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Cite this: Chem. Commun., 2013,
49, 9824
Received 23rd July 2013,
Accepted 5th September 2013
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Detection of salt bridges to lysines in solution in
barnase†
Mike P. Williamson,*a Andrea M. Hounslow,a Joe Ford,a Kyle Fowler,a
Max Hebditcha and Poul Erik Hansenb
DOI: 10.1039/c3cc45602a
www.rsc.org/chemcomm
We show that salt bridges involving lysines can be detected by
deuterium isotope effects on NMR chemical shifts of the sidechain
amine. Lys27 in the ribonuclease barnase is salt bridged, and
mutation of Arg69 to Lys retains a partially buried salt bridge.
The salt bridges are functionally important.
Salt bridges in proteins are formed by the interaction of
positively and negatively charged sidechains.2 Isolated charges
within the hydrophobic interior of proteins are of high energy,
and therefore buried salt bridges are rare, confer significant
stabilisation energy to the protein, and are usually of functional
importance.3 By contrast, surface-exposed salt bridges are generally
weaker, more variable in their contribution to stability, and more
difficult to predict:4 we showed recently that three salt bridges
exposed on the surface of the protein GB1, which are well-defined
in crystal structures, are not present in solution.1 Since all analyses
of salt bridges are based on crystal structures (because NMR is
usually unable to define the positions of surface-exposed sidechains), it is difficult to assess the real importance of exposed
salt bridges. However when present, exposed salt bridges are
usually important for function, not least because of their strong
geometrical dependence,5 for example in directing coiled-coil
formation;6 directing the folding pathway;7,8 or stabilising
proteins from thermophiles.9 Intermolecular salt bridges are
also important determinants of specificity,10 where for example
a salt bridge between two kinase monomers was shown to be
essential for kinase activity.11
It is therefore important to be able to identify salt bridges in
solution. Currently two experimental approaches are used for
the detection of salt bridges in solution. One is to measure the
pKa of one or both of the residues involved: the acidic partner
should have a lower pKa than normal, whereas the basic partner
should have a higher pKa than normal.12,13 Site-specific measurements of pKa are normally done by NMR, by observation of Asp or
Glu, since measurement of Lys or Arg sidechains is difficult,
especially at high pH (typically >10).14 The magnitude of the
change in pKa provides a good estimate of the free energy of the
salt bridge.12 The second method is to mutate the residues
involved. This is typically done as a double mutant cycle, in which
both residues are mutated, individually and together, and the
stability to unfolding of each mutant is measured.12 The difference in free energy between the double mutant and the sum of the
two individual mutations is the coupling free energy, which is
assumed to equal the free energy of the salt bridge. Both of these
methods have drawbacks. Measurement of pKa requires the
folded protein to be stable at an extreme of pH, while the double
mutant cycle assumes no sidechain interactions in the unfolded
state. Therefore we present here an alternative, more direct, and
less laborious method.
As a test system, we have used the ribonuclease, barnase
(Fig. 1). This is a small and well characterized enzyme that
hydrolyses RNA. Salt bridges in barnase have been studied by
both the methods described above, which revealed two salt
bridges involving arginines (R83-D75 and R69-D93) plus a third
a
Dept of Molecular Biology and Biotechnology, University of Sheffield, Firth Court,
Western Bank, Sheffield S10 2TN, UK. E-mail: m.williamson@sheffield.ac.uk;
Fax: +44 (0)114 2224243; Tel: +44 (0)114 2224224
b
Dept of Science, Systems and Models, 18.1, Roskilde University, Universitetsvej 1,
PO Box 260, DK-4000 Roskilde, Denmark. E-mail: [email protected];
Fax: +45 46743011; Tel: +45 46742432
† Electronic supplementary information (ESI) available: Lysine assignments,
NMR spectra, experimental methods. See DOI: 10.1039/c3cc45602a
9824
Chem. Commun., 2013, 49, 9824--9826
Fig. 1 The structure of barnase (PDB 1a2p), showing the three salt bridges
described here.
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involving a lysine (K27-D54). Wild-type barnase is stable to
below pH 2, allowing the measurement of the pKas of the
aspartates involved (D75, D93 and D54) as 3.1, 1.5, and 2.2
respectively, compared to approximately 3.9 in the denatured
state.15 From the relationship DDG = DpKa 2.303RT, this
corresponds to free energies of approximately 4.6, 13.8 and
9.6 kJ mol 1, respectively. The stabilities of mutants of salt
bridge residues are reduced: by 20.9 kJ mol 1 for D75N,
10.9 kJ mol 1 for D54N, 13.8 kJ mol 1 for D54A, and 1.7 kJ mol 1
for K27A,15–17 giving an estimated coupling free energy of
12.6–14.6 kJ mol 1 for the R83-D75 and R69-D93 salt bridges.7
The salt bridges are usually present in crystal structures. All
three salt bridges are thus well defined.
The method that we present here to detect salt bridges relies
on observation of deuterium isotope effects on NMR signals
from the amine group at the end of the lysine sidechain. Lysine
amino protons are broadened by rapid exchange with water,
and are consequently difficult to observe. We therefore used
low pH and temperature to slow down the exchange (pH 4.8,
3 1C) and used a modification of the HSQC experiment named
HISQC that was developed for application to lysine sidechains,
in which 15N transverse magnetization is kept in-phase with
respect to 1H during t1 evolution.18 Fig. 2 shows the HISQC
spectrum of barnase, in 10 mM sodium acetate. All eight lysine
sidechain signals are observed. In 20% D2O–80% H2O (Fig. 2b)
one expects the relative concentrations of NH3+, NH2D+ and
NHD2+ to be 0.51 : 0.38 : 0.10. In agreement with these ratios,
the NH3+ and NH2D+ peaks are of comparable intensity, while
the NHD2+ peak is much weaker.
Lysine sidechain signals were assigned by using a CCH-TOCSY
experiment to assign sidechain 1H and 13C signals from the backbone out to Ce, combined with 2D H3NCECD18 and HSQC-TOCSY
experiments to go from the amino group towards the backbone
(ESI†). Chemical shift assignments are given in Table 1, and show
that the highest field 15N signal is from K27.
The deuterium isotope effect is the difference in chemical
shift between NH3+ and NH2D+ peaks, or equivalently NH2D+
and NHD2+ peaks; the difference in 15N shift is called 1D15N,
while the difference in 1H shift is called 2D1H. The isotope
effects are defined as 1D15N = dN(H)
dN(D). The deuterium
Fig. 2 HISQC spectrum of lysine sidechains in barnase: (a) in 100% H2O (b) in
20% D2O–80% H2O. In (b), for each lysine, the lower signal (larger 15N chemical
shift) is from the NH3+ form, while the upper one is NH2D+. For three of the
residues the NHD2+ peak is also visible, with the NH3+–NH2D+ spacing being
equal to the NH2D+–NHD2+ spacing. The residue indicated in green is the saltbridged K27.
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Table 1 Chemical shifts and isotope effects for lysine residues in barnase
(in ppm)
15
1
32.72
31.82
32.46
32.30
32.82
32.48
32.60
32.38
32.75
7.68
7.88
7.73
7.60
7.92
7.67
7.52
7.60
7.91
N shift
K19
K27
K39
K49
K62
K66
K98
K108
K69a
H shift
1 15
D N
2 1
0.357
0.348
0.359
0.363
0.362
0.363
0.358
0.354
0.356
DH
0.0233
0.0335
0.0239
0.0274
0.0260
0.0217
0.0284
0.0260
0.0318
Chemical shifts are given in ppm relative to DSS, and were measured in
10 mM acetate, pH 4.8, 3 1C. For exchange of two deuteriums the total
effect is twice as large (see Fig. 2b). a In the R69K mutant. All others are
means from wild-type and R69K.
isotope effect is expected to be different in salt-bridged and
non-salt-bridged amines, because the formation of an N–H O
salt bridge makes the N–H bond longer and weaker, and therefore changes the perturbation of nuclear shielding caused by the
introduction of the heavier 2H nucleus. Both calculation19,20 and
experiment21 suggest that there should be significant differences
in isotope effects as a result of salt bridging.
The isotope effects are readily measured by studying samples in
H2O–D2O mixtures (Fig. 2) and are listed in Table 1. Because the
isotope effects are differences between pairs of peaks in the same
spectrum, they can be measured with high accuracy. The isotope
effect for K27 is different from those of all other lysines: 1D15N is
smaller while 2D1H is larger (more negative). Measurements of pKas
of all 12 acidic residues in barnase15 showed that four (D54, E73,
D93 and D101) have pKas lowered significantly compared to typical
‘random coil’ values. Crystal structures suggest that D54 has a salt
bridge to K27 and D93 to R69, while E73 forms a hydrogen bond to
Y103 OH, and D101 to T105. Thus, previous data suggest that the
only lysine to be involved in a salt bridge is K27. The unusual
isotope effect for K27 is therefore most obviously explained as
arising from the salt bridge.
In order to check this hypothesis, we generated another saltbridged lysine by mutation of R69 to lysine. R69 forms a strong and
partially buried salt bridge to D93, as evidenced by a pKa for D93 of
approximately 1.5, a coupling free energy of 13.8 kJ mol 1, and
consistent formation of salt bridges in crystal structures.7,15 The
mutation was made using PCR and verified by sequencing, and
the mutated protein was purified using the standard protocol for
wild-type barnase.22 The HISQC spectrum of lysine sidechains in
the mutant shows the presence of an extra signal (ESI†).
In order to verify that K69 in the mutant is still forming a salt
bridge to D93, we measured the pKa of D93 by following the
chemical shift of the sidechain carboxyl resonance as a function
of pH, as detected in 2D H(CA)CO spectra modified for aspartate
sidechains.23 The results are shown in Fig. 3, and show that D93 has
a pKa of approximately 2.3, with the other aspartates measured
having pKas in the normal non-hydrogen-bonded range of 3.2–3.8.
Thus, D93 remains strongly salt bridged in the mutant. The R69K
mutant is however significantly less stable at low pH than is the wildtype: the wild-type protein unfolds at pH 2.15,15 whereas R69K
unfolds at pH 2.85, making it difficult to obtain data at low pH.
Chem. Commun., 2013, 49, 9824--9826
9825
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phosphate oxygen.25 In most crystal structures of free barnase,
K27 has an unusual folded-back conformation and forms a salt
bridge to D54, but in almost all the structures of barnase bound
to substrate analogues or inhibitors, K27 is extended and interacts with the ligand. The loss of the intramolecular salt bridge
and formation of an alternative salt bridge to the cleaved
phosphate oxygen therefore seems to be a crucial part of the
structural rearrangements needed to form and stabilize the
transition state. The salt bridge to Arg69 is also essential for
directing the folding pathway and stabilising the folded protein.7
In general there is good evidence that salt bridges in proteins,
where present, are functionally important. The ability to detect
such salt bridges is thus crucial.
We thank SURE (Sheffield Undergraduate Research Experience) for funding (KF).
Notes and references
Fig. 3 Aspartate sidechain carboxylate chemical shifts as a function of pH in the
R69K mutant. Black, D93; brown, D8; blue, D54; orange, D44; green, D12. The
curves were fitted to single pH dependences.1 For D93, the fits were less reliable
because less of the titration curve could be sampled. The curve shown was fitted
by fixing the chemical shift change on protonation to be similar to those seen for
the other aspartates.
This provides a good illustration of the difficulties of detecting
salt bridges using pKa measurements: if the protein is unstable
at low pH, then chemical shifts cannot be followed to the end of
the pH titration, and pKa measurements will be less precise.
In the R69K mutant, the isotope effects for K69 are similar to
those observed for K27, though slightly less extreme (Table 1).
There is thus a small but clear difference in isotope effects
between salt bridged and non-salt bridged lysines, providing a
simple indicator of the presence of a salt bridge: the 1D15N and
2 1
D H are respectively 0.363 0.004 and 0.026 0.003 in the
absence of a salt bridge, and 0.352 0.004 and 0.033 0.002 in
the presence of a salt bridge, a highly significant difference for
both (even using non-parametric tests), though more obvious for
2 1
D H. The limited data available suggests that isotope effects from
stronger salt bridges are more different. We note that the change
in both 1D15N and 2D1H on salt bridge formation is smaller than
that predicted from calculation,19,20 although it is in the direction
predicted. The magnitudes of 1D15N are also generally less than
predicted, but similar to those observed previously for protein
G1 and HoxD9 homeodomain.18 We have previously suggested
that this difference in 1D15N may be due to solvation of the amine;
it is likely that solvation is also responsible for reducing the
changes in isotope effects in salt bridges.
In summary, we have shown that salt bridges in solution can
be detected using deuterium isotope effects. The salt bridge
formed by K27 in barnase is functionally important. Mutation
of lysine 27 to alanine produces an enzyme with kcat only 0.03%
of wild-type activity.24 It is suggested that in the transition state
for RNA hydrolysis, K27 forms a salt bridge with the cleaved
9826
Chem. Commun., 2013, 49, 9824--9826
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