Effects of salt on the kinetics and thermodynamic
stability of endonuclease I from Vibrio salmonicida
and Vibrio cholerae
Laila Niiranen
1
, Bjørn Altermark
2
, Bjørn O. Brandsdal
2
, Hanna-Kirsti S. Leiros
2
, Ronny Helland
2
,
Arne O. Smala
˚
s
2
and Nils P. Willassen
1
1 Department of Molecular Biotechnology, Institute of Medical Biology, Faculty of Medicine, University of Tromsø, Norway
2 Norwegian Structural Biology Centre (NorStruct), Department of Chemistry, Faculty of Science, University of Tromsø, Norway
Extracellular and periplasmic enzymes of marine
organisms are exposed to environments in which
large variations in temperature and salinity can
occur. Such conditions require the proteins to fold
effectively and maintain their stability in spite of the
stresses they face [1]. At the same time, enzymatic
activity is dependent on fine-tuned structural flexibi-
lity [2]. How do enzymes cope with these contradict-
ing demands?

cat
. In differential scanning
calorimetry, salt stabilized both enzymes, but the effect on the calorimetric
enthalpy and cooperativity of unfolding was larger for VsEndA, indicating
salt dependence. Mutation of DNA binding site residues (VsEndA, Q69N
and K71N; VcEndA, N69Q and N71K) affected the kinetic parameters.
The VsEndA Q69N mutation also increased the T
m
value, whereas other
mutations affected mainly DH
cal
. The determined crystal structure of
VcEndA N69Q revealed the loss of one hydrogen bond present in native
VcEndA, but also the formation of a new hydrogen bond involving residue
69 that could possibly explain the similar T
m
values for native and N69Q-
mutated VcEndA. Structural analysis suggested that the stability, catalytic
efficiency and salt tolerance of EndA were controlled by small changes in
the hydrogen bonding networks and surface electrostatic potential. Our
results indicate that endonuclease I adaptation is closely coupled to the
conditions of the habitats of natural Vibrio, with VsEndA displaying a
remarkable salt tolerance unique amongst the endonucleases characterized
so far.
Abbreviations
DSC, differential scanning calorimetry; EndA, endonuclease I; VcEndA, Vibrio cholerae endonuclease I; VsEndA, Vibrio salmonicida
endonuclease I; Vvn, Vibrio vulnificus endonuclease I.
FEBS Journal 275 (2008) 1593–1605 ª 2008 The Authors Journal compilation ª 2008 FEBS 1593
proteins is thought to be a result of extensive intra-
molecular networks and compact packing that restrict

poses a problem when conclusions are to be drawn
about the mechanisms of cold adaptation. Are the
observed adjustments a result of true adaptation to
low temperature, or a combination of cold and salt
adaptation? Choosing non-marine (freshwater) psychro-
philes as study targets has been proposed as a
solution [7]. The interplay between the two types of
adaptation is, however, interesting in itself, and it is
possible to design experiments in a manner that facili-
tates the separation of the two effects. The first steps
towards this approach have been taken. In a compara-
tive study of a marine psychrophilic and an estuarine
mesophilic endonuclease I (EndA, EC 3.1.30) [11], the
different salt optima of the enzymes were taken into
consideration when the temperature-dependent enzy-
matic properties were characterized. In the discussion,
the authors stressed the importance of performing
measurements in buffers that were as physiological as
possible. Similar to psychrophilic EndA, marine car-
rageenase was found to display an activity optimum
around the salt concentration of seawater [12]. It
appears that the choice of buffer and the determina-
tion of the salt dependence of the activity are impor-
tant in comparative experiments on extracellular
enzymes.
EndA is a periplasmic or extracellular sugar non-
specific endonuclease. Its physiological function is not
known, but it has been proposed to be involved in the
prevention of the uptake of foreign DNA, the degrada-
tion of intestinal mucus to facilitate colonization, and

pointed out by Altermark et al. [18]. The first is loop
51–54 which contains two more positive charges in
VsEndA than in VcEndA, but is unlikely to contact
DNA. The second is residues 69 and 71 which partici-
pate in the formation of the substrate binding site.
These residues are Gln and Lys, respectively, in
VsEndA, but both are replaced by Asn in VcEndA.
Intuitively, such changes in charge and steric effects
may alter substrate binding and salt sensitivity.
In this study, the effect of NaCl concentration on
the kinetic constants and thermodynamic stability of
VsEndA and VcEndA was investigated. In addition,
the effects of reciprocal mutations of two non-con-
served DNA binding site residues (VsEndA, Q69N and
K71N; VcEndA, N69Q and N71K) on the kinetics,
thermostability and salt dependence of these enzymes
Effects of salt on Vibrio endonucleases L. Niiranen et al.
1594 FEBS Journal 275 (2008) 1593–1605 ª 2008 The Authors Journal compilation ª 2008 FEBS
were examined. Although salt stabilizes both native
enzymes equally, VsEndA is adapted to retain activity
at much higher salt concentrations than VcEndA. The
relationship between these observations and the struc-
ture determined for the VcEndA N69Q variant, as well
as the previously published native EndA structures, is
discussed. A thorough decomposition of the thermo-
dynamic data, together with mutational and structural
investigations, was used to gain an insight into halotol-
erant adaptation.
Results
Protein production and thermal stability

state. The increases in T
m
from 0.175 to 1 m NaCl
were 10.1 and 9.0 °C for VsEndA and VcEndA,
respectively (Table 1). The DH
cal
values increased with
salt concentration, except for VcEndA above 0.425 m
NaCl, although DH
eff
also increased in this case.
VsEndA Q69N showed a higher T
m
value, but DH
cal
was unchanged. All other mutants showed T
m
values
comparable with the native enzyme, but a lower DH
cal
.
The accordance between DH
cal
and the model-depen-
dent van’t Hoff enthalpy (DH
eff
) was best at moderate
salt concentrations, decreasing at high extreme concen-
trations and for the mutants. The denaturation heat
capacity increment could not be determined because of

in a buffer
containing 0.175, 0.425 and 1.00
M NaCl for native VsEndA, and
also 0.050
M NaCl for native VcEndA. For VsEndA and VcEndA
mutants, 0.425 and 0.175
M NaCl, respectively, were used.
Thermograms were baseline-subtracted and normalized for protein
concentration.
L. Niiranen et al. Effects of salt on Vibrio endonucleases
FEBS Journal 275 (2008) 1593–1605 ª 2008 The Authors Journal compilation ª 2008 FEBS 1595
enzymes (0.175 and 0.1 m for VsEndA and VcEndA,
respectively), but the optimum was much broader for
VsEndA.
The reciprocal mutations of the two residues partici-
pating in creating the substrate binding site affected
both kinetic constants (Table 2). The variants dis-
played higher K
m
and k
cat
values, especially at high
salt concentrations, except for the VcEndA N71K
mutation which showed a decreased k
cat
value and a
minimal effect on K
m
. The catalytic efficiency of all
variants was decreased compared with the native

was also rotated, interacting only with a symmetry
related molecule with a hydrogen bond to Glu179 O
and a water-mediated bond to Gln180 O.
Electrostatic calculations
The electrostatic surface potentials of the enzyme vari-
ants were calculated at the optimal salinity of the
native enzymes (Fig. 4). The effects of the mutations
on the overall potentials were small, but some local
changes were observed. The mutations of VsEndA
appeared to result in a less positively charged surface
by increasing the exposure of a negatively charged
patch (VsEndA Q69N, Fig. 4B) or through the loss of
a positive charge (VsEndA K71N, Fig. 4C). VcEndA
N69Q mutation (Fig. 4E) led to the rotation of a
neighbouring positive charge, Arg67, whereas, in
Table 1. Thermodynamic parameters of the thermal unfolding of
VsEndA and VcEndA as a function of NaCl concentration deter-
mined by DSC.
NaCl
(
M)
T
m
(°C)
DH
cal
(kJÆmol
)1
)
DH

a
Molecular masses: VsEndA, 25 005 Da; VcEndA, 24 732 Da;
VsEndA mutants, 24 645 Da; VcEndA mutants, 24 991 Da.
b
Mini-
mal values as a result of aggregation.
Fig. 2. Plot of the kinetic parameters K
m
(A) and k
cat
(B) for native
VsEndA (d) and VcEndA (s) in 0–0.6
M NaCl. The error bars repre-
sent maximum and minimum values.
Effects of salt on Vibrio endonucleases L. Niiranen et al.
1596 FEBS Journal 275 (2008) 1593–1605 ª 2008 The Authors Journal compilation ª 2008 FEBS
VcEndA N71K (Fig. 4F), an increased positive surface
potential was observed.
Discussion
For marine organisms and their extracellular proteins,
adaptation to environmental conditions can be
assumed to be somewhat more complex than simple
temperature or salt adaptation. Previous studies of the
two secreted endonucleases VsEndA (marine psychro-
philic) and VcEndA (estuarine mesophilic) have shown
that their activity is strongly dependent on tempera-
ture, but also on NaCl concentration [18]. We studied
how different salt concentrations and mutations affect
the stability and kinetic constants of VsEndA and
VcEndA, and found the effects to be striking, espe-

direct relationship between salinity and the upshift in
the thermal unfolding temperature T
m
[26–28]. This
agrees with our finding of a nearly equal increase in
the T
m
values of the two enzymes when salt is added,
although the salt-induced increase in enthalpy is more
pronounced in VsEndA than in VcEndA.
A salt-induced increase in T
m
with a simultaneous
decrease in DH
cal
has been proposed to result from
stronger but less cooperative intramolecular interac-
tions [29]. In this context, cooperativity means that the
protein structure unfolds as a single unit (one single
transition), as opposed to several more or less inde-
pendent units (several transitions). The increase in T
m
and DH
cal
of EndA with increasing salt concentration
Table 2. Kinetic constants for native and mutant VsEndA and VcEndA at 0–0.6 M NaCl.
[NaCl] (
M) VsEndA VsEndA Q69N VsEndA K71N VcEndA VcEndA N69Q VcEndA N71K VsEndA ⁄ VcEndA
K
m

0.175 213 133 105 50.1 10.7 32.3 4.2
0.250 200 103 122 14.5 3.11 8.25 14
0.350 123 78.0 101 2.09 0.786 2.23 59
0.425 112 35.1 38.5 0.717 160
0.500 62.8 21.0 23.6
0.600 16.1
L. Niiranen et al. Effects of salt on Vibrio endonucleases
FEBS Journal 275 (2008) 1593–1605 ª 2008 The Authors Journal compilation ª 2008 FEBS 1597
could therefore be interpreted, conversely, as increased
cooperativity of unfolding and a more compact struc-
ture as a result of stronger intramolecular interactions.
These salt-induced effects on DH
cal
and DH
eff
are
stronger in VsEndA, possibly because of the increased
number of solvent-exposed charged and hydrophobic
residues relative to VcEndA, as found when viewing
the molecular surfaces and their amino acid properties.
This indicates a certain degree of salt dependence of
VsEndA stability, but is in disagreement with the
observation that the T
m
values for both enzymes are
equally affected by salt addition. It has been suggested
that salt stabilizes halophilic proteins to a greater
extent than non-halophilic proteins, and that halophilic
proteins are destabilized by low salt concentrations [9].
Both effects may originate from the characteristic high

provides a positive entropic effect that drives substrate
binding. This effect is dependent on both temperature
and salt concentration [32,33]. At elevated salt concen-
trations or low temperatures, the gain in entropy on
release of ions is reduced and substrate binding is
therefore weaker [33]. This makes binding of highly
charged DNA very challenging for marine enzymes.
DNA binding to non-halophilic proteins has been
found to be inversely dependent on salt concentration
[32,34], whereas the binding efficiency of halophilic
proteins appears to actually increase with increasing
salt concentration [35,36]. A halophilic nuclease from
Micrococcus varians [37] with maximal activity in
3–4 m NaCl displays an excess of acidic residues char-
acteristic of many halophilic enzymes. It is possible
that this enzyme has a binding mechanism involving
counterion uptake, similar to that proposed for the
halophilic Pyrococcus woesei TATA-box binding pro-
tein [36]. Contrary to these halophilic proteins,
VsEndA displays an excess of basic residues contacting
the negatively charged substrate, and the K
m
value
increases with increasing salt concentration, although
this occurs at a much higher salinity than for VcEndA.
In our previous study of EndA temperature adapta-
tion, the more positively charged surface of VsEndA
was considered not to decrease the K
m
value relative

a
6.5 (35.6)
Wilson B-factor (A
˚
2
) 20.5
Refinement
PDB entry 2VND
Resolution (A
˚
) 15.00-1.70
R-factor (all reflections) (%) 19.7
R-free (%)
b
25.9
No. of atoms 1928
No. of water molecules 223
No. of other molecules 1 Mg
2+
,1Cl
)
rmsd bond lengths (A
˚
) 0.017
rmsd bond angles (deg) 1.520
Average B-factor (A
˚
2
)
All atoms 17.1

R-free calculations.
Effects of salt on Vibrio endonucleases L. Niiranen et al.
1598 FEBS Journal 275 (2008) 1593–1605 ª 2008 The Authors Journal compilation ª 2008 FEBS
same magnitude. This can be explained by the similar
or slightly lower electrostatic surface potential of
VsEndA compared with VcEndA at the respective
optimal salinities (Fig. 4A,D). The results of the pres-
ent study show that the K
m
values are strongly affected
by the NaCl concentration, similar to the surface
charge of the enzyme. The higher positive charge of
VsEndA therefore decreases K
m
, but this is a method
of coping with the charge shielding of buffer solutes
rather than low temperatures. The higher charge may
allow VsEndA to retain sufficient charge, even at rela-
tively high salinity, to enable tight substrate binding,
contrary to VcEndA.
The salt adaptation of kinetic constants as striking
as that observed in the present study has not been pre-
sented previously. Only two comparative studies of the
salt-dependent kinetics of a non-halophilic and a salt-
adapted enzyme have been published to date. In the
comparison of halotolerant Dunaliella salina carbonic
anhydrases dCA I and dCA II and the human homo-
logue in 0–0.5 m NaCl, the largest differences were
found in the K
m

is a typical mechanism
[7]. A high k
cat
value may also be a feature of salt tol-
erance, but more studies on halotolerant enzymes are
required to verify this. The addition of salt may cause
the EndA substrate binding cleft to reach a more opti-
mal configuration for enzyme catalysis, thereby affect-
ing k
cat
. The DSC thermograms indicate that salt
constricts the structural fluctuations of the enzyme. At
a certain concentration, these fluctuations may become
optimal for enzymatic turnover, whereas, at salt con-
centrations above the optimum, the structure becomes
too rigid and will function less optimally. If the stabi-
lizing effect of salt is caused mainly by the weakening
Fig. 3. (A) Electron density (2F
o
– F
c
at 1r contoured in blue) and omit (F
o
– F
c
at 3r contoured in green) maps illustrating the orientation of
Asn71 and the N69Q mutation in the VcEndA N69Q structure. (B) Superposition of the VcEndA N69Q mutant (red), native VcEndA (blue)
and VsEndA (green) structures. (C) A partial sequence alignment of VsEndA and VcEndA. The asterisks indicate the non-conserved residues
selected for mutagenesis, and the plus sign denotes the catalytically important His80. Sequence numbering follows that of Vvn [20].
L. Niiranen et al. Effects of salt on Vibrio endonucleases

F
Fig. 4. Electrostatic surface potentials in
the DNA binding groove of VsEndA with a
modelled DNA (A), VsEndA Q69N (B) and
VsEndA K71N (C) all in 0.425
M NaCl, and
VcEndA (D), VcEndA N69Q (E) and VcEndA
N71K (F) all in 0.175
M NaCl. The black
arrows show the mutated residues. The sur-
face potential is coloured from )10 kT ⁄ q
(red) to 10 kT ⁄ q (blue).
Effects of salt on Vibrio endonucleases L. Niiranen et al.
1600 FEBS Journal 275 (2008) 1593–1605 ª 2008 The Authors Journal compilation ª 2008 FEBS
N69Q is slightly altered as both residues 71 and 69 are
moved in the crystal structure (Fig. 3), and the current
orientation of Gln69 is different from the Vvn–DNA
structure (Fig. 3B) and very close to a modelled DNA
backbone, possibly explaining the higher K
m
values
(Table 2). Gln69 in VsEndA is poorly defined in the
native crystal structure, and the characterization of the
VsEndA Q69N mutant (Table 2) reveals poorer DNA
binding and an increased k
cat
with a maximum at
0.5 m NaCl. The Gln69 side chain in the Vvn–DNA
structure (PDB 1OUP) is less than 3.2 A
˚

and increased k
cat
compared with VsEndA, indicating
that the positive charge is more important for DNA
binding in VsEndA than in VcEndA.
No side chain contacts are seen for residue 71 in
the native structures or models of the mutants. As the
longer side chain of lysine has more rotamers, the
N71K substitution in VcEndA may stabilize the struc-
ture by increasing the rotational entropy, whilst retain-
ing the backbone interactions. An increase of 1 °Cis
observed for the T
m
value of this mutant. In VsEndA
K71N, both DH
cal
and the cooperativity of unfolding
are decreased, possibly indicating changes in the
hydrogen bonding networks. The increase in K
m
may
be the result of a slightly enlarged binding site or less
positive charge. Indeed, the changes seen in the electro-
static surface potential of each of the mutants (Fig. 4)
match surprisingly well with their kinetic results. Both
VsEndA mutants and the VcEndA N69Q mutant show
more dispersed or less positive charge, and, accord-
ingly, display higher and more salt-sensitive K
m
values.

the catalytic cleft [22,41]. The identity of the rate-limit-
ing step in the endonuclease reaction mechanism is not
known. However, the high catalytic efficiency of both
VsEndA and VcEndA (k
cat
⁄ K
m
in the region of
10
8
s
)1
Æm
)1
) shows that the reaction is nearly diffusion
controlled, suggesting that the rate-limiting step is
either substrate binding or dissociation. As all muta-
tions affect K
m
, especially at high salt concentrations,
the optimization and salt tolerance of binding interac-
tions are most probably hampered by the mutations
by electrostatic, steric or flexibility effects. The less
tight binding of DNA may enable the enzymes to
release the products more easily, thus leading to the
observed increase in the k
cat
values of three of the vari-
ants. Similarly, the seven-fold higher k
cat

effect indicates the formation of a more compact struc-
ture through the strengthening of intramolecular inter-
actions or the weakening of intramolecular repulsive
forces, and the salt dependence of VsEndA stability.
The higher positive electrostatic surface potential of
VsEndA compared with VcEndA plays a key role in
adaptation. On the whole, the characteristics of
VsEndA and VcEndA illustrate the fine-tuned adapta-
tion to their natural environments.
Materials and methods
Site-directed mutagenesis and plasmid
purification
Residue targets for mutagenesis were selected on the basis
of the sequence and structural alignments of Vvn, VsEndA
and VcEndA. The selected residues 69 and 71 were non-
conserved between VsEndA and VcEndA, located in the
DNA binding region and close to the active site. Site-direc-
ted mutagenesis was performed using a QuikChange Site-
Directed Mutagenesis Kit (Stratagene, Cedar Creek, TX,
USA), as described in the manual. The oligonucleotides
were synthesized by Sigma-Aldrich (St Louis, MO, USA).
Mutated plasmids were transformed into E. coli TOP10
cells (Invitrogen, Carlsbad, CA, USA), and plasmid extrac-
tion was performed using QIAprep minipreps (Qiagen,
Hilden, Germany) or the alkaline lysis method [43].
Expression and purification
The expression and purification of recombinant VsEndA
and VcEndA native enzymes and mutants were performed
as described previously [11] with a few modifications. Cells
were cultured in either shake-culture flasks or a Techfors S

values were calculated by fitting the
velocity data to the Michaelis–Menten equation.
Differential scanning calorimetry
Differential scanning calorimetry experiments were con-
ducted on a Nano-Differential Scanning Calorimeter III,
model CSC6300 (Calorimetry Sciences Corporation, Lin-
don, UT, USA). Preparations of the native enzymes were
first filtered with a 0.45 lm Spin-X centrifuge tube filter
(Corning, Corning, NY, USA), and then dialysed overnight
at 4 °C against 1 L of dialysis buffer (50 mm Hepes, 5 mm
MgCl
2
, pH 8.0) containing 0.050, 0.175, 0.425 or 1.00 m
NaCl. Slide-A-Lyzer dialysis discs from Pierce (Rockford,
IL, USA) with a 2 kDa cut-off were used. The protein con-
centration of the dialysed enzyme solution was determined
using BioRad Protein Assay Dye Reagent Concentrate
(BioRad, Hercules, CA, USA) with bovine serum albumin
(Sigma) as standard. The dialysates were used as blank ref-
erences in DSC runs. Reference buffers and samples were
carefully degassed before loading into the DSC cells. The
scans were performed at a constant pressure of 304 kPa in
the range 15–75 °C or 20–80 °C with a heating rate of
1 °CÆmin
)1
. Thermograms were analysed according to a
single non-two-state transition model in which T
m
, DH
cal

mosflm [44], scaled with scala, and the structure factors
obtained with truncate in the ccp4 program suite [45].
The structure was solved by molecular replacement using
the program molrep [46] in ccp4 and the structure of
native VcEndA (PDB code 2G7E) as a search model. The
structure was refined in refmac5 [47] interspersed with
rounds of manual model building in O [48] based on
r
A
-weighted 2F
o
– F
c
and F
o
– F
c
electron density maps.
The final model was validated using procheck [49].
Molecular modelling and electrostatic calculations
Continuum electrostatic calculations were carried out using
the delphi program package [50,51]. The parse3 set of
atomic radii [52], together with formal charges, was used in
all calculations. The electrostatics were determined using
the linear Poisson–Boltzmann equation and a three-dimen-
sional grid with a size of 165 · 165 · 165. Stepwise focus-
ing was used to increase the accuracy [53]. Initially, a rough
grid was calculated with Coulombic boundary conditions,
and the resulting grid was adopted as the boundary condi-
tion for one further focused calculation. The protein mole-

D, Feller G, Georlette D, Gratia E, Hoyoux A, Meuwis
MA et al. (2001) Did psychrophilic enzymes really win
the challenge? Extremophiles 5, 313–321.
6 Hochachka PW & Somero GN (1984) Biochemical
Adaptation. Princeton University Press, Princeton, NJ.
7 Siddiqui KS & Cavicchioli R (2006) Cold-adapted
enzymes. Annu Rev Biochem 75, 403–433.
8 Mevarech M, Frolow F & Gloss LM (2000) Halophilic
enzymes: proteins with a grain of salt. Biophys Chem
86, 155–164.
9 Elcock AH & McCammon JA (1998) Electrostatic con-
tributions to the stability of halophilic proteins. J Mol
Biol 280, 731–748.
10 Premkumar L, Greenblatt HM, Bageshwar UK, Sav-
chenko T, Gokhman I, Sussman JL & Zamir A (2005)
Three-dimensional structure of a halotolerant algal car-
bonic anhydrase predicts halotolerance of a mamma-
lian homolog. Proc Natl Acad Sci USA 102, 7493–
7498.
11 Altermark B, Niiranen L, Willassen NP, Smalas AO &
Moe E (2007) Comparative studies of endonuclease I
from cold-adapted Vibrio salmonicida and mesophilic
Vibrio cholerae. FEBS J 274, 252–263.
12 Ohta Y & Hatada Y (2006) A novel enzyme, lambda-
carrageenase, isolated from a deep-sea bacterium.
J Biochem (Tokyo) 140, 475–481.
13 Focareta T & Manning PA (1991) Distinguishing
between the extracellular DNases of Vibrio cholerae and
development of a transformation system. Mol Microbiol
5, 2547–2555.

20 Li CL, Hor LI, Chang ZF, Tsai LC, Yang WZ & Yuan
HS (2003) DNA binding and cleavage by the periplas-
mic nuclease Vvn: a novel structure with a known active
site. EMBO J 22, 4014–4025.
21 Wang YT, Yang WJ, Li CL, Doudeva LG & Yuan HS
(2007) Structural basis for sequence-dependent DNA
cleavage by nonspecific endonucleases. Nucleic Acids
Res 35, 584–594.
22 D’Amico S, Gerday C & Feller G (2003) Temperature
adaptation of proteins: engineering mesophilic-like
activity and stability in a cold-adapted alpha-amylase.
J Mol Biol 332, 981–988.
23 Georlette D, Damien B, Blaise V, Depiereux E, Uver-
sky VN, Gerday C & Feller G (2003) Structural and
functional adaptations to extreme temperatures in psy-
chrophilic, mesophilic, and thermophilic DNA ligases.
J Biol Chem 278, 37015–37023.
24 Nishimura C, Uversky VN & Fink AL (2001) Effect of
salts on the stability and folding of staphylococcal
nuclease. Biochemistry 40, 2113–2128.
25 Lanyi JK (1974) Salt-dependent properties of proteins
from extremely halophilic bacteria. Bacteriol Rev 38,
272–290.
26 Dragan AI, Li Z, Makeyeva EN, Milgotina EI, Liu Y,
Crane-Robinson C & Privalov PL (2006) Forces driving
the binding of homeodomains to DNA. Biochemistry
45, 141–151.
27 Waldron TT, Schrift GL & Murphy KP (2005) The
salt-dependence of a protein–ligand interaction:
ion–protein binding energetics. J Mol Biol 346, 895–

368–381.
34 Record MT Jr, Zhang W & Anderson CF (1998) Anal-
ysis of effects of salts and uncharged solutes on protein
and nucleic acid equilibria and processes: a practical
guide to recognizing and interpreting polyelectrolyte
effects, Hofmeister effects, and osmotic effects of salts.
Adv Protein Chem 51, 281–353.
35 Britton KL, Baker PJ, Fisher M, Ruzheinikov S, Gil-
mour DJ, Bonete MJ, Ferrer J, Pire C, Esclapez J &
Rice DW (2006) Analysis of protein solvent interactions
in glucose dehydrogenase from the extreme halophile
Haloferax mediterranei. Proc Natl Acad Sci USA 103,
4846–4851.
36 Bergqvist S, Williams MA, O’Brien R & Ladbury JE
(2003) Halophilic adaptation of protein–DNA interac-
tions. Biochem Soc Trans 31, 677–680.
37 Kamekura M & Onishi H (1978) Properties of the
halophilic nuclease of a moderate halophile, Micro-
coccus varians subsp. halophilus. J Bacteriol 133, 59–
65.
38 Bageshwar UK, Premkumar L, Gokhman I, Savchenko
T, Sussman JL & Zamir A (2004) Natural protein engi-
neering: a uniquely salt-tolerant, but not halophilic,
alpha-type carbonic anhydrase from algae proliferating
in low- to hyper-saline environments. Protein Eng Des
Sel 17, 191–200.
39 Asgeirsson B & Cekan P (2006) Microscopic rate-con-
stants for substrate binding and acylation in cold-adap-
tation of trypsin I from Atlantic cod. FEBS Lett 580,
4639–4644.

48 Jones TA, Zou JY, Cowan SW & Kjeldgaard M (1991)
Improved methods for building protein models in elec-
tron density maps and the location of errors in these
models. Acta Crystallogr A 47, 110–119.
49 Laskowski RA, Moss DS & Thornton JM (1993) Main-
chain bond lengths and bond angles in protein struc-
tures. J Mol Biol 231, 1049–1067.
50 Rocchia W, Alexov E & Honig B (2001) Extending the
applicability of the nonlinear Poisson–Boltzmann equa-
tion: multiple dielectric constants and multivalent ions.
J Phys Chem B 105, 6507–6514.
51 Rocchia W, Sridharan S, Nicholls A, Alexov E,
Chiabrera A & Honig B (2002) Rapid grid-based
construction of the molecular surface and the use of
induced surface charge to calculate reaction field
energies: applications to the molecular systems and
geometric objects. J Comput Chem 23, 128–137.
52 Sitkoff D, Sharp KA & Honig B (1994) Accurate
calculation of hydration free-energies using macroscopic
solvent models. J Phys Chem 98, 1978–1988.
53 Moreira IS, Fernandes PA & Ramos MJ (2005)
Accuracy of the numerical solution of the Poisson–
Boltzmann equation. J Mol Struct-Theochem 729,
11–18.
L. Niiranen et al. Effects of salt on Vibrio endonucleases
FEBS Journal 275 (2008) 1593–1605 ª 2008 The Authors Journal compilation ª 2008 FEBS 1605