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Salt bridges in proteins are bonds between oppositely charged residues that are sufficiently close to each
other to experience electrostatic attraction. They contribute to protein structure and to the specificity of
interaction of proteins with other biomolecules, but in doing so they need not necessarily increase a proteins
free energy of unfolding. The net electrostatic free energy of a salt bridge can be partitioned into three
components: chargecharge interactions, interactions of charges with permanent dipoles, and desolvation of
charges. Energetically favorable Coulombic chargecharge interaction is opposed by often unfavorable
desolvation of interacting charges. As a consequence, salt bridges may destabilize the structure of the folded
protein. There are two ways to estimate the free energy contribution of salt bridges by experiment: the pKa
approach and the mutation approach. In the pKa approach, the contribution of charges to the free energy of
unfolding of a protein is obtained from the change of pKa of ionizable groups caused by altered electrostatic
interactions upon folding of the protein. The pKa approach provides the relative free energy gained or lost
when ionizable groups are being charged. In the mutation approach, the coupling free energy between
interacting charges is obtained from a double mutant cycle. The coupling free energy is an indirect and
approximate measure of the free energy of chargecharge interaction. Neither the pKa approach nor the
mutation approach can provide the net free energy of a salt bridge. Currently, this is obtained only by
computational methods which, however, are often prone to large uncertainties due to simplifying assumptions and insufficient structural information on which calculations are based. This state of affairs makes the
precise thermodynamic quantification of salt bridge energies very difficult. This review is focused on
concepts and on the assessment of experimental methods and does not cover the vast literature. Copyright
# 2004 John Wiley & Sons, Ltd.
Keywords: protein electrostatics; protein thermodynamics; pKa determination; double mutant cycle; NMR
spectroscopy; salt bridge
Received 9 October 2003; revised 7 November 2003; accepted 17 November 2003
INTRODUCTION
The stability of a protein results from a delicate balance
between opposing forces. The folded native protein structure is maintained at the edge of thermodynamic stability,
the free energy of unfolding being in the range of at most a
few tens of kilojoules per mol (Pace, 1975; Privalov, 1979).
Probably the major contributor to stability is the hydrophobic effect, a term coined to describe the energetically
H. R. BOSSHARD ET AL.
Figure 1. Classification of salt bridges in a protein whose polypeptide chain is represented as a heavy line. Salt bridges are
exposed on the protein surface in a high dielectric environment
(a and b), or fully buried in the protein interior in a low dielectric
environment (c, stippled), or half-buried (d). Salt bridges can be
affected by other charges such as helix dipoles (e). Charge
charge distances are variable and geometries differ (geometry
is defined as the angle between the vectors from the C atom to
the centre of the interacting charges). Most salt bridges are built
from only two opposite charges (ae), but several charges may
form a network in a complex salt bridge (f).
discussed shortly. For example, the value of GijU obtained if GU;no sb refers to protonation of a negatively
charged side chain of a salt bridge (GluO ! GluOH) is
not the same as GijU obtained if GU;no sb refers to the
mutation of a negatively charged to an uncharged residue,
for example Glu ! Ala. In other words, eqn (3) is a logical
and commonsensical definition of the free energy contribution of a salt bridge yet it cannot provide a single and unique
net free energy contribution for a given salt bridge without
defining how the salt bridge is being removed. The free
energy relationships defined by eqns (2) and (3) are presented schematically in Fig. 2.
According to eqn (3), GijU is positive if the salt bridge
stabilizes the folded conformation of the protein. This is an
arbitrary definition. Some authors prefer to change the sign
on the right hand side of eqn (3) so that a stabilizing salt
bridge has a negative free energy. GijU comprises the
direct and the indirect effects contributing to the salt bridge.
GijU is sometimes called Gele , or only Gele called
the free energy of a salt bridge. Note, however, that the
free energy of a salt bridge is a misnomer as it is not a
single free energy difference as defined by eqn (2) but
always a double difference as defined by eqn (3).
Two ways to measure the free energy contribution of a
salt bridge
To determine GijU , the salt bridge has to be broken or
removed. To remove a salt bridge without distorting the
conformation of the rest of the protein is no simple task. In
Copyright # 2004 John Wiley & Sons, Ltd.
H. R. BOSSHARD ET AL.
Figure 3. The two experimental approaches to determine the contribution of a salt bridge to stability. Left: in
the pKa approach chargecharge interaction is disrupted by protonation of acidic groups or deprotonation of
basic groups. However, electrostatic interactions (H-bonding) between side chains may remain. Right: in the
mutation approach one or both charges are replaced by mutation (mutation to Ala is just an example).
Mutation does not only remove the charge but also changes the structure and chemical nature of the side
chains. This effect can be partly circumvented by a thermodynamic double-mutation cycle (see Fig. 10).
Table 1. Relationship between pKa in folded and unfolded protein, stability contribution of charged and
uncharged side chain, and sign of GUi
GiU is positive
GiU is negative
i
GU
2:3RTqiU qiF pH
4a
pH
1
1
i
i pH or q
i
pK
a
1 10
1 10pHpKa
4b
2:3RTQU QF pH
5a
GU
pH
Pn
P
where QU i1 qiU and QF ni1 qiF .
Integration of eqn (5a) over the entire pH range yields the
stability difference between the fully protonated and the
fully deprotonated protein molecule:
GU GU;dp GU;p 2:3RT
n
X
pKaU;i pKaF;i
i1
5b
Figure 4. Thermodynamic cycle of the relationship between the
proton association constants KF;i and KU;i of residue i in the
folded (F) and unfolded (U) protein and the free energies of
U;i
unfolding GU;i
p and Gdp of the protein carrying the protonated
U;i
(p) or the deprotonated (dp) residue i. GF;i
prot and Gprot are the
free energies of protonation of residue i in the folded and
unfolded protein, respectively. The contribution of group i to
stability results from different pKa values in the folded and
unfolded protein and is calculated according to eqn (4c) of the
text, which is shown at the bottom of the figure.
where qi refers to qiU and qiF , and pKai to pKaU;i and pKaF;i ,
respectively. The left equation is for negative charges
(acidic residues) and the right one for positive charges
(basic residues). Integration of the combined eqns (4a)
and (4b) over the entire pH range yields the stability
difference between the protein with the fully protonated
(subscript p) and the fully deprotonated (subscript dp) group
i:
GiU GiU;dp GiU;p 2:3RT pKaU;i pKaF;i
4c
Equation (4c) says that the contribution of charged residue i
to protein stability is directly related to the pKa shift of
residue i between the unfolded and folded protein. This is
shown graphically by the thermodynamic cycle of Fig. 4.
From Table 1 it is seen that GiU is positive if the
deprotonated form of the side chain (charged acidic or
uncharged basic group) is stabilizing, and negative if the
protonated side chain makes the protein more stable (uncharged acidic or charged basic group).
It is very important to note that GiU and GiU defined
by eqns (4a) and (4c) are not the total contribution of group i
to the stability of a protein. As already mentioned above, it
is the relative stability contribution due to electrostatics with
regard to a reference pH value. Hence, GiU is sometimes
called Gi;ele
U . For example, the carbon atoms of the side
chain of an ionizable residue may contribute to molecular
packing in the folded protein but this effect on the free
energy of unfolding cannot be deduced from the change of
pKa and is not included in GiU and GiU (Bashford and
Karplus, 1990; Yang and Honig, 1993). That means, the
starting value for integration of eqn (4a) is arbitrary, for
example pH 0. GiU of eqn (4a) then provides the free
energy difference in the interval pHpH 0, which is the
relative electrostatic contribution to the overall free energy
change obtained from the titration of group i.
Copyright # 2004 John Wiley & Sons, Ltd.
H. R. BOSSHARD ET AL.
Figure 6. Upper part: sequence of leucine zipper ABSS composed of an acidic and a basic chain connected by a disulfide
bridge (vertical line at right). Six interhelical salt bridges observed by NMR spectroscopy are indicated by double headed
arrows. Dashed numbers refer to sequence positions in the basic
chain. Lower part: ensemble of 25 NMR structures of ABSS. Only
the backbone trace and the side chains of acidic and basic
residues are shown. Salt bridges Glu8Lys130 , Glu15Lys200
and Glu22Arg270 are encircled. The remaining three salt
bridges are on the back of the molecule and are not seen in
this representation. NMR data from Marti et al. (2000).
pKaF
pKaU
Glu8
Glu13
Glu15
4.45
4.34
3.96
4.33
4.36
4.31
0.71
0.12
2.08
Destabilizing
Negligible
Stabilizing
Glu20
Glu22
4.41
4.86
4.61
4.53
1.19
1.96
Stabilizing
Destabilizing
Glu27
Sum of GiU
4.65
4.35
1.78
1.1
Destabilizing
Destabilizing
Salt bridge
partner residue
Lys130
Arg80
Lys200
pKaF 10:4a
Arg150
Arg270
pKaF 12:7a
Lys220
From computation.
H. R. BOSSHARD ET AL.
Figure 8. Model according to Bashford and Karplus (1990) depicting the partitioning of the electrostatic free energy difference
[GUi, eqns (4a) and (4c)] of the ionizable group i (black circle)
between the unfolded and the folded protein. Moving group i
from a high dielectric environment in the unfolded protein
(dotted circle at right) to a low dielectric environment in the
folded protein (stippled area at left) is accompanied by unfavorable desolvation of group i and leads to GUi,desolv . Electrostatic
interactions of group i with partial charges of the peptide backbone and polar, non-ionizable side chains (open circles, dashed
arrows) are different in the unfolded and folded state and lead
to the term GUi,backgrd . Direct electrostatic interactions between charged group i and other charged groups (grey circles,
solid arrows) are formed in the folded protein leading to
GUi,bridge .
water-accessible, high dielectric environment to an environment of low dielectricity. As a consequence, the groups have
to be partly or fully desolvated. (ii) In the folded as well as
the unfolded protein, charged groups are interacting with
permanent partial charges such as dipoles of peptide bonds
or polar non-ionizable side chains. These so-called background interactions differ in the unfolded and folded state.
(iii) In the folded protein, charges are interacting with other
full charges. Such direct chargecharge interactions are
attractive (e.g. salt bridges) or repulsive. No direct charge
charge interactions are expected to occur in the unfolded
protein if it is considered to be a fully extended polypeptide
chain. Note, however, that his assumption may not be
justified since some residual direct chargecharge interaction
can exist in the denatured state (see the section Electrostatic
Interactions in the Denatured State).
Accordingly, the electrostatic free energy difference can
be decomposed into three components:
Gi;backgrd
Gi;bridge
6a
GiU Gi;desolv
U
U
U
Gi;desolv
and Gi;backgrd
are the indirect energy conU
U
is the direct contribution.
tributions and Gi;bridge
U
is the free energy difference caused by desolvaGi;desolv
U
is the
tion of charge i. It is an unfavorable term. Gi;backgrd
U
free energy difference due to background interactions of
charge i with permanent dipoles of the peptide backbone, of
helices, or of non-ionizable polar side chains. Gi;backgrd
U
can be favorable or unfavorable. The indirect energy con Gi;desolv
been analyzed in terms of direct and indirect energy contributions using the program DelPhi (Nicholls and Honig,
1991). Figure 9 shows the results as bar plots in which the
contribution of each single residue (bars 14) is presented
together with the net free energy contribution of the salt
bridge (bar 5). According to this calculation, both salt
bridges are destabilizing the coiled coil conformation by
about 5 kJ/mol. This means that the favorable direct charge
charge interactions are outbalanced by unfavorable desolvation [the background terms are small or even negligible
(Marti and Bosshard, 2003)]. In the case of the Glu15
Lys200 salt bridge, only the negative charge on Glu15 adds
favorably when placed opposite to charged Lys20 (bar 1 in
the top panel of Fig. 9). When the charge on Lys200 is
removed, the lonely negative charge on Glu15 becomes
slightly destabilizing (bar 2 of top panel). The positive
charge of Lys200 is destabilizing whether or not juxtaposed
to a negative charge on Glu15 (bars 3 and 4 of top panel). In
the case of the salt bridge Glu22Arg270 , only the positive
charge of Arg270 is stabilizing when next to the negative
charge of Glu22 (bar 3 of bottom panel). Charged Glu22 is
destabilizing irrespective of the presence of a counter
charge (bars 1 and 2 of bottom panel). Even though opposing stability, the two salt bridges are nevertheless
present in the folded structure of ABSS as shown by
NMR spectroscopy and by the results from mutation
(see below).
J. Mol. Recognit. 2004; 17: 116
10
H. R. BOSSHARD ET AL.
7b
11
12
H. R. BOSSHARD ET AL.
ELECTROSTATIC INTERACTIONS
IN THE DENATURED STATE
Experimental and computational approaches to estimate the
energetic contribution of salt bridges are based on thermodynamic cycles involving the unfolded state [eqns (2) and
(3), Figs 2 and 4]. Throughout the preceding discussion all
effects of salt bridges have been implicitly attributed to the
folded protein. This is a reasonable but not a necessary
assumption. The denatured protein is produced by the
addition of chemical denaturants or by heat and constitutes
a large ensemble of states in rapid equilibrium. Residual
electrostatic interactions may occur in some members of
this ensemble since secondary and tertiary contacts may be
retained. Indeed, the importance of electrostatic effects in
the denatured state has been demonstrated experimentally
and predicted by computation (Oliveberg et al., 1995;
Swint-Kruse and Robertson, 1995; Tan et al., 1995; Khare
et al., 1997; Schaefer et al., 1997; Elcock et al., 1999;
Copyright # 2004 John Wiley & Sons, Ltd.
13
CONCLUDING REMARKS
There have been innumerable attempts to determine the
energy of salt bridges since the first measurement of the
strength of a salt bridge in chymotrypsin (Fersht, 1971).
Good computational methods permit to calculate the net
free energy of salt bridges based on high resolution protein
structures. However, the actual experiment remains the
benchmark. Therefore, our aim has been to compare the
merits and shortcomings of the two experimental methods
presently in use, the pKa approach and the mutation approach. The following points have been raised and
discussed:
(1) The free energy of unfolding of a salt bridge is defined
as a difference of differences. It is the change in the free
energy between the unfolded and the folded protein,
once with and once without the salt bridge [eqn (3)].
This difference is thermodynamically related to the
change of pKa of the ionizable groups in the unfolded
and folded protein [eqn (4c)].
(2) The net free energy contribution of a salt bridge is
composed of direct and indirect components. The direct
component comprises of the direct chargecharge inter, which are dominated by the single
actions, Gbridge
U
direct interaction between the oppositely charged
groups of the salt bridge; but interactions with other
. The indirect
nearby charges may also add to Gbridge
U
component comprises of desolvation and background
Gbackgrd
. Desolvation is
interactions, Gdesolv
U
U
unfavorable if the charges are buried and immobilized
in the folded protein. Background interactions with
permanent dipoles can be favorable or unfavorable.
The summed indirect effects tend to be unfavorable
since they are dominated by desolvation.
(3) The pKa approach provides the electrostatic contribution of charged groups relative to the corresponding
J. Mol. Recognit. 2004; 17: 116
14
H. R. BOSSHARD ET AL.
Acknowledgements
We thank Alemajehu Gorfe, Andrey Karshikoff, Heinz Ruterjans and Jim
Warwicker for many helpful discussions.
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