseudocontact
contribution to the observed shifts is negligible and
para-magnetic shifts arise only from through-bond spin density
delocalization from the metal to the ligands Therefore, in Type I copper centers, pseudocontact shifts can not be used for structural purposes, unlike many other classes of metalloproteins [41–43] As a partial compensation of such
a drawback, the shifts can be safely interpreted on the basis
of the chemical shift index [44]
It was recently shown that solution structures of copper(II) proteins can be obtained [45] To this end, the standard protocol for solution structure of biomacromole-cules has been substantially augmented by a number of non conventional strategies for resonances assignments and by the use of paramagnetism-based constraints for structure calculations [46] This approach often requires specific expertise in the field of electron relaxation and hyperfine interaction [42,46–53] and, in some case, specific hardware [54] As a consequence, NMR structural characterization of paramagnetic metalloproteins is routinely performed only
in a limited number of laboratories [31,55–63]
We would like to present here a different perspective of the NMR study of paramagnetic proteins and to emphasize the fact that paramagnetic proteins should not necessarily
be considered as a different field with respect to mainstream biomolecular NMR We will discuss the information content of basic 2D and 3D experiments when they are collected using a different choice of experimental parameters with respect to the standard ones The additional experi-ments that we propose are deliberately restricted to simple modifications of the pulse sequences that are routinely used for resonance assignment, like CBCA(CO)NH [64,65] and CBCANH [66,67], in such a way that their implementation does not require any special expertise This approach should extend significantly the detectability of resonances that sense the hyperfine interaction and therefore should substantially increase the number of assignments in the proximity of the paramagnetic center that can be obtained within a standard protocol [68,69] The modifications discussed here
to CBCA(CO)NH and CBCANH experiments do not substantially alter the coherence transfer pathway with
Correspondence to M Piccioli, Via L Sacconi 6,
50019 Sesto Fiorentino, Florence, Italy.
Fax: + 39 055457 4253, Tel.: + 39 055457 4265,
E-mail: piccioli@cerm.unifi.it
Abbreviations: INEPT, insensitive nuclei enhanced by polarization
transfer; PFG, pulsed field gradients.
(Received 12 June 2002, revised 25 October 2002,
accepted 27 November 2002)
Trang 2respect to the scheme originally proposed by Bax and
coworkers [64], but they make the two sequences much
more effective in the presence of contributions to relaxation
arising from the hyperfine interaction
The test system chosen is the blue copper protein
plastocyanin from the cyanobacterium Synechocystis sp
PCC6803 It contains a typical Type I center extensively
spectroscopically characterized [70–72] Previous NMR
studies showed that this is an excellent system to address
the efficiency of nonconventional NMR approaches to
obtain structural information [45,73]
In the present work we will demonstrate that an approach
which does not require any hardware or software dedicated
to paramagnetic systems can substantially improve the
available assignments close to the copper (II) ion without
recourse to metal substitution
Materials and methods
Protein expression and purification
The expression and purification of Synechocystis sp
PCC6803 plastocyanin in Escherichia coli was performed as
previously described [74] Uniformly13C,15N-labeled
over-expressed plastocyanin was obtained from M9 minimal
medium containing (15NH4)2SO4as the sole nitrogen source
and [13C6]D-glucose as the sole carbon source Samples for
NMR spectroscopy (2 mM) were prepared in 50 mMsodium
phosphate buffer (either in 90% H2O, 10% D2O or in 100%
D2O) at pH 5.2 Complete oxidation of the protein was
achieved using a slight excess of ferricyanide, subsequently
removed by gel filtration The samples were kept at 4C in
between measurements
NMR Spectroscopy
Experiments were performed at 295 K on Bruker Avance
spectrometers operating at 700 and 800 MHz Diamagnetic
1H-13C HSQC and 1H-15N HSQC [75] experiments were
performed The number of real data points acquired were 512
in the t1dimension (13C and15N), and 2048 in acquisition (t2
dimension) Spectral widths of 11 p.p.m for1H dimension,
80 p.p.m for13C dimension and 50 p.p.m for15N
dimen-sion were used For both experiments, 4 scans and a recycle
delay of 800 ms were used Echo-antiecho acquisition [76]
was used to perform quadrature detection in t1dimension
Sensitivity improvement [77,78] and crush gradients during
the insensitive nuclei enhanced by polarization and transfer
(INEPT) and inverse INEPT mixing were also used
Two dimensional tailored 1H-13C HSQC and 1H-15N
HSQC experiments were performed to detect fast relaxing
signals [79] All delays (INEPT transfer and recycle) were
shortened to 1.6 ms and 100 ms, respectively, in order to
detect resonances near the paramagnetic center Two
dimensional nonselective inversion-recovery1H-15N HSQC
experiments (15N IR-HSQC) were performed to measure
nonselective longitudinal relaxation rates of protons [79] In
order to measure T1values of very fast relaxing protons,
INEPT transfer and relaxation delays were shortened to
1.6 ms and 200 ms, respectively Eight points were collected
to fit T1values, with the following inversion recovery delays:
2, 4, 8, 16, 32, 64, 128 and 256 ms
A three dimensional HNCO experiment [80] was per-formed to assign backbone resonances For the above experiment spectral windows of 11 p.p.m for1H, 50 p.p.m for 15N, and 30 p.p.m for13C dimensions were typically used The number of real data points acquired were 128 in the t1 dimension (13C), 64 in the t2 dimension (15N), and
1024 in acquisition (t3 dimension) Three dimensional CBCA(CO)NH [64,65] and CBCANH [66,67] experiments were carried out to sequentially assign 13C resonances Spectral widths of 11 p.p.m for 1H dimension, 76 p.p.m for 13C dimension and 41 p.p.m for15N dimension were used The number of real data points acquired were 64 points in the15N dimension, 256 in the13C dimension, and
1024 in acquisition (t3dimension) for both experiments A recycle delay of 800 ms was used and 8 scans per increment were collected
All the data were zero-filled in the indirect dimensions and apodized using cosine squared functions Linear prediction was always applied in the indirect dimension All NMR data were processed with the BrukerXWINNMR software packages The programSPARKY3 (T D Goddard and D G Kneller, University of California, San Francisco, USA) was used for the analysis of all NMR spectra
Theory
A classical approach toward structure determination in a paramagnetic metalloprotein does not provide information
in the proximity of the metal center [81,82], even when careful and extensive studies are performed using double and triple labeled samples [61,83]
CBCA(CO)NH and CBCANH are among the most popular experiments for sequential assignment of macro-molecules in solution [65,66] CBCA(CO)NH spectra con-nect HN(i) with Cb(i-1) and Ca(i-1) resonances, while CBCANH spectra connect HN(i) with Cb(i), Ca(i), Cb(i-1) and Ca(i-1) resonances, the inter residue peaks being lower
in intensity than the intra residue peaks The standard versions of both experiments make use of several INEPT transfer delays, crush gradients, flip back pulses, sensitivity improvement schemes and echo-antiecho gradient selection Each of the above building blocks requires coherence transfer delays during which the magnetization of interest is relaxed In the case of paramagnetic molecules, the presence
of the unpaired electron makes large contributions to nuclear relaxation for nuclei nearby As a consequence, CBCA(CO)NH and CBCANH are expected to be unsuit-able for the study of such systems However, a series of modifications can be planned that make the two sequences exploitable
The optimization of polarization transfer and recycle delays in heteronuclear experiments has been extensively discussed elsewhere, as well as the choice of the number of scans and data points in t1, t2and t3dimensions [46,84] On such bases, the NH reverse INEPT and the CH INEPT transfer delays were shortened to 1.6 ms and 1.8 ms, respectively, in the CBCA(CO)NH experiment, while only the NH transfer delay was shortened to 1.6 ms in the CBCANH The building blocks of the pulse sequences related to the coherence transfers pathway Cb-Ca-CO-N or
Cb-Ca-N were not modified with respect to the standard version of the sequence A recycle delay of 300 ms was used
Trang 3and 64 scans were collected for both experiments With
respect to the diamagnetic version of the experiments, the
number of data points in the15N and13C dimensions were
reduced from 64 to 48 and from 256 to 128, respectively
Besides the choice of INEPT transfer delays, other
modifications can be introduced with respect to the
diamagnetic version of the sequences
The sensitivity improvement scheme (SI) [78] makes use,
during the reverse INEPT, of a double spin-echo which
allows the detection of both antiphase components NxHz
and NyHzcreated during15N evolution, thus giving a 21/2
improvement of the signal to noise ratio [78] This scheme
has twice the duration of a normal reverse INEPT, and
different relaxation mechanisms are operative on the
various coherence transfer pathways that transform the
two above components to observable magnetizations Even
if the transfer delays are shortened, as already extensively
discussed [84], the occurrence of a strong contribution to
relaxation may be such that, for fast relaxing signals, the
elimination of sensitivity improvement scheme gives a better
S/N
The use of pulsed field gradients (PFGs) within a pulse
sequence to detect paramagnetic signals may be critical In
general, their use to clean observable magnetization from
spurious peaks has no drawbacks, provided that PFG do
not entail additional delays [85] However, in the case of
echo-antiecho detection schemes [76], the gradient selection
requires that two additional gradients are placed in the
sequences, together with two additional 180 pulses and
refocusing delays Because hyperfine relaxation depends on
c2X, where X is the involved nucleus, the loss of signal
intensity is critical in those coherence transfer steps in which
1H R2relaxation is involved [86] This is of course the case of
the period immediately preceding t3 acquisition Similar
considerations hold for the use of crush gradients during the
INEPT and reverse INEPT steps In this case, the loss due
to relaxation depends on R1 Therefore the use of crush
gradients for fast relaxing signals is less destructive that the
gradients needed in the echo-antiecho scheme Of course, a
major drawback expected from the elimination of gradient
selection and crush gradients is that there is no efficient
water suppression scheme left in the sequence To overcome
this problem, a Watergate scheme, with short gradients in
order to be compatible with the short delays of the reverse
INEPT step [87] can be reintroduced in the final reverse
INEPT step
The calculated effects of the stepwise removal of the crush
gradients, echo-antiecho and sensitivity improvement
schemes are shown in Fig 1 The calculations are
per-formed for the transfer function from a15N nucleus to a
bound proton in either the CBCANH or CBCA(CO)NH
pulse schemes The proton is considered to be at 6 A˚ from
the copper(II) center, assuming a ss¼ 0.5 ns [73] and a
sr¼ 5.9 ns [74] Under these conditions R2 c.¼ 600 s)1,
while R1 is about 5 times smaller If we use the standard
values for duration and recovery of gradients of 1 ms and
0.5 ms, respectively, the transfer function has a maximum at
about 1 ms (Fig 1A) Its intensity is about 2% of the
intensity expected for the corresponding peak in a normal
reverse INEPT when relaxation is neglected Elimination of
the crush gradients, during which1H R1relaxation occurs,
leads to a gain in intensity of about 15% (Fig 1B)
The most important effect arises from the elimination of the antiecho scheme The effect of removing the echo-antiecho building block is observed in the calculated transfer functions shown in Fig 1C Considering as a test case the signal discussed above, the replacement of the echo-antiecho block with any other quadrature detection scheme that does not rely on gradient selection of coherences, increases signal intensity by about a factor of five Of course the relative gain
in intensity is reduced when, in the diamagnetic version of the sequence, shorter gradients and recovery delays are used When gradient and recovery delays in the diamagnetic experiment are shortened down to 150 ls and 100 ls, respectively, the gain of signal intensity under the above conditions is still of about a factor of two This shows that even if very short values of gradient and recovery delays are used within the diamagnetic version of CBCA(CO)NH and CBCANH (and this would not be the ÔdefaultÕ choice in the absence of fast relaxation), the use of echo-antiecho quadrature detection is not recommended with respect to States-TPPI [88] or any other quadrature detection scheme methods that does not rely on gradient selection of coherences
Finally the effects of the replacement of the sensitivity improvement step with the usual reverse INEPT step is illustrated in the transfer function shown in Fig 1D It can
be seen that the single reverse INEPT step, not only gives about a 10% increase in the maximum of the transfer function with respect to the sensitivity improvement scheme but also it gives a transfer function which is much less sensitive to optimization of the transfer delay, as observed in Fig 1 when transfer delays longer than 1.8–2 ms are considered
Fig 1 Calculated transfer functions for the NH reverse INEPT transfer step of CBCA(CO)NH or CBCANH experiments with: (A) diamagnetic pulse sequence, using sensitivity improvement detection scheme and echo-antiecho quadrature detection (all applied gradients were 1 ms with a recovery delay of 0.5 ms); (B) same as (A) without the use of crush gradient occurring in between the 90° pulses; (C) same as (B) without the echo-antiecho detection, i.e with the elimination of the additional delays needed for the gradients of the echo-antiecho; (D) same as (C) with the removal of the SI scheme All transfer functions are normalized with respect to a normal reverse INEPT under optimized condition for the transfer delay and neglecting losses due to 1 H- 15 N relaxation Transfer functions have been calculated for a1H signal of a proton at about 6 A˚ from the metal center (R 2 ¼ 600 s)1, R 1 ¼ 120 s)1 assuming a
s s ¼ 0.5 ns and a s r ¼ 5.9 ns).
Trang 4Results and discussion
Spectral assignment of oxidized plastocyanin:
the standard approach
Synechocystissp PCC6803 plastocyanin was overexpressed
in E coli to obtain large amounts of 13C,15N-enriched
protein The already available assignment of1H and 15N
resonances [45] was extended to13C resonances of backbone
and side chains by a combination of classic triple resonance
experiments 3D HNCO [89], CBCA(CO)NH [64,65] and
CBCANH [66,67] were collected at 700 and 800 MHz
spectrometers The analysis of these spectra has lead to the
assignment of 73% of C¢, 81% of Ca and 79% of Cb
Because of broadening effects induced by the paramagnetic
center, no sequential backbone assignment is available [45]
in the loop regions encompassing residues 7–8, 38–42,
61–62, and 82–88
Detection of fast relaxing signals:15N- and13C-HSQC
experiments
Tailored versions of1H-15N HSQC [90],1H-13C HSQC [79],
CBCA(CO)NH and CBCANH were used to detect
reso-nances in the proximity of Cu(II)
The comparison of two1H-15N HSQC spectra recorded
with different recycle and polarization transfer delays allows
to identify 14 resonances that clearly experience a
substan-tial gain in signal intensity when comparing a diamagnetic
HSQC experiment with a tailored experiment The overlay
of the two spectra is shown in Fig 2, and the 14 resonances
are highlighted Of these, 7 are observed with much lower
intensity in the diamagnetic experiment while 7 were
completely missing in the diamagnetic experiment The
former 7 signals were already assigned in a previous study
[45], and correspond to residues Leu14, Phe16, Asn34,
Lys35, Ser37, Ile41 and Ala89 The seven new signals are
listed in Table 1
In order to measure the proton T1 values of the
previously unobserved fast relaxing signals detected in the
tailored 1H-15N HSQC, a series of two dimensional nonselective inversion-recovery1H-15N HSQC experiments was performed [79] As our present interest is focused on relatively fast relaxing signals, we used for the inversion recovery experiment a recycle delay of 200 ms Therefore the inversion recovery experiment gave fully reliable results only for those resonances having a T1values < 60 ms The
T1values obtained for the above signals, together with the
1H and15N shifts, are also reported in Table 1
Similarly to the1H-15N HSQC experiment, the compari-son of two1H-13C HSQC spectra recorded with different recycle and polarization transfer delays allows to identify 11 resonances that clearly experience a substantial gain in signal intensity when comparing a diamagnetic HSQC experiment with a tailored experiment Of these, four belong
to Cas peaks 1–4 in Table 2 and 7 to Cbs peaks 5–11 in Table 2 They are also highlighted in Fig 3A and 3B
Detection of fast relaxing signals: tailored CBCA(CO)NH and CBCANH
A 3D version of these experiments tailored as discussed above to optimize the detection of fast relaxing signals has been performed The new peaks identified through1H-15N HSQC were monitored in CBCA(CO)NH While in the diamagnetic version of CBCA(CO)NH experiment only
Fig 2 Overlay of diamagnetic and tailored
1
H-15N HSQC spectra Fourteen resonances
are highlighted The seven signals completely
missing in the diamagnetic experiment are
labelled A-G, while the seven observed with
much lower intensity in the diamagnetic
experiment are labelled with their
corres-ponding assignment.
Table 1 Previously unobserved signals found in the tailored 1H-15N HSQC.
dN (p.p.m) dHN (p.p.m) T 1 (ms)
Trang 5one of the peaks (signal C) listed in Table 1 was observed,
the tailored CBCA(CO)NH has allowed us to observe
connectivities with previous amino acid for 5 out of 7
residues, as reported in Table 3 As far as signal C is
concerned, the very weak connectivities observed in the
diamagnetic version of CBCA(CO)NH are observed with
much larger intensity (a factor of 2) in the tailored
experiment
Similar considerations hold for CBCANH The
sensiti-vity of CBCANH is expected to be smaller than CBCA
(CO)NH, as already proven extensively in diamagnetic
systems None of the peaks listed in Table 1 was observed in
the diamagnetic experiment while four out of seven gave
connectivities in the tailored CBCANH, as reported in
Table 3, which summarizes the information obtained using
modified CBCA(CO)NH and CBCANH
Assignment of fast relaxing signals
The assignment of the new signals found in the tailored
1H-15N HSQC can be performed considering the following:
(a) limited number of missing assignments in the1H-15N HSQC spectrum (14, listed in Table 4); (b) Cb and Ca chemical shifts provide substantial information on the nature of the amino acid under investigation [44,91,92] Of course, such assignment is feasible only under the assump-tion that contribuassump-tions arising form pseudocontact shifts are negligible with respect to the chemical shift index tolerance [93] As outlined above, this is a very reasonable assumption
as shown by the available literature on Cu(II) proteins [39,94]
Let us consider signal A [Table 3]: the intra residue Ca peak at 43.8 p.p.m shows unambiguously that signal A belongs to a Gly residue, while inter residue Ca and Cb peaks at 58.3 and 27.9 p.p.m are primarily consistent with Met, Arg or His residues Therefore the only possible assignment is Gly8, preceded by Met7 In previous works only some sparse1H assignments were available for residues
7, 8, 61 and 62 [45]
No assignment can be performed for signal B, for which
no connectivities are found in both CBCA(CO)NH and CBCANH
Signal C shows no connectivities in the CBCANH spectrum, but the inter residue Ca peak found in the CBCA(CO)NH at 43.1 p.p.m is only consistent with a Gly
as preceding residue Given the limited number of missing assignments, this is in agreement only with the assignment
of signal C as the HN of Leu61, preceded by Gly60 Signal D shows in the CBCA(CO)NH spectrum inter residue peaks at 56.7 and 36.3 p.p.m., while among the intra residue peaks only the Ca is found in the CBCANH at 59.2 p.p.m These connectivities perfectly fit the assignment
of signal D as NH of Val42, preceded by Ile41 The identification of Val42 is also confirmed by the pattern observed in the CBCA(CO)NH for Phe43, which presents inter residue connectivities at 59.2 and 30.8 p.p.m
As far as signal E is concerned, the four peaks corres-ponding to intra and inter residue Caand Cbdo not permit a fully consistent assignment Inter and intra residue Ca’s are observed at 52.2 and 55 p.p.m., respectively, and they match with His86-Arg87 residues This assignment is supported by
Table 2 Signals that experience a substantial gain in signal intensity in
the tailored1H-13C HSQC compared with the diamagnetic experiment.
d1H (p.p.m) d13C (p.p.m) Assignment
1 5.44 56.2 Val15 (Ca-Ha)
2 5.14 58.0 Met7 (C a -H a )
3 4.92 42.3 Gly88 (C a -H a )
4 4.67 43.0 Gly8 (Ca-Ha)
5 1.27 37.3 Tyr 81 (C b -H b )
6 0.93 37.9 Arg87 (C b -H b ) a
7 0.72 37.3 Tyr 81 (Cb-Hb)
8 2.66 33.8 Val15 (C b -H b )
9 1.00 31.2 Val42 (C b -H b )
11 ) 0.30 11.6 Ile41 (Cd-Hd)
a Tentative assignment.
Fig 3 Overlay of diamagnetic and tailored
1 H- 13 C HSQC spectra (A) C a region (B) C b
region The 11 resonances that substantially increase their intensity in the tailored experi-ment are highlighted and labelled 1–11.
Trang 6the inter residue Cb, which is observed at 35.6 p.p.m (a
typical His region), but does not fit with the intra residue Cb
that is observed at 38 p.p.m., i.e out of the region where Cb
of Arg residues are expected to fall Therefore, we assign
signal E as the HN of Arg 87 only tentatively
The15N shift of signal F is only consistent with a Gly
residue As Gly8 has been already identified as signal A,
signal F can be safely assigned as the only other glycine
residue missing, i.e Gly88, even if no connectivities are
found in both CBCA(CO)NH and CBCANH
For signal G, in the CBCANH spectrum only the intra
residue connectivities are observed while those with the
previous residue are observed only in CBCA(CO)NH The
intra residue peaks at 48.9 p.p.m for the Caand 17.3 p.p.m
for Cbare only consistent with an Ala residue, while side
chain carbons observed from signal G in CBCA(CO)NH
(53.2 and 40.3 p.p.m) are only consistent with an Asn or a
Leu residues The only possible assignment for signal G is
thus Ala62 NH, preceded by Leu61
In summary, the tailored experiments described above
allowed us to detect and assign six new HN signals that were
previously completely unobserved Another seven signals
showed a sizable increase in their S/N ratio With the only
exception of signal B, all these newly identified signals in the tailored1H-15N HSQC could be assigned
Figure 4 shows, as an example, comparison of diamag-netic and tailored CBCA(CO)NH as far as signal D is concerned As observed, the two spectra are processed and displayed with the same resolution While the two peaks arising from signal D are unambiguously detected in the paramagnetic spectrum, there is no evidence of them in the diamagnetic experiment
Some of the13C resonances that were identified as arising from the proximity of the paramagnetic center can be also identified in the tailored1H-13C HSQC This is the case of the Ha-Capeaks 1–4 shown in Fig 3A, whose shifts match with the Caresonances of Val15, Met7, Gly88 and Gly8 Analogous considerations hold for the 7 Hb-Cbresonances identified (Fig 3B), which are assigned on the basis of the already available1H assignment [45] The only exception to this criterion is peak 6 which has a Cbshift that corresponds
to the identified Arg87 Cband for which no.1H assignment
is available Therefore, we tentatively assign peak 6 as
Table 3 Connectivities found for signals A-G in tailored
CBCA(CO)NH and CBCANH spectra.
HN(i)
dC a (i-1)
(p.p.m)
dC b (i-1) (p.p.m)
dC a (i) (p.p.m)
dC b (i) (p.p.m)
B
F
Table 4 New assignments obtained for oxidized plastocyanin from
Synechocystis sp PCC6803 Copper(II) ligands are highlighted in bold.
In the right column N-Cu and H N -Cu distances are reported for each
amino acid.
dN
(p.p.m)
dNH
(p.p.m)
dH a
(p.p.m)
dC a
(p.p.m)
dH b
(p.p.m)
dC b
(p.p.m)
N, HN distances (A˚) Met7 5.14 58.3 27.9 7.9–8.4
Gly8 108.29 8.80 4.67 43.8 6.7–7.0
Val15 5.44 56.3 2.68 33.1 7.9–8.5
Val42 126.57 8.6 59.2 30.8 8.1–7.3
Leu61 121.62 8.1 53.2 40.3 10.3–11.3
Ala62 125.02 9.23 48.9 17.3 8.6–7.7
Arg87 128.23 8.28 55 38 7.2–7.5
Gly88 107.45 8.1 4.92 42.3 8.9–8.9
Fig 4 Strip plot of tailored (left) and diamagnetic (right) CBCA (CO)NH spectra in the region corresponding to signal D While inter-residue Caand Cbpeaks are present in the tailored spectrum, no correlation is found in the diamagnetic one.
Trang 7Arg87 Cb-Hband we identify an Hb 1H signal of Arg87 at
0.93 p.p.m No assignment is proposed for peak 10 All the
new assignments are summarized in Table 4
A simple NMR protocol is an important tool to study
paramagnetic proteins
Plastocyanin is a good model system to address features of
paramagnetic copper proteins in terms of assignment
strategy Indeed, the previously available assignment on
oxidized plastocyanin from Synechocystis sp PCC6803 [45]
was obtained through a combination of methods which
basically rely on saturation transfer techniques [73] Within
such a frame, dedicated experiments overcome the
difficul-ties arising from the presence of the paramagnetic center
and, eventually, permit the assignment for most of the
amino acids, including those directly bound to the copper
ion Such nonconventional experiments include saturation
transfer [95,96] from signals broadened beyond detection
[97], mono dimensional NOEs over1H signals very broad
and shifted in the region 100/)50 p.p.m [98,99], NOESY
and TOCSY experiments that allowed several1H
assign-ment only on the basis of relative line broadening (i.e based
on a metal-to proton distance predictable by means of
relaxation rates) [79], NOESY cross peaks between protons
that were not identified in a classical sequential assignment
work [100], the occurrence of signals with unusual chemical
shift behaviour [39]
The above approach, which had lead to extensive
assignment of paramagnetic copper proteins even in the
first coordination sphere, required the occurrence of
favourable exchange rates between the oxidized form and
the reduced diamagnetic form Of course, such requirements
limit the application of the approach Therefore we have
designed experiments to extend the assignment of
plasto-cyanin without relying on its reduced state and without any
specific a priori knowledge
A standard approach to resonance assignment, i.e
CBCA(CO)NH, and CBCANH, applied on plastocyanin
permitted the identification of 80 out of 94 non proline
residues [85%] with 14 amino acid for which no information
were available All missing residues belong to the northern
loops of the protein surrounding the copper ion and fall
within a 11 A˚ sphere from the metal center The protocol
proposed in the present work allowed assignment of 9 out of
the above 14 residues Indeed, no information was obtained
only for two of the three strong ligands of copper(II) (His39
and Cys83), for Asn40, whose HN group is directly involved
in a hydrogen bond with the copper-bound Cys83 Scatom
[45,101–103], and for Tyr82 and Glu84 It is noteworthy
that both Caand Cbresonances of the binding residue His86
can be assigned This permits the identification of
reso-nances as close as 3.6 A˚ from the copper center without
relying on any knowledge on the electron-nucleus coupling
Missing residues also provide a picture of the electron
spin density delocalization on the ligands Experimental
evidence and theoretical calculations show that a larger
amount of spin density is expected on Cys83 [97,104–106]
Consistently, not only Cys83 but also the surrounding
residues (Tyr82, Glu84) are missing in the present
assign-ment Electron spin density is delocalized also through the
H-bond between Cys83 Sc and Asn40 This makes Asn40
unobservable The missing assignment of Asn40 prevents, in turn, the identification of the preceding residue His39 Indeed both14N ENDOR [107] and1H NMR data [45] on plastocyanin indicate that metal bound imidazoles from His86 and His39 experience a similar spin density delocali-zation, thus supporting the hypothesis that the H-bond between Cys83–Asn 40 is indeed responsible for the non identification of His39 with this approach
In summary, such an approach allows identification, in a sequence specific fashion, 89 out of 94 non proline residues (95%) providing 89%, 87% and 92% of the assignment of
Ca, Cband N–H, respectively With the above approach we can reach metal-to-nucleus distances of 7.2, 3.6, and 7.5 A˚, for H, Caand N, respectively
Conclusions
In the case of the oxidized plastocyanin from Synechocystis
sp PCC6803, an NMR approach based on classical two and three dimensional experiments for sequential assign-ment leaves unobserved 14 residues out of 98 amino acids A protocol that simply makes use of tailored version of 2D HSQC and 3D CBCA(CO)NH and CBCANH leads to the identification of 9 of the above 14 residues Although it is clear that such improvement does not circumvent all the limitations arising from the presence of an oxidized copper center and actually still prevents the complete characteriza-tion of the first coordinacharacteriza-tion sphere, we should stress that the approach proposed allows those structural biologists that are not experts nor familiar with paramagnetic proteins
to substantially increase their knowledge
Acknowledgements
We are grateful to Prof Ivano Bertini for his advice and support The expression system of Synechocystis sp PCC6803 plastocyanin was a generous gift of Prof S Ciurli This work was supported by the European Union Research and Training Network (RTN) Project ÔCross correlation between the fluctuations of different interactions: a new avenue for biomolecular NMRÕ (Contract no HPRN-CT-2000– 00092) I.G is a Fellow of the Marie Curie Training Site ÔNMR in Inorganic Structural BiologyÕ, contract no HPMT-2000–000137 Support from PARABIO (HPRT-CT-00009) is acknowledged.
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Supplementary material
The following material is available from http://www blackwellpublishing.com/products/journals/suppmat/EJB/ EJB3400/EJB3400sm.htm
Table S1.13C assignment obtained for oxidized plastocya-nin from Synechocystis sp PCC6803 (BMRB accession number: 5584)