J. Biol. Macromol., 7(1) 3-8 (2007)

ArticleIncreased thermodynamic stability by hydrophilic amino acid substitutions on the surface ofStreptomyces subtilisin inhibitor
Masayuki Oda1, Kenji Kanaori2, and Kazuyuki Akasaka3
1

Graduate School of Agriculture, Kyoto Prefectural University, 1-5 Hangi-cho, Shimogamo, Sakyo-ku,
Kyoto 606-8522, Japan, 2 Graduate School of Science and Technology, Kyoto Institute of Technology,
Goshokaido-cho, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Japan, 3 School of Biology-Oriented Scienceand Technology, Kinki University, 930 Nishimitani, Kinokawa-shi, Wakayama 649-6493, Japan.
Received January 21, 2007; Accepted January 30, 2007
Streptomyces subtilisin inhibitor (SSI) is a homodimer composed of 113 amino acid residues in a
subunit and contains three Met residues at the positions of 70, 73, and 103. Met73 is the P1 site
residue on the surface of SSI and is crucial for inhibitory activity. We have studied the relationshipbetween the thermal stability and hydrophobicity of the P1 site residue. Mutant proteins wereprepared by the substitution of Met73 with Gly, Ala, Val, Leu, Ile, Lys, Asp, or Glu. The melting
temperatures of the mutants were determined by monitoring the temperature dependence of the 1H
NMR signal intensities of Met70 and Met103. The results showed that mutants whose P1 residue ishydrophilic are more stable than the wild-type SSI. A good correlation was obtained between theincrease of the thermal stability and the decrease of the hydrophobicity of the side-chain of the P1
site, which would be a “reverse hydrophobic effect”, as first reported by Pakula and Sauer (Nature
344, 363-364, 1990).
Keywords: NMR, protein engineering, Streptomyces subtilisin inhibitor, thermal stability
Introduction
Streptomyces subtilisin inhibitor (SSI) was
first isolated from Streptomyces albogriseolus S3253, and strongly inhibits alkaline serine
proteases such as subtilisin BPN’ [1]. SSI has aunique homodimeric structure with 11.5 kDa
subunits and has been the subject of thoroughstudies, especially as the target of protein
engineering in the 1980s and 1990s [2,3]. The
three-dimensional structures of both free SSI and
its complex with subtilisin BPN’ show that the two
subunits face each other through predominantly
hydrophobic residues on four-stranded β-sheets,
which are tightly bound and make almost a single
large hydrophobic core (Fig. 1) [4,5]. This
arrangement of the two subunits makes the protein
quite stable, causing the thermal transition of thewild-type to be over 80°C at neutral pH [6,7]. SSI
contains three Met residues, which are all locatedCorrespondence to Masayuki Oda.
Tel & Fax: +81-75-703-5673
E-mail: oda@kpu.ac.jp

at crucial sites for its structure and function (Fig.
1). Met73 constitutes the P1 site in the reactivesite of the inhibitor, and Met70 constitutes the P4
site of the enzyme-binding segment, in which, inboth cases, the side-chains are well exposed to thesolvent. The effects of the Met70 and Met73
mutations on the inhibitory activity and structureof SSI have been studied in some detail [8-13]. In
contrast to the surface residues of Met70 and
Met73, Met103 is deeply buried in thehydrophobic core of each subunit and is located at
a crucial position to define the stability of the core.
We previously reported the effects of Met103
replacement with other amino acids on the
The abbreviations used are: SSI, Streptomyces
subtilisin inhibitor; DSC, differential scanningcalorimetry; δ, chemical shift (in ppm) down fieldfrom the internal reference, sodium 3trimethylsilylpropionate-2,2,3,3-d4; Tm, midpointtransition temperature for denaturation: ∆G, Gibbs
free energy change; ∆HvH, van’t Hoff enthalpy
change for denaturation; OMH, optimal matchinghydrophobicity

Increased thermodynamic stability of SSI

4

Met73
Met70

Met103Figure 1. Backbone structure of an SSI subunitwith the three Met residues, based on the
crystallographic coordinates (PDB code, 3SSI).
The side-chain of Met73 is represented as aspacefill model, and those of Met70 and Met103
are indicated as stick models.
inhibitory activity, structure, and stability,
analyzed by fluorescence, 1H NMR, anddifferential scanning calorimetry (DSC) [7,14]. In
this study, we analyzed the effects of amino acidresidues at the P1 site, the hyper-exposed surfaceof SSI, on the thermal stability by 1H NMR, andre-evaluated the hydrophobic effects on the protein
surface with regard to stability using a wellanalyzed protein, SSI.
Materials and Methods
Protein preparation
The SSI proteins were expressed and purified
as described previously [7]. The purified proteins
were lyophilized and redissolved in 40 mM
sodium deuterated phosphate buffer (pH 7.0)
containing 100 mM NaCl. Here the name of eachmutant protein is indicated as, for example, M73G
for the mutation that replaces Met73 with Gly.
NMR measurements
1
H NMR spectra were measured on a JEOL
GX-400 spectrometer with standard 5 mm probes.
The lyophilized proteins were redissolved to a
final protein concentration of 5 mg ml-1. Chemical
shifts (δ) were measured relative to an internalreference, sodium 3-trimethylsilylpropionate2,2,3,3-d4. The midpoint transition temperature fordenaturation (Tm) is defined as the temperature atwhich the peaks of the native and denatured
proteins have equal intensities. The van’t Hoffenthalpy change for denaturation (∆HvH) was
calculated from the conventional linear van’t Hoffplot, using a two state model for the scheme N2 ↔
2D [7].
Results
The 1H NMR spectra of the mutant proteins at
30°C were similar to that of wild-type SSI,
indicating that the three-dimensional structures ofmutants, other than at the mutation site itself, arethe same as that of the wild-type, which is also
supported by X-ray crystallography analysis[11,12]. The thermal stabilities were analyzed
based on the methyl proton signals of Met residues
obtained as a function of the temperature at pH
7.0. The three methyl proton signals of Met70,
Met73, and Met103 of the native wild-type SSI are
separately observed and were previously assigned
as follows: Met103 in the native state at δ = 1.78,
Met70 in the native state at δ = 2.08, Met73 in thenative state at δ = 2.13, Met73 and Met103 in the
denatured state at δ = 2.04, and Met70 in the
denatured state at δ = 2.06 [15]. In the denatured
state, the signals of Met73 and Met103 overlap,
but the signal of Met70 is separately observed andcan be used to monitor the denatured fraction[7,15]. The chemical shifts of Met70 and Met103of the mutants, M73V, M73I, M73K, M73E, and
M73D, were similar to those of the wild-type inboth the native and denatured states (Fig. 2). The
transition could be analyzed based on either the
Met70 or Met103 signal, and the Tm and ∆HvH
values were determined from the van’t Hoffanalysis based on the respective signals. For
M73G, M73A, and M73L mutants, because thesignals of Met70 and Met103 in the denatured
state overlap, the Tm and ∆HvH values were
determined on the basis of the sum of these signals(Fig. 3). Table 1 summarizes the thermodynamic
parameters of the wild-type and eight mutant
proteins studied at pH 7.0. The Tm values of
M73G, M73V, M73L, and M73I are lower thanthat of the wild-type, while those of M73A,
M73K, M73D, and M73E are higher than that of
the wild-type. The mutant with higher Tm tends tohave higher ∆HvH.

M. Oda et al.

5

(a)

(a)
Temperature(°C)

Temperature
(°C)

86.0

83.0

87.0

84.0

88.0

85.0

89.0

86.0

90.0

87.0
δ (ppm)δ (ppm)2.5

1.5(b) 100(b) 100
Fraction denatured (%)

2.0

Fraction denatured (%)

2.5

80
60
40
20
0

1.5

80
60
40
20
0

85

86

87 88 89 90oTemperature ( C)

91

82

(c)

(c) 3

2

1

1

0

-1

-2

-2
2.76

2.77
2.78
-3 -1
1/T (x 10 K )2.79

Figure 2. Thermodynamics of M73E mutant for
denaturation at pH 7.0. (a) Temperaturedependence of the 1H NMR spectra in the methyl
proton region. (b) Denatured fraction analyzed
based on Met70 (○) and Met103 (∆) signals. (c)van’t Hoff plots on the basis of the data in (b).

84 85 86 87oTemperature ( C)88

89

2.78 2.79 2.8-3 -1
1/T (x 10 K )

2.81

0

-1

-3
2.75

83

3

2

ln K

ln K

2.0

-3
2.76

2.77

Figure 3. Thermodynamics of M73G mutant for
denaturation at pH 7.0. (a) Temperaturedependence of the 1H NMR spectra in the methyl
proton region. (b) Denatured fraction analyzed
based on the sum of Met70 and Met103 signals.
(c) van’t Hoff plots on the basis of the data in (b).

6

Increased thermodynamic stability of SSI

Table 1. Thermodynamic parameters of wild-type SSI and mutant proteins∆Tm b (°C)
∆HvH (kcal mol-1)
T1/2 c (°C)
ProteinTm (°C)awild-type86.2 ± 0.2
0177.1 ± 5.5
82.2
M73G
85.6
-0.6
185.3
82.0
1.0
203.6
83.4
M73A
87.2
85.1 ± 0.1-1.1
231.1 ± 19.5
81.0
M73V a
-0.6
164.4
81.8
M73L
85.6
a
84.8 ± 0.1-1.4
206.2 ± 12.8
80.4
M73I
M73K a87.1 ± 0.1
0.9
221.7 ± 21.6
82.9
M73D a89.0 ± 0.2
2.8
283.5 ± 47.0
85.3
a
M73E
88.6 ± 0.1
2.4
271.5 ± 28.5
84.2
a
Tm and ∆HvH are the average values for the methyl proton signals of Met residues, as mentioned
in the Results.
b∆Tm is the change in Tm relative to that of the wild-type.
c
Calorimetric data in 1H2O taken from Tamura and Sturtevant [16].
thermal stability of each mutant is comparable forboth measurements (Table 1). Because the solventsused for DSC and NMR were 1H2O and 2H2O,
respectively, the higher Tm values determined by
NMR would be due to the solvent isotope effects
between 1H2O and 2H2O. Several reports haveshown the relatively higher stability in 2H2O anddiscussed the solvent isotope effects [21,22]. In thepresent case of Met73 mutants, the Tm values in2
H2O at pH 7.0 were 4.0 ± 0.4°C higher than thosein 1H2O; these results are similar to those of
Met103 mutants, reported previously [7]. Whilecalorimetric analyses can provide precise
thermodynamic parameters, NMR analyses canmonitor the transitions of the respective residues.
90
89

D
E

88
T m (oC)

Discussion
The thermal stability of each mutant protein
could be correlated with the hydrophobicity of thesubstituted amino acid at the position of 73 (Fig.
4). This would be a “reverse hydrophobic effect”
observed in the mutational study in λ Cro [17].
The “reverse hydrophobic effect” means that theprotein stability can be increased by the aminoacid substitution with decreasing side-chainhydrophobicity at a protein surface. The
hydrophobic group of the hyper-exposed residue
can alter the equilibrium ratio of native todenatured protein. The replacement of Met73 byAsp, which has the lowest optimal matchinghydrophobicity (OMH) [18], generates the mutant,
M73D, with the highest thermal stability among
the mutants analyzed in this study. The stability of
M73G seems to be lower than that predicted fromthe Gly hydrophobicity. The coefficient ofdetermination for the linear fitting to the data in
Fig. 4 actually changes from 0.70 for all data to
0.90 for the data except that for M73G. The lower
stability of M73G should be due to the increased
entropy in the denatured state. Gly lacks β-carbon
and has more backbone conformational flexibility.
In order to increase protein stability in general, it
will be effective to replace Gly with other aminoacids [19]. Furthermore, this can also be applied tothe enhanced binding affinity for protein
interaction [20].
The thermal stabilities of Met73 mutants werealso analyzed by means of DSC [16]. Although theT1/2 values determined by DSC are lower than theTm values determined by NMR, the relative

K

87

A
M

86

L
G

85

V
I

84
-1.5

-1

-0.5

0
0.5
OM H

1

1.5

Figure 4. The correlation of thermodynamic
stability, Tm, with hydrophobicity of substituted
amino acid at 73. The values at x-axis are theoptimal matching hydrophobicity (OMH), as
reported by Sweet and Eisenberg [18]. The nameof introduced amino acid into position 73 is shownin the figure as one-letter symbol.

M. Oda et al.

The respective NMR signals, Met70 and Met103,
located at the surface and in the hydrophobic core,respectively, transit from the native to thedenatured states concomitantly (Fig. 2 and Table1), indicating that the unfolding transition of SSI is
cooperative not only within the core partconsisting of the five-stranded β-sheet and two
short helices but also within the reactive segment
carrying the P1 and P4 sties.
Protein stability is affected not only by theprotein structure itself but also by hydration. While
the residue at 103 of SSI contributes to the native
structure [7], the residue at 73 can determine thestability without changing the native structure.
Although the residue 73 is critical for the
inhibitory function, the M73K mutant, forexample, showed almost the same inhibitory
activity as the wild-type [10,13]. The design
strategy to replace surface residues with morehydrophilic ones will be effective to increase theprotein stability with causing very little
perturbation to the protein structure and function.
Acknowledgements
The authors thank Dr. Shuichi Kojima and
Dr. Kin-ichiro Miura of The University of Tokyo
for the guidance of protein engineering, and Dr.
Atsuo Tamura of Kobe University and Dr. Rieko
Ishima of University of Pittsburgh for helpful
discussion. The authors greatly acknowledge theleadership of the late Prof. Keitaro Hiromi, who
enthusiastically guided the research group on SSI
in the 1970s and 1980s, and was deceased in
March 2004. We owe much to him for ourscientific activities today and dedicate this work tohim.
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Increased thermodynamic stability of SSI

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Determinationofthethermodynamicooparameters, ∆H , ∆S , and ∆Cpo, andevaluation of solvent isotope effects. Protein
Sci. 7, 2405-2412.
Communicated by Shin-ichi Tate