Destabilization of psychrotrophic RNase HI in a localized
fashion as revealed by mutational and X-ray
crystallographic analyses
Muhammad S. Rohman
1
, Takashi Tadokoro
1
, Clement Angkawidjaja
1
, Yumi Abe
1
,
Hiroyoshi Matsumura
2,3
, Yuichi Koga
1
, Kazufumi Takano
1,3
and Shigenori Kanaya
1
1 Department of Material and Life Science, Graduate School of Engineering, Osaka University, Japan
2 Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Japan
3 CREST, JST, Osaka, Japan
Psychrophiles and psychrotrophs are defined as micro-
organisms that can grow even at around 0 °C [1].
Enzymes from these microorganisms are usually less
stable than those from mesophiles and thermophiles
[2–4]. It has been reported that a decreased number of
ion pairs and hydrogen bonds, decreased hydrophobic
interactions and packing at the core, an increased
fraction of nonpolar surface area, a decreased surface

2
O). These mutations also stabilized the So-RNase HI derivative
(4·-RNase HI) with quadruple thermostabilizing mutations in an additive
manner. As a result, the resultant sextuple mutant protein (6·-RNase HI)
was more stable than the wild-type protein by 28.8 °CinT
m
and 27.0
kJÆmol
)1
in DG (H
2
O). To analyse the effects of the mutations on the pro-
tein structure, the crystal structure of the 6·-RNase HI protein was deter-
mined at 2.5 A
˚
resolution. The main chain fold and interactions of the
side-chains of the 6·-RNase HI protein were basically identical to those of
the wild-type protein, except for the mutation sites. These results indicate
that all six mutations independently affect the protein structure, and are
consistent with the fact that the thermostabilizing effects of the mutations
are roughly additive. The introduction of favourable interactions and the
elimination of unfavourable interactions by the mutations contribute to the
stabilization of the 6·-RNase HI protein. We propose that So-RNase HI is
destabilized when compared with its mesophilic and thermophilic coun-
terparts in a localized fashion by increasing the number of amino acid
residues unfavourable for protein stability.
Abbreviations
4·-RNase HI, So-RNase HI derivative with Asn29 fi Lys, Asp39 fi Gly, Met76 fi Val and Lys90 fi Asn mutations; 5·-RNase HI, 4·-RNase
HI derivative with additional Arg97 fi Gly mutation; 6·-RNase HI, 5·-RNase HI derivative with additional Asp136 fi His mutation; D136H-
RNase HI, So-RNase HI derivative with Asp136 fi His mutation; Ec-RNase HI, E. coli RNase HI; GdnHCl, guanidine hydrochloride; PDB,

by 3.6–6.7 °CinT
m
and 1.7–5.2 kJÆmol
)1
in DG(H
2
O)
[13]. They include Asn29 fi Lys, Asp39 fi Gly,
Met76 fi Val and Lys90 fi Asn. The effects of these
mutations are roughly additive, and a combination of
these mutations strikingly increases the stability of
So-RNase HI to a level similar to that of Ec-RNase HI.
These results suggest that Asn29, Asp39, Met76 and
Lys90 are not optimal for the stability of So-RNase HI
and their replacement with other residues increases
stability. However, the stabilization mechanisms of the
protein with these mutations remain to be understood.
In addition, it remains to be determined whether the
four residues mentioned above are the only ones that
are not optimal for the stability of So-RNase HI.
It has been reported that Ec-RNase HI is stabilized
by the Lys95 fi Gly [14] or Asp134 fi His [15] muta-
tion by approximately 7 °CinT
m
at pH 5.5. Because
Lys95 and Asp134 are conserved as Arg97 and
Asp136, respectively, in So-RNase HI, and the struc-
tures around these residues are conserved in So-RNase
HI [9], the Arg97 fi Gly and Asp136 fi His mutations
are also expected to increase the stability of So-RNase

The far-UV CD spectra of these mutant proteins were
similar to that of the wild-type protein (data not
shown), suggesting that these mutations do not
seriously affect the conformation of the protein. The
specific activities of R97G-RNase HI and D136H-
RNase HI were 99% and 65%, respectively, of that of
the wild-type protein (Table 1).
The stabilities of R97G-RNase HI and D136H-
RNase HI against thermal denaturation were analysed
at pH 5.5 in the presence of 1 m guanidine hydrochlo-
ride (GdnHCl) by monitoring the change in the CD
values at 220 nm. Thermal denaturation of these
mutant proteins was fully reversible in this condition.
The thermodynamic parameters characterizing the
thermal denaturation curves of the wild-type and
mutant proteins are summarized in Table 1. The tem-
perature of the midpoint of the transition, T
m
, was
30.4 °C for the wild-type protein, 35.8 °C for R97G-
RNase HI and 40.1 °C for D136H-RNase HI. Thus,
R97G-RNase HI is more stable than the wild-type
protein by 5.4 °CinT
m
and 3.9 kJÆmol
)1
in DDG
m
.
D136H-RNase HI is more stable than the wild-type

and 0.7 m, respectively, for D136H-RNase HI. Thus,
the stabilities of the mutant proteins against urea-
induced denaturation show good agreement with those
against thermal denaturation.
Stabilization of 4
·
-RNase HI with Arg97
fi
Gly
and Asp136
fi
His mutations
To examine whether the Arg97 fi Gly and Asp136 fi
His mutations stabilize the quadruple mutant protein
of So-RNase HI (4·-RNase HI), in which the four
thermostabilizing mutations identified by directed evo-
lution are combined, the quintuple (5·-RNase HI) and
sextuple (6·-RNase HI) mutant proteins of So-RNase
HI were constructed. The 5·-RNase HI and 6·-RNase
HI proteins represent the 4·-RNase HI derivatives
with additional Arg97 fi Gly mutation and additional
Arg97 fi Gly and Asp136 fi His mutations, respec-
tively. These mutant proteins were overproduced in
E. coli and purified to give a single band on SDS-
PAGE like the wild-type protein (data not shown).
The far-UV CD spectra of these mutant proteins were
similar to that of the wild-type protein (data not
shown), suggesting that the quintuple and sextuple
mutations do not seriously affect the protein confor-
mation. The specific activities of the 5·-RNase HI and

)
DH
m
b
(kJÆmol
)1
)
So-RNase HI 7.8 100 30.4 – – 217
R97G-RNase HI 7.7 99 35.8 5.4 3.9 264
D136H-RNase HI 5.1 65 40.1 9.7 7.0 267
4·-RNase HI 5.5 70 49.1 18.7 13.5 359
5·-RNase HI 5.1 65 52.5 22.1 15.9 366
6·-RNase HI 3.4 43 59.2 28.8 20.7 433
Ec-RNase HI
c
9.1 120 52.8 22.4 – 325
a
The enzymatic activity was determined at 30 °C using M13 DNA ⁄ RNA hybrid as a substrate, as described in Experimental procedures.
Each experiment was carried out at least twice and the average value is shown. Errors are within 15% of the values reported.
b
Parameters
characterizing the thermal denaturation of So-RNase HI and its derivatives. The thermal denaturation curves of these proteins were mea-
sured at pH 5.5 in the presence of 1
M GdnHCl. The thermal denaturation of these proteins was reversible in this condition. The melting
temperature (T
m
) is the temperature of the midpoint of the thermal denaturation transition. The difference in the melting temperature
between the wild-type and mutant proteins (DT
m
) was calculated as T

m
values. Each experiment was carried out at least twice and the average value is shown. Errors are within ± 0.3 °C for T
m
, ± 26 kJÆmol
)1
for DH
m
, ± 0.12 kJÆmol
)1
ÆK
)1
for DS
m
and ± 0.3 kJÆmol
)1
for DDG
m
.
c
Data from Tadokoro et al. [11].
Table 2. Parameters characterizing the urea-induced denaturation
of So-RNase HI and its derivatives
a
.
Protein
C
m
a
(M)
M

4.3 8.2 34.8 12.5
a
The urea-induced denaturation curves of these proteins were
measured at pH 5.5 and 20 °C. Urea-induced denaturation of these
proteins was reversible in this condition. The urea concentration of
the midpoint of the urea-induced denaturation curve (C
m
), the mea-
surement of the dependence of DG on the urea concentration (m),
and the free energy change of unfolding in H
2
O[DG(H
2
O)] were
calculated from the urea-induced denaturation curves. The differ-
ence in DG(H
2
O) [DDG(H
2
O)] between the wild-type and mutant
proteins was calculated using the equation: DDG(H
2
O) = m
av
DC
m
,
where m
av
represents the average m value (8.7 kJÆmol

So-RNase HI or 4·-RNase HI.
The stabilities of the 4·-RNase HI, 5·-RNase HI
and 6·-RNase HI proteins against thermal denatur-
ation were analysed as described for R97G-RNase HI
and D136H-RNase HI. Thermal denaturation of these
proteins was fully reversible in this condition. The
thermodynamic parameters characterizing the thermal
denaturation curves of these proteins are summarized
in Table 1. The temperature of the midpoint of the
transition, T
m
, was 49.1 °C for 4·-RNase HI, 52.5 °C
for 5·-RNase HI and 59.2 °C for 6·-RNase HI. Thus,
the 5·-RNase HI protein is more stable than the wild-
type and 4·-RNase HI proteins by 22.1 and 3.4 °C,
respectively, in T
m
, and 15.9 and 2.4 kJÆmol
)1
, respec-
tively, in DDG
m
. The 6·-RNase HI protein is more
stable than the wild-type, 4·-RNase HI and 5·-RNase
HI proteins by 28.8, 10.1 and 6.7 °C, respectively, in
T
m
, and 20.7, 7.2 and 4.8 kJÆmol
)1
, respectively, in

5·-RNase HI and 6·-RNase HI proteins against urea-
induced denaturation show good agreement with those
against thermal denaturation, although the DDG(H
2
O)
and DDG
m
values are significantly different from each
other for these proteins.
Overall structure of 6
·
-RNase HI
The crystal structure of the 6·-RNase HI protein with
the sextuple thermostabilizing mutations was deter-
mined at 2.5 A
˚
resolution. The asymmetric unit of the
crystal structure consists of four protein molecules
(A–D). The structures of these four protein molecules
are virtually identical with one another with rmsd val-
ues of 0.73 A
˚
between molecules D and A, 0.59 A
˚
between molecules D and B, and 0.61 A
˚
between mole-
cules D and C for 148 Ca atoms. In the structures of
these protein molecules, however, three N-terminal
(Met1–Glu3) and four C-terminal (Gln155–Ser158) res-

W122 are shown. For the structure around residues 90 and 97 (E), the side-chains of K89, residue 90 and residue 97 are shown. The dis-
tances between the e-amino groups of K89 and K90, and between the e-amino group of K90 and the guanidino group of R97, in the wild-
type protein are shown. For the structure around residue 136 (F), the side-chains of D12, E50, D72, H126, E133 and residue 136 are shown.
A p-stacking interaction between His126 and His136 in 6·-RNase HI is shown as a gold broken line, together with the distance.
Destabilization mechanism of psychrotrophic RNase HI M. S. Rohman et al.
606 FEBS Journal 276 (2009) 603–613 ª 2008 The Authors Journal compilation ª 2008 FEBS
A
BC
D
F
E
M. S. Rohman et al. Destabilization mechanism of psychrotrophic RNase HI
FEBS Journal 276 (2009) 603–613 ª 2008 The Authors Journal compilation ª 2008 FEBS 607
helices, and around Gly128 in a loop between the aV
helix and bE strand. The shifts around Gly17 and
Asn18 are probably a result of fluctuations rather than
perturbations caused by the mutations, because any
mutation site is located close to this region. The shifts
around Ser95 are probably a result of the
Lys90 fi Asn and ⁄ or Arg97 fi Gly mutations, and
those around Gly128 are probably caused by the
Asp136 fi His mutation. The details of these shifts are
described in the Discussion section.
The solvent accessibilities of the amino acid residues
that are located around the mutation sites, including
the parent and mutated residues at these sites, were
calculated on the basis of their accessible surface areas
in a native and extended structure. Comparison of
these values for the wild-type and 6 ·-RNase HI
proteins indicated that the solvent accessibilities of all

mational change caused by each mutation.
Asn29
fi
Lys
The 6·-RNase HI structure around residue 29 is com-
pared with that of the wild-type protein in Fig. 1B.
Asn29 and Lys29 are located in the bB strand and are
partially exposed to the solvent by 20 and 39%, respec-
tively. In the wild-type protein, Asn29 forms hydrogen
bonds with Thr34 and Glu131, which are located in the
bC strand and aV helix, respectively. In 6·-RNase HI,
Lys29 forms an ion pair with Glu131. The distances
between the Nf atom of Lys29 and the Oe2 atom of
Glu131 are 2.7, 4.1, 3.3 and 3.3 A
˚
for molecules A, B,
C and D, respectively. Thus, by the Asn29 fi Lys
mutation, one ion pair is introduced and two hydrogen
bonds are eliminated at the mutation site. Both the
hydrogen bond and ion pair have been reported to
contribute to protein stabilization [16,17]. However, the
finding that So-RNase HI is stabilized by the
Asn29 fi Lys mutation by 3.6 °CinT
m
and 3.5
kJÆmol
)1
in DG(H
2
O) [13] suggests that the stabilization

Fig. 2. Displacement of the Ca coordinates between the 6·-RNase
HI and wild-type proteins (full line) and between molecules C and D
(broken line). a Helices and b strands are indicated by bars.
Destabilization mechanism of psychrotrophic RNase HI M. S. Rohman et al.
608 FEBS Journal 276 (2009) 603–613 ª 2008 The Authors Journal compilation ª 2008 FEBS
Asp39 is changed to Ala (Ala37) in Ec-RNase HI,
which is buried inside the protein molecule by 83%.
Therefore, the Asp39 fi Gly mutation stabilizes the
protein, probably because hydrophobic interactions
around the mutation site increase. In the wild-type
protein, Asp39 forms hydrogen bonds with Gln149.
However, these hydrogen bonds may not seriously
contribute to the stabilization of the protein, because
the hydrogen bond partner, Gln149, can form hydr-
ogen bonds with water molecules.
Met76
fi
Val
So-RNase HI is stabilized by the Met76 fi Val muta-
tion by 6.7 °CinT
m
and 5.2 kJÆmol
)1
in DG(H
2
O)
[13]. The 6·-RNase HI structure around residue 76 is
compared with that of the wild-type protein in
Fig. 1D. Met76 and Val76 are located in the aII helix
within a hydrophobic core and almost fully buried

m
[23]. These results suggest that the fill-
ing of a cavity with Met is not as effective as the filling
of a cavity with Val with respect to protein stabiliza-
tion. The Met residue at the hydrophobic core is less
preferable than Val for protein stability, probably
because its solvation free energy is higher than that of
Val [24], and its linear side-chain is rotated more freely
than the branched one of Val [25].
Lys90
fi
Asn and Arg97
fi
Gly
The 6·-RNase HI structure around residues 90 and 97
is compared with that of the wild-type protein in
Fig. 1E. Lys90 and Arg97 are located in the C-terminal
region of the aIII helix and a long loop between the aIII
and aIV helices, respectively. In the vicinity of Lys90,
Lys89 is located. Lys89, Lys90 and Arg97 are well
exposed to the solvent by 80%, 69% and 95%, respec-
tively. So-RNase HI is stabilized by the Lys90 fi Asn
mutation by 4.1 °CinT
m
and 1.7 kJÆmol
)1
in DG(H
2
O)
[13]. It has been reported that the avoidance of unfa-

because the (/, w) values of Gly97 in the 6·-RNase HI
structure are (54.0°, 66.3°). The reason why the effects
of this mutation on the thermal stabilities of the wild-
type and 4·-RNase HI proteins are not consistent with
those on the conformational stabilities (stabilities
against urea denaturation) remains to be clarified.
It should be noted that a loop region (residues
94–97) is shifted towards the aIII helix at most by 0.5,
3.4, 1.5 and 3.0 A
˚
in the structures of molecules A, B,
C and D, respectively, of 6·-RNase HI when
compared with that in the structure of the wild-type
protein. As shown in Fig. 2, the largest shift is
observed for the Ca atom of Ser95. Elimination of the
positive charge repulsions among Lys89, Lys90 and
Arg97 may be responsible for this shift. However, the
mutation sites at residues 90 and 97 are close to the
protein–protein contacts in the crystal packing, which
may account for the large deviation in the loop shift
among the molecules A–D.
Asp136
fi
His
The Asp136 fi His mutation stabilizes the wild-type
and 5·-RNase HI by 9.7 and 6.7 °C, respectively in
M. S. Rohman et al. Destabilization mechanism of psychrotrophic RNase HI
FEBS Journal 276 (2009) 603–613 ª 2008 The Authors Journal compilation ª 2008 FEBS 609
T
m

action (Fig. 1F). The distances of this interaction are
3.9 and 3.7 A
˚
for molecules C and D, respectively. A
p-stacking interaction has been reported to contribute
to protein stabilization [28]. However, this interaction
may not be a major stabilization factor of the mutant
protein with the Asp136 fi His mutation, because this
interaction is not observed in the structures of the
Ec-RNase HI variants with the corresponding mutation
[31,32]. According to the crystal structures of these
Ec-RNase HI variants, the position of His124, which
corresponds to His126 of So-RNase HI, varies for dif-
ferent proteins, because of the intrinsic flexibility of the
loop containing His124 and the crystal packing effect.
Destabilization mechanism of So-RNase HI
A combination of the six thermostabilizing mutations
increases the stability of So-RNase HI by 28.8 °Cin
T
m
and 27.0 kJÆmol
)1
in DG(H
2
O). Five of the six
substituted residues in the resultant sextuple mutant
protein (6·-RNase HI) are found in the corresponding
positions of at least one of the amino acid sequences
of its mesophilic and thermophilic counterparts. Lys29
is conserved as Arg27 in Ec-RNase HI and Arg31 in

pET500M [11] and pET500M4x [13] for the overproduction
of So-RNase HI and 4·-RNase HI, respectively, were also
previously constructed in our laboratory.
Mutagenesis
The genes encoding R97G-RNase HI, D136H-RNase HI,
5·-RNase HI and 6·-RNase HI were constructed by site-
directed mutagenesis using PCR as described previously
[33]. Plasmid pET500M or pET500M4x was used as tem-
plate. The mutagenic primers were designed such that the
codons for Arg97 (CGT) and Asp136 (GAT) were changed
to those for Gly (GGT) and His (CAT), respectively. The
nucleotide sequences of the genes encoding the mutant pro-
teins were confirmed using a Prism 310 DNA sequencer
(Applied Biosystems, Tokyo, Japan). Overproduction and
purification of the wild-type and mutant proteins were
carried out as described previously [11]. The protein concen-
tration was determined from the UV absorption at 280 nm,
assuming that the absorption coefficient at this wavelength
(2.1 for 0.1% solution) was not changed by the mutation.
Enzymatic activity
The RNase H activity was determined at 30 °C and pH 8.0
by measuring the radioactivity of the acid-soluble digestion
product from
3
H-labelled M13 DNA ⁄ RNA hybrid, as
described previously [34]. The reaction mixture contained
10 pmol of the substrate and an appropriate amount of
enzyme in 20 lLof10mm Tris ⁄ HCl (pH 8.0) containing
10 mm MgCl
2

and 2 mm,
respectively. The temperature of the protein solution was
increased linearly by approximately 1.0 °CÆmin
)1
. Thermal
denaturation of these proteins was reversible in the presence
of 1 m GdnHCl. The temperature of the midpoint of the
transition, T
m
, was calculated from curve fitting of the
resultant CD values versus temperature data on the basis of
a least-squares analysis. The enthalpy (DH
m
) and entropy
(DS
m
) changes for thermal denaturation at T
m
were calcu-
lated by van’t Hoff analysis.
Urea-induced denaturation
Urea-induced denaturation curves of So-RNase HI and its
derivatives were measured at 20 °C as described previously
[11]. The proteins (0.1–0.2 mgÆmL
)1
) were dissolved in
10 mm sodium acetate (pH 5.5) containing 100 mm NaCl
and the appropriate concentrations of urea. The protein
solution was incubated for at least 2 h at 20 °C before the
measurement. The urea-induced denaturation of these pro-

mounting for X-ray diffraction.
Structure determination and refinement
X-Ray diffraction data sets of the 6·-RNase HI crystal
were collected at 100 K using synchrotron radiation at the
BL44XU station in SPring-8, using a DIP6040 multiple
Table 3. Data collection and refinement statistics for 6·-RNase HI.
Beamline BL44XU
Wavelength (A
˚
) 1.0
Resolution (A
˚
) 50.0–2.49 (2.59)2.49)
Observations 329 997
Unique reflections 23 782
Completeness (%) 100 (100)
R
merge
(%)
a
14.6 (52.5)
Average I ⁄ r(I) 28.4 (6.39)
Refinement
Resolution limit (A
˚
) 47.52–2.49
Space group P4
1
2
1

R
merge
=
P
I
hkl
) <I
hkl
> ⁄
P
I
hkl
, where I
hkl
is the intensity measure-
ment for reflections with indices hkl and <I
hkl
> is the mean inten-
sity for multiply recorded reflections.
b
R
free
was calculated using
5% of the total reflections chosen randomly and omitted from
refinement.
M. S. Rohman et al. Destabilization mechanism of psychrotrophic RNase HI
FEBS Journal 276 (2009) 603–613 ª 2008 The Authors Journal compilation ª 2008 FEBS 611
imaging plate diffractometer (Bruker AXS Inc., Madison,
WI, USA). These data sets were indexed, integrated and
scaled using the hkl2000 program [36]. The crystal struc-

approval of the Institute for Protein Research, Osaka
University, Osaka, Japan (2008A6909). This work was
supported in part by a Grant-in-Aid for Scientific
Research on Priority Areas ‘Systems Genomics’ from
the Ministry of Education, Culture, Sports, Science,
and Technology of Japan, and by an Industrial Tech-
nology Research Grant Program from the New Energy
and Industrial Technology Development Organization
(NEDO) of Japan.
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