- Email: [email protected]
Surface electrostatic interactions contribute little to stability of barnase
J. Mol. Biol. (1991)
220, 779-788
Surface Electrostatic
Interactions Contribute of Bamase
Little to Stability
Da&a Salit, Mark Bycroft and Alan R. Fershtt MRC Unit for Protein Function and Design Cambridge IRC for Protein Engineering University Chemical Laboratory Cambridge CBl ZEW, U.K. (Received 4 February
1991; accepted 16 April
1991)
Electrostatic interactions are believed to play an important role in stabilizing the native structure of proteins. We have quantified the contribution to stability of an interaction between two oppositely charged side-chains on the surface of barnase. Using site-directed mutagenesis, glutamate 28 and lysine 32 were introduced onto the solvent-accessible side of the second a-helix in barnase. These two residues are separated by one turn of the helix, and so are ideally situated for their opposite charges to interact. Double mutant cycle analysis reveals that the interaction between Glu28 and Lys32 contributes only approximately 92 kcal/mol to stability of the protein. All other interations between exposed charged sidechains in barnase examined so far also contribute little to stability. We explain this low value by their location on the surface, rather than in the interior, of the protein. Keywords: protein stability;
protein folding; electrostatics;
1. Introduction Electrostatic interactions between point charges are ubiquitous in proteins; they play an essential role in molecular recognition, binding, catalysis, assembly of macromolecular structures and co-operativity of allosteric transition (Horovitz et al., 1990; Perutz, 1987; Russell & Fersht, 1987; Warshel & Russel, 1984). It has also been suggested that salt bridges, together with hydrogen bonds between uncharged groups, are a major source of the greater thermal stability of ferredoxins from thermophilic organisms compared with their less stable homologues from mesophilic organisms (Perutz t Raidt, 1975). Although the interactions between oppositely charged side-chains and polypeptide chain termini are generally thought to stabilize proteins, the magnitude of this stabilization has been estimated in only a few instances, and no evidence is available so far that surface salt bridges contribute significantly to protein stability. Marqusee t Baldwin (1987) examined the interaction between oppositely charged pairs of residues 7 Present address: National Institute for Medical Research, The Ridgeway, Mill Hill, London EW7 1AA. U.K. $ Author to whom all correspondence should be addressed.
salt bridges
in a synthetic polypeptide a-helix; they found that the greatest contribution to stability of the helix was obtained when the two residues were four positions apart, with a negatively charged glutamate closer to the N terminus of the helix and a positively charged lysine closer to the C terminus. Since these peptides have a relatively high helix content at extreme pH values, Perutz & Fermi (1988) argued that this stabilization may be due to the non-polar rather than electrostatic interactions between the glutamate and lysine residues. In a different set of synthetic a-helical polypeptides, however, Bradley et al. (1990) found no evidence of stabilizing interactions between Glu, Lys (i,i + 3) or (i,i + 4) ion pairs. Anderson et al. (1990) suggest that the half-buried salt bridge between His31 and Asp70 in phage T4 lysozyme contributes 3 to 5 kcal/ mol (1 cal = 4184 J) to stability of the enzyme. Recently, several electrostatic interactions on the surface of barnase, the extracellular ribonuclease from Bacillus amyloliquefacien.s, have been examined in detail in this laboratory. Barnase is a monomeric enzyme of 110 amino acid residues, with an M, value of 12,382 (Hartley & Barker, 1972). Its gene has been cloned and expressed in Escherichia coli (Paddon & Hartley, 1986). The X-ray crystal structure of barnase has been solved at high resolution (Mauguen et al., 1982), and two-dimensional
779 0022-2836/91/150779-10
$03.00/O
0
1991 Academic
Press Limited
D. gali et al.
780
Table 2 Contacts between Ala32 and other residues observed in the crystal structure of barnuse
S28 A32
Ala32 atom
Figure 1. Ribbon diagram of barnase, showing the sidechains of Ser28 and Ala32.
n.m.r.t has been used to assign resonances for proteins in all its residues (Bycroft et al., 199%) and to determine its structure in solution (M.B., S. Ludvigsen, A.R.F. 8z F. Poulsen, unpublished results). Despite its small size, barnase has extensive secondary structure, including two a-helices and a five-stranded antiparallel /?-sheet (Fig. 1). It undergoes reversible, two-state denaturation induced in vitro by solvent or high temperature (Hartley, 1968; t Abbreviations used: n.m.r., nuclear magnetic resonance; COSY, 2D correlated spectroscopy; NOESY, 2D nuclear
Overhauser
correlated spectroscopy; enhancement.
spectroscopy;
TOCSY,
2D total
NOE, nuclear Overhauser
Table 1 Contacts between Ser28 and other residues observed in the crystal structure of barnase Other atom
Distance (A)
N
C Thr26 0 Thr26 Oyl Thr26 CB Lys27
33 33 35 33
(!
Cp Glu29 N Ala30 N Gln31
3.7 34 39
0
N Ala30 N Gln31 C’ Gln31 C Gln31 Cfi Gln31 N Ala32 c” C@
36 37 40 39 39 30 38 33
C’
Cyl Thr26 N Glue9 C Lys27
40 3.1 3.7
07’
c Lys27
40
Ser28 atom
At.oms up to 4 A apart are listed.
Other atom
Distance (A)
N
c 0 C 0 C’
Oh29 Glu29 Ala30 Ala36 Glu31
38 30 3.3 3.3 3.2
C”
0 Glu29
3.5
C
C8 Leu33 N Gly34 0 Glu29 0 Ala30
3.7 34 36 39
0
N Gly34
37
P
0 C N 0
36 37 35 33
Glu28 Gln31 Leu33 &x28
Atoms up to 4 A apart are listed.
Kellis et al., 1989), allowing for straightforward thermodynamic measurements of protein stability. As barnase contains no cysteine residues, there are no complications due to presence of disulfide bridges in the folded and unfolded state. Barnase has been used extensively in investigating the importance of specific interactions for barnase stability and in the folding pathway (Bycroft et al., 1996a; Horovitz et al., 1990; Kellis et al., 1988, 1989; Matouschek et aZ., 1989, 1996; Sali et al., 1988; Serrano & Fersht, 1989; Serrano et al., 1999). The general approach has been to make conservative site-directed mutants of the enzyme in order to disrupt, or, more rarely, add, a particular interaction and then to compare thermodynamic or kinetic properties of wild-type and mutant enzymes. Here we present a study of a surface ion pair that was introduced onto the surface of the second a-helix in barnase (residues 26 to 34) by sitedirected mutagenesis (Fig. 1). The residues mutated are Ser28 and Ala32. Since these two residues are located on the side of the helix that is fully accessible to the solvent and make no contacts with residues outside the helix (Tables 1 and 2), their substitution with charged residues was not expected to disrupt the structure of the enzyme. Ser28 was mutated to glutamic acid and Ala32 to lysine, mimicking the (i+4) Glu, Lys ion pairs from the experiments on model peptides by Marqusee & Baldwin (1987). In addition to the double mutant, the two single mutants were also made and the contribution of the interaction between Glu28 and Lys32 to stability of the protein was determined using double mutant cycles. The mutations were shown to be non-disruptive and the orientation of the mutant side-chains explored using two-dimensional ‘H n.m.r.
Protein
Electrostatic
2. Materials and Methods (a) Materials Molecular biological reagents were obtained from New England Biolabs and Boehringer-Mannheim, the radioand International, from Amersham chemicals SP-Trisacryl from IBF. Urea was highly purified AristaR grade from BDH Limited, and the buffer used in the denaturation experiments was 2-(iV-morpholino)ethanesulfonic acid (Mes) from Sigma. All other reagents were purchased from Sigma, Aldrich or BDH Limited. (b) Expression and purijication
of barnase
The vector used was pTZ18U containing the EcoRT-Hind111 fragment with the genes for barnase and bantar (Serrano et al., 1990). In this construct, the barnase gene is preceded by the promoter and signal sequence of the E. coli alkaline phosphatase gene; therefore, barnase was expressed in low-phosphate medium (Serpersu ul al., 1986). The enzyme was purified to homogeneity as described (Kellis et al., 1989; Mossakowska et al.. 1989). (v) Site-directed mutagenesis Site-directed mutants were made using the Kunkel method (Kunkel et al., 1987). The mutagenic primers used were: for Ser28-+GIu, 5’-GAGGGCTTGT(:C:TTCTT*C*TTTTGTA-3’: for Ala32+Lys. 5’.CCACCCAGCCGAGT*T*T*TTGTGCTTCTG-3’: and for Ser28-+Glu, Ala32+Lys, 5’.CAGCCGAGT*T*T*TTGTGCTTCTT*C*TTTTGTA-3’; where asterisks follow the mismatched bases. The mutants were identified by DNA sequencing. (cl) I Trua.denatura.tion Urea denaturation was observed using fluorescence intensity as the probe, with excitation at 290 nm and emission at 315 nm. Experiments were performed at 25( +@05)“C. pH 6.3. Mes acid/base buffer was used at concentrations of 7 mM, 10 mM and 50 mM. NaCl was added for high salt experiments to final concentrations of 250 mM and 556 mM. The concentration of barnase was 1 PM. The denaturation curves were analyzed as described (Kellis et al., 1988; Serrano & Fersht, 1989). The free energy of unfolding of the protein is linearly proportional to the concentration of the denaturant according to t.he following equation (Pace. 1986): A(&, = AC&,, -m
[urea].
The free energy of unfolding in water (AGH1o), the slope (m) and the half point of denaturation ([urea15c0,,) were obtained for each protein. The difference in free energy of unfolding between different forms of the protein is given by : AA(;,,2
= !!!$?t?
(lureaho:2
- [urealso~oI).
(e) Nuclear magnetic resonance Barnase was dissolved in 65 ml of 90% H,O/lO% ‘H,O to a final concentration between 4 and 6 mM. pH
781
Interactions
was adjusted to 4.5. n.m.r. spectra were acquired at 37 “C on a Bruker AM 500 spectrometer with an Aspect, 3000 computer. COSY (Aue et al., 1976; Bax & Freeman, 1981). KOESY (Jeener el al., 1979; Kumar et al.. 1980). and TOCSY (Braunschweiler & Ernst. 1983) experiments were recorded with 2048 data points in 1, and 512 t, increments, with a spectral width of 8000 Hz in both dimensions. A relaxation delay of 1.5 s was used. Suppression of the solvent signal was achieved by presaturation of the water resonance during the relaxation delay.
3. Results (a) Stability of wild-type and /mutant barnase and determination of the free energy of interaction between Glu.28 and Lys32
Table 3 lists the free energies of unfolding of the wild-type, the two single mutants and the double mutant under different conditions of ionic strength. The SE28 mutant is more stable than wild-type barnase at most ionic strengt,hs, although the difference in stability between the two proteins decreases with increasing salt concentration. ln 7 mM-MeS, the AAGu value between the wild-type and SE28 is 0.52 kcal/mol; in 10 mM-Mes it is 639 kcal/mol; in 50 mrvr-Mes, 0.23 kcal// mol; and in 50 mM-Mes with 250 mM-Nacl, 606 kcal/mol. In 50 mM-Mes, 556 mM-NaCl, the wild-type is more stable than the mutant, the AAG, value being -0.04 kcal/mol. The AK32 is less stable than the wild-type under all conditions examined; the AAG, value between the wild-type and this mutant is -919 kcal/mol in 7 mM-Mes, -018 kcal/mol in 10 mM-Mes, -914 kcal/mol in 50 mM-Mes, -622 kcal/mol in 50 mM-Mes with 250 mM-NaCl, and -024 kcal/mol in 50 mhr-Mes with 556 m&r-NaCl. The stability of the double mutant, SE28AK32, follows a pattern similar to that of the single SE28 mutant, with its AAG, value from the wild-type of 048 kcal/mol in 7 m&r-Mes, 0.33 kcal/mol in 10 m&r-Mes, 625 kcal/mol in 50 mM-Mes, 004 kcal/mol in 50 mM-Mes with 250 mM-NaCl, and -618 kcal/mol in 50 mM-Mes, 556 m&r-NaCl. Urea concentrations at the half-point of denaturation were the most accurate values measured, with the error of &@Ol M, and were therefore used to calculate the differences in free energies of denaturation. The resulting errors in AAG, were kO.03 kcal/mol. The errors in the measurement of AG,,, were much larger, at approximately 1 kcal/mol. The simplest way of estimating the magnitude of the interaction between Glu28 and Lys32 is to compare the stabilities of wild-type and the double mutant, SE28AK32. The problem with this approach is that it does not distinguish between the contribution to stability from the interaction between the two charges introduced by mutation and any contribution due to the interaction of either of these side-chains with the rest of the protein. For example, it is clear from Table 3 that the single mutation from Ser28 to Glu already stabilizes the protein under most conditions, while the single mutation from Ala32 to Lys makes barnase less
782
D. gali et al. Table 3 Free energies of unfolding for wild-type and the mutants at positions 28 and 32, at pH 6.3 Protein
Wsl
[NaCI]
(mM)
(mW
A’%,,
(kcal/mol)
m
PJ~al,O,P~l ABC, (M)
(kcal/mol)
A%, (kcal/mol)
~~28 AK32 SE28AK32
7I 7 7
0 0 0
1021 848 922 10.24
2.16 1.89 2.10 2.17
448 473 4.39 471
+052 -019 + @48
-0.15
G28 AK32 SE28AK32
10 10 10
0 0 0
944 896 8.85 8.95
1.99 1.97 1.99 190
454 4.74 445 4.71
+w39 -@18 +w33
-618
wt SE28 AK32 8E28AK32
50 50 50 50
0 0 0 0
10.25 10.14 1069 lo-63
2.23 216 2.36 2.26
459 469 453 4.70
+023 -014 +0.25
-619
G28 AK32 SE28AK32
50 50 50
250 250 250
1096 9.93 1021 993
2.01 200 2.10 I .99
497 500 486 499
+ 0.06 -0.22 + 0.04
-0.15
%28 AK32 SE28AK32
50 50 50
556 556 556
10.24 10.73 lo-42 lo-70
2.05 1-95 203 2.07
525 523 513 516
- 0.04 - 0.24 -0.18
-914
AAG, values were calculated for each ionic strength as a difference between wild-type mutant. There are small rounding errors in the last column.
stable. Both these effects have to be taken into account when measuring the interaction between Glu28 and Lys32. This problem can be overcome by using double mutant cycles (Carter et al., 1984; Serrano et al., 1990). Such cycles consist of the form of the enzyme in which neither of the partners in the interaction is present (in this case, the wild-type), the form in which both partners are present (SE28AK32) and the two forms in which only one of them is present (SE28 and AK32) (Fig. 2). The free energy of the interaction between the two residues is determined by measuring the unfolding free energies of each of the proteins in the cycle. The difference between the change in the free energy of unfolding upon mutation of the first residue when the second
X
AGE+E-Y
\
/
-
‘E’
E
AGE-E-X
AGE-x--E-XY
I Y
Y
x )
‘E’
(wt) and each
residue is present and absent is the apparent free energy of the interaction between the two residues. If the two mutations are not independent, i.e. there is an interaction between the two residues in the double mutant, the following inequalities are true (using the symbols defined in Fig. 2): A%,.,
Z A G,.,,,.,,,
(1)
A%s., ZAG,,,,,,. (2) The free energy of interaction between X and Y is given by:
By using double mutant cycles, the favorable free energy of interaction between Glu28 and Lys32 was kcaljmol in 7 m&r-Me& found to be -615 -0.18 kcal/mol in 10 mM-Mes, -0.19 kcal/mol in 50 m&r-Mes, -615 kcal/mol in 50 m&r-Mes with 250 mM-NaCl, and -0.14 kcal/mol in 50 m&i-Mes containing 556 mM-NaCl. These values are all within 0.03 kcal/mol of their average, 0.16 kcal/mol. Since the errors in AGint are approximately f604 kcal/mol, the variation of the value with ionic strength is not significant.
\/ E
AGE-Y-N-XY
Figure 2. The scheme for a double mutant cycle. X and Y are the 2 residues being introduced by mutations and AGE,,.,, AcfE.x,E.xy and AGE.y,E.xy the changes AG,+,x> in free energy of unfolding for the appropriate transitions in the cycle.
(b) Comparison of two-dimensional n.m.r. spectra of wild-type and the mutants Fingerprint regions of mutant two-dimensional n.m.r. spectra were assigned, starting with the similarities between the spectra of wild-type barnase
Protein
Electrostatic
Interactions
783
--~-____
Table 4
:
0 ‘I “f
-3.2 -3.4
Chemical shifts of backbone and side-chain protons between residues 27 and 32 in wild-typp. position 28 and 32 mutants Position
Proton
wt
KP7
NH CH VBH ( @H’ (‘?H (“H’ C6H (“H’ (“H ( “H’ (‘“NH
IO.21 398 2.11
SZS/E2S
8.6
I
i’.-
NH (‘“H (‘OH
x79 393 121
c/31
NH (‘“H (‘BH (‘#H’ CqYH (“H’
9.29
9.0
,--W.--v, 9.6 9.4
9.2
9.0 8-8 ppm
, 8.6
SH C”H ( @H (‘BH’ (‘?H (VH’ ( “H (‘6H’ (“H (“H’ C”NH
7.95 4.32 I -tiu
IO.0
I 8.4
0.2
. 8.0
7.8
Figure 3. Sequential connectivities in the second CIhelix of the mutant SE28AK32. p.p.m.. parts per million.
and the mutants. About 75% of the COSY crosspeaks corresponding to the main-chain protons in the mutant,s could be assigned by analogy with the wild-type spectra, with another 15 to 20% of assignments obtained from sequential connectivities (Wiithrich, 1986). The plots of chemical shifts of equivalent main-chain protons in the mutants and the wild-type (Fig. 4) show that the only differences occurred at the position of the mutation or at the immediately adjacent positions. The patterns of NOE connectivities between backbone protons which characterize specific secondary structure elements, i.e. d,,, d,,(i,i + 3) and daN(iri + 4) connectivities for the ol-helices (Fig. 5), and strong d,, and inter-strand NOE contacts for the o-sheets (Fig. 6), were very similar for the wild-type and the three mutants throughout the molecule, including the connectivities in the 26 to 34 cc-helix (Fig. 3). The small differences in the NOE patterns that did occur resulted from collecting data for the mutants at a lower temperature than for the wild-type barnase,
3.99 3+x)
-430
9.6 9.8
9.10 $40
7.74 4.14 IGI 2.45 SA Nl
9.2
A32/K32
SE28AK32
XA I ,46 NA 1.16 2.98 NA NA
SH (‘“H CqBH (“OH’ CYH CqYH
9.0
AK32
i?@2
E19
E 6.8 a Q
9,4
SH (‘“H (‘“H CflH’ CfYH (‘?H’
SE32
367
2.56 1.36
NA NA
-, Proton not present in side-chains; NA. proton not assigned. The wild-type (wt) data are from Psycroft rt ml. (I99Oh).
due to their changed stability. Through-bond connectivities given by TOCSY and COSY experiments were also used to determine resonances of protons in the side-chains that are found on the surface of the second u-helix in barnase. The results for the three mutants and the wild-type are shown in Table 4. Although it is not possible to interpret quantitatively changes in chemical shifts of nuclear resonances, it is known that. the chemical shifts are extremely sensitive to the local magnetic environment of the nuclei. This magnetic environment is in
784
D. gali et al. wt
and
SE28(NH1
wt
7E 4 ,o
and
SE28
(C’H)
/ 6
/
I
I
7
6
9
8 wt
chemical
wt
and
shift
IO
3
4
(p.p.m.1
wt
wt
AK32(NH)
5
chemical
and
6
shift
AK32
7
(p.p.m.)
(Can)
*
I
L
6
7 wt
wt
9
8 chemical
and
shift
SE28AK32
0
IO
3
(p.p.m.!
4
5
wt chemical
wt
(NH)
I
and
shift
SE28AK32
6
7
6
7
(p.p.m.)
(C
Ii)
I
’
I
T26
J 6
7
9
8 wt chemical
shift
IO
3
(p.p.m.1
Figure 4. Chemical shifts of amide and C” protons in the mutants plotted against The wild-type assignment is from Bycroft et al. (1990b). p.p.m., parts per million.
wt
4
5
chemical
shift
the chemical
(p&m.)
shifts
in wild-type
(wt).
785
Protein Electrostatic Interactions SE28 d.Nli, j+4)
-
dpN
-
-
-
duu
d UN
%N duu
_--
---
-
---
dPN
-
-
__30
!O
-
d UN d ou
--
-
---
---
dpN duu
-
-
--
-
-
d au
-
----
% duu d ON
-
d au
SE28AK32
AK32 d aNlbj+4)
d UN d ON
_ -
--
abN
---
d ON
d UN
____
~o”,N~FDG”*D”Lor”“K~CDNYI!KEEIO*
daNl,,j+41
-
_--
dON
d (LN
% duu d (IN
--
d.Nti,j+3) dpN
d aN(i,j+OJ
--
----
-
d.Ntisj+3)
---
~O”,N~FD~“*D”LOrVKKLPDNVlTKsE~~Kl 10
---
--
-
dsN
-
-
-
d ULI
--.
--
--
10’
dpN duu
dPN
d UN d [IN
d(IN
-
---
--
--
L~~VA~KGN~*DV”‘GKS~GGO!FSNREGKL
.o
dpu O’,,,,
dPN duu
w
60
--
-
-
--
--
-
d au
Figure 5. Sequential and secondary (Bycroft et al.. 199Ob) and the mutants.
structure
connectivities
defined by both distances and angles between magnetic nuclei. It is, therefore, assumed that, when chemical shifts of the analogous protons in the wild-type and the mutant spectra are the same, the two protons are in exactly the same environment in the two proteins. As the chemical shifts of backbone protons in the wild-type and in the three mutants are different only at the positions of the mutations and those immediately adjacent (Fig. 4), the two proteins must have largely identical structures. The fact that the NOE patterns characteristic of regular structure elements are unchanged secondary between the wild-type and the mutant (Figs 5 and 6) shows that the mutations did not perturb the turn
between main-chain
protons of t*he wild-type
(WT)
secondary structure of barnase, not even in the second a-helix, where the mutations and the changes in chemical shift occur. Chemical shifts of the assigned side-chain protons of the second a-helix are also found to be very similar between the mutants and the wild-type, except at the positions where the covalent structure has been changed (Table 4), suggesting that the environment of the side-chains was not significantly altered. Unfortunately, the overlap in the region of the twodimensional spectra containing resonances from C?, Cy, Cd and C” protons of the side-chains meant that the through-space NOE connectivities between these protons could not be assigned unambiguously
786
D. #ali WT
et al.
SE28
SE28AK32
K32
H
0
H
H
6
H
ti
6
Figure 6. NOE contacts between /?-strand main-chain protons of wild-type mutants.
MI,
barnase (Bycroft et al.. 19906) and of the
hydrogen bond; - - - - - -. NOE connectivity.
in order to find the relative side-chains.
conformation
of the
4. Discussion Stabilization energies listed in Table 3 indicate that the SE28 mutation by itself stabilizes barnase by about as much as the double mutant (SE28AK32). This may be due to the introduction of a favorable interaction between Glu28 and the a-helix dipole of the 26 to 34 a-helix. Th’_s type of interaction has been observed in barnase (Sali et al., 1988). The interaction energy between two oppositely on the barnase surface charged side-chains measured here is more than an order of magnitude
lower than that reported for buried, hydrogenbonded salt bridges (Anderson et al., 1990), but comparable to the interaction energies that have been measured for other ion pairs and salt bridges on the barnase surface. Serrano et al. (1990) examined the interaction between two oppositely charged side-chains on the first a-helix (residues 6 to 18), Asp12 and Argl6. Asp12 is present in the wild-type enzyme, while the was introduced by the ThrlS-+Arg arginine mutation. Argl6 is also present in the wild-type form of binase, the RNase from Bacillus intermedius which is highly homologous to barnase. In barnase, the TR16 mutant is more stable than the wild-type by 6.5 kcal/mol, and 633 kcal/mol of this free energy can be attributed to the interaction between
Protein Electrostatic Interactions Asp12 and Argl6 Structural evidence suggests that the two residues do not form a hydrogen-bonded salt bridge; instead, Argl6 interacts with the protein backbone and Asp12 forms salt bridge with ArgllO (Serrano et al., 1990). Studies by Horovitz et al. (1990) focused on the interactions between the C-terminal Argl 10 and two residues on the first a-helix of barnase, Asp8 and Aspl2. The side-chains of these residues are in the ideal orientation to form two salt bridges. They found that the interaction energy between Asp8 and ArgllO is 998 kcal/mol with the Aspl2-ArgllO salt and 921 kcal/mol when the bridge present Aspl2-ArgllO salt bridge is absent. Similarly, the free energy of interaction between Asp12 and ArgllO is 1.25 kcal/mol with the Asp&ArgllO salt bridge present and 0.48 when the Asp8-Argl lO salt bridge is removed by mutation. When Asp8, Asp12 and ArgllO are mutated to alanine residues, the resulting mutant is more stable than the wild-type with the two salt bridges intact. That the interaction energies between ion pairs on the protein surface measured in this laboratory are lower than the values reported for buried salt bridges may be explained by the dielectric constant in a protein core being much lower than in water or at the surface of a protein (Warshel & Russel, 1984), so that the enthalpy of interaction between two buried charges should be larger than that between two charges separated by the same distance at the surface of the molecule. In addition, as side-chains on the surface of a protein have more conformational freedom than those in the center of the protein, they lose entropy when being constrained in a salt, bridge. The difference between the results of Marqusee & Baldwin (1987), who observed a stabilizing (i&+4) Glu, Lys interaction in polypeptides largely made of alanine residues, and the work of Bradley et al. (1990), who detected no such interaction between (i,i+4) Glu and Lys in polypeptides containing a variety of residues, suggests that the two oppositely charged side-chains may only interact in the rare instance when the steric hindrance from neighboring side-chains is completely eliminated (Bradley et at., 1990). The local environment of Glu28 and Lys32 in the mutant is more similar to the situation in the synthetic polypeptide described by Bradley et al. than of that by Marqusee et al.; it is possible that a stronger interaction between the two charged sidechains may be observed if the other residues on the helix surface were all mutated to alanine residues. Our conclusion that the interaction between (Glu28 and Lys.32 is weak in the native form of the double mutant is based on the assumption that t,heir side-chains do not interact in the unfolded stat,e; this is a reasonable expectation for a pair of residues four positions apart in a random coil. The relative stability of wild-type barnase and the mutants can. however, also be explained by a model where Glu28 and Lys interact both in the unfolded and the folded state, with the interaction only slightly more favorable in the folded enzyme.
787
have no direct evidence to distinguish between these two possibilities. In either case, though, the presence of the (i,i+4) Glu, Lys ion pair does not contribute significantly to stability of the native protein relative to the denatured state.
5. Conclusion We have shown that the interaction between two oppositely charged side-chains on the surface of the second a-helix of barnase, which are ideally situated to form a surface salt bridge, contributes only approximately 615 kcal/mol to the stability of the protein. This value is an order of magnitude lower than that reported for a partially buried salt bridge (Anderson et al., 1990), but it agrees with other studies of surface electrostatic interactions in barnase (Horovitz et al., 1990; Serrano et al., 1990). We conclude that electrostatic interactions between point charges are strongly context-dependent, with interactions between single ion pairs on the protein surface generally contributing little to protein stability.
D.S. was a recipient of the Sir Hans Krebs Scholarship administered by the Biochemical Society.
References Anderson, D. E., Becktel, W. J. 8: Dahlquist, F. W. (1990). pH-induced denaturation of proteins: a single salt bridge contributes 3-5 kcal/mol to the free energy of folding of T4 lysozyme. Biochemistry, 29, 2403-2408. Aue, W. P., Bartholdi, E. & Ernst, R. R. (1976). Two-dimensional spectroscopy. Application to nuclear magnetic resonance. J. Chem. Phys. 64, 2229-2246. Bax, A. t Freeman. R. (1981). Investigation of complex networks of spin-spin coupling by two-dimensional n.m.r. J. Magn. Reson. 44, ,542-561. Bradley, E. K.. Thomason, J. F.. Cohen, F. E., Kosen! P. A. & Kuntz, I. D. (1990). Studies of synthetic helical peptides using circular dichroism and nuclear magnetic resonance. J. Mol. Biol. 215. 607-622. Braunschweiler, L. & Ernst, R. R. (1983). Coherence transfer of isotropic mixing-application to proton correlation spectroscopy. J. Magn. Reson. 53, 521-528. Bycroft, M.. Matouschek, A.. Kellis, J. T. & Fersht, A. R. (1990a). Detection and characterization of a folding intermediate in barnase by PU’MR. ~Vaturu (London), 346,488-490. Bycroft, M., Sheppard, R. N.. Lau. F. T.-K. & Fersht, A. R. (1990b). Sequential assignment of the ‘H nuclear magnetic resonance spectrum of barnase. Biochemistry, 29, 7425-7432. Carter, P. J., Winter. G.. Wilkinson. A. .I. & Fersht. A. R. (1984). The use of double mutants t.o detect structural changes in the active site of the tyrosyltRNA synthetase (Batiks stuaroth,prm,ophilus). C’rll. 38. 83.5840. Hartley, R. W. (1968). A reversihle thermal t,ransition of the extracellular ribonuclease of Bacillus nmyloliquqfaciens. Biochemistry, 7. “40 I-- "408.
788
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Hartley, R. W. & Barker, E. A. (1972). Amino-acid sequence of extracellular ribonuclease (barnase) of Bacillus amyloliquefaeiens. Nature (London), 235, 15-16. Horovitz, A., Serrano, L., Avron, B., Bycroft, M. & Fersht, A. R. (1990). Strength and co-operativity of contributions of surface salt bridges to protein stability. J. Mol. Biol. 216, 1031-1044. Jeener, J., Meier, B. H., Bachmann, P. & Ernst, R. R. (1979). Investigation of exchange processes by twodimensional NMR spectroscopy. J. Chem. Phys. 71, 45464553. Kellis, J. T., Jr, Kyberg, K., gali, D. & Fersht, A. R. (1988). Contribution of hydrophobic interactions to protein stability. Nature (London), 333, 784-786. Kellis, J. T., Nyberg, K. & Fersht, A. R. (1989). Energetics of complementary side-chain packing in a protein hydrophobic core. Biochemistry, 28, 49144922. Kumar, A., Ernst, R. R. & Wiithrich, K. (1980). Two-dimensional nuclear Overhauser experiment (2D EjOE) for the elucidation of complete protonproton cross relaxation networks in biological macromolecules. B&hem. Biophys. Res. Commun. 95, l-6. Kunkel, T. A., Roberts, J. D. & Zakour, R. A. (1987). Rapid and efficient site-specific mutagenesis without selection. Methods Enzymol. 154, phenotypic 367-382. Marqusee, S. & Baldwin, R. L. (1987). Helix stabilization by Glu- Lys+ salt bridges in short peptides of de novo design. Proc. Nat. Acad. Sci., U.S.A. 84, 8898-8902. Matouschek, A., Kellis, J. T., Jr, Serrano, L. & Fersht, A. R. (1989). Mapping the transition state and pathway of protein folding by protein engineering. Nature (London), 340, 122-126. Matouschek, A., Kellis, J. T., Serrano, L., Bycroft. M. & Fersht, A. R. (1990). Transient folding intermediates characterized by protein engineering. Nature (London), 346, 440-445. Mauguen, Y., Hartley, R. W., Dodson, E. J., Dodson. G. G., Bricogne, G., Chothia, C. & Jacks, A. (1982). Molecular structure of a new family of ribonucleases. Nature (London), 297, 163-164.
Edited
et al.
Mossakowska, D. E., Nyberg, K. $ Fersht, A. R. (1989). characterization of the recombinant Kinetic from Bacillus amyloliquefaciena ribonuclease (barnase) and investigation of key residues in catalysis by site-directed mutagenesis. Biochemistry. 28, 3843-3850. Pace, C. K’. (1986). Determination and analysis of urea and guanidinium hydrochloride denaturation curves. Methods Enzymol. 131, 26G-279. Paddon, C. J. & Hartley, R. W. (1986). Cloning, sequencing and transcription of an inactivated copy extracellular Bacillus amyloliquefaciens of ribonuclease (barnase). Gene, 40, 231-239. Perutz, M. F. (1987). Electrostatic effects in proteins. Science, 201, 1187-1191. Perutz, M. F. & Fermi, G. (1988). Stereochemistry of saltbridge formation in a-helices and b-sheets. Proteins: Struct. Fund. Genet. 4, 294-295. Perutz, M. F. & Raidt, H. (1975). Stereochemical basis of in bacterial ferredoxins and heat stability haemoglobin A2. Nature (London), 255, 256-258. Russell, A. J. & Fersht, A. R. (1987). Rational modification of enzyme catalysis by engineering surface charge. Nature (London), 328, 49&500. Sali, D., Bycroft, M. & Fersht, A. R. (1988). Stabilization of protein structure by interaction of a-helix dipole with a charged side chain. Nature (London). 335. 740-743. Serpersu, E. H., Shortle, D. & Mildvan, A. S. (1986). Kinetic and magnetic resonance studies of effects of genetic substitutions of a Ca2 +-1iganding amino acid in staphylococcal nuclease. Biochemistry, 25, 68-77. Serrano, L. & Fersht, A. R. (1989). Capping and a-helix stability. Nature (London), 342, 296-299. Serrano, L., Horovitz, A., Avron, B., Bycroft, M. & Fersht, A. R. (1990). Estimating the contribution of engineered surface electrostatic interactions to protein stability using double mutant cycles. Biochemistry, 29, 9343-9352. Warshel, A. 6 Russel, S. T. (1984). Calculations of electrostatic interactions in biological systems and in solutions. Quart. Rev. Biophys. 17, 283422. Wiithrich, K. (1986). NMR of Proteins and Nucleic Acids Wiley, Pu’ew York.
by A. Klug
220, 779-788
Surface Electrostatic
Interactions Contribute of Bamase
Little to Stability
Da&a Salit, Mark Bycroft and Alan R. Fershtt MRC Unit for Protein Function and Design Cambridge IRC for Protein Engineering University Chemical Laboratory Cambridge CBl ZEW, U.K. (Received 4 February
1991; accepted 16 April
1991)
Electrostatic interactions are believed to play an important role in stabilizing the native structure of proteins. We have quantified the contribution to stability of an interaction between two oppositely charged side-chains on the surface of barnase. Using site-directed mutagenesis, glutamate 28 and lysine 32 were introduced onto the solvent-accessible side of the second a-helix in barnase. These two residues are separated by one turn of the helix, and so are ideally situated for their opposite charges to interact. Double mutant cycle analysis reveals that the interaction between Glu28 and Lys32 contributes only approximately 92 kcal/mol to stability of the protein. All other interations between exposed charged sidechains in barnase examined so far also contribute little to stability. We explain this low value by their location on the surface, rather than in the interior, of the protein. Keywords: protein stability;
protein folding; electrostatics;
1. Introduction Electrostatic interactions between point charges are ubiquitous in proteins; they play an essential role in molecular recognition, binding, catalysis, assembly of macromolecular structures and co-operativity of allosteric transition (Horovitz et al., 1990; Perutz, 1987; Russell & Fersht, 1987; Warshel & Russel, 1984). It has also been suggested that salt bridges, together with hydrogen bonds between uncharged groups, are a major source of the greater thermal stability of ferredoxins from thermophilic organisms compared with their less stable homologues from mesophilic organisms (Perutz t Raidt, 1975). Although the interactions between oppositely charged side-chains and polypeptide chain termini are generally thought to stabilize proteins, the magnitude of this stabilization has been estimated in only a few instances, and no evidence is available so far that surface salt bridges contribute significantly to protein stability. Marqusee t Baldwin (1987) examined the interaction between oppositely charged pairs of residues 7 Present address: National Institute for Medical Research, The Ridgeway, Mill Hill, London EW7 1AA. U.K. $ Author to whom all correspondence should be addressed.
salt bridges
in a synthetic polypeptide a-helix; they found that the greatest contribution to stability of the helix was obtained when the two residues were four positions apart, with a negatively charged glutamate closer to the N terminus of the helix and a positively charged lysine closer to the C terminus. Since these peptides have a relatively high helix content at extreme pH values, Perutz & Fermi (1988) argued that this stabilization may be due to the non-polar rather than electrostatic interactions between the glutamate and lysine residues. In a different set of synthetic a-helical polypeptides, however, Bradley et al. (1990) found no evidence of stabilizing interactions between Glu, Lys (i,i + 3) or (i,i + 4) ion pairs. Anderson et al. (1990) suggest that the half-buried salt bridge between His31 and Asp70 in phage T4 lysozyme contributes 3 to 5 kcal/ mol (1 cal = 4184 J) to stability of the enzyme. Recently, several electrostatic interactions on the surface of barnase, the extracellular ribonuclease from Bacillus amyloliquefacien.s, have been examined in detail in this laboratory. Barnase is a monomeric enzyme of 110 amino acid residues, with an M, value of 12,382 (Hartley & Barker, 1972). Its gene has been cloned and expressed in Escherichia coli (Paddon & Hartley, 1986). The X-ray crystal structure of barnase has been solved at high resolution (Mauguen et al., 1982), and two-dimensional
779 0022-2836/91/150779-10
$03.00/O
0
1991 Academic
Press Limited
D. gali et al.
780
Table 2 Contacts between Ala32 and other residues observed in the crystal structure of barnuse
S28 A32
Ala32 atom
Figure 1. Ribbon diagram of barnase, showing the sidechains of Ser28 and Ala32.
n.m.r.t has been used to assign resonances for proteins in all its residues (Bycroft et al., 199%) and to determine its structure in solution (M.B., S. Ludvigsen, A.R.F. 8z F. Poulsen, unpublished results). Despite its small size, barnase has extensive secondary structure, including two a-helices and a five-stranded antiparallel /?-sheet (Fig. 1). It undergoes reversible, two-state denaturation induced in vitro by solvent or high temperature (Hartley, 1968; t Abbreviations used: n.m.r., nuclear magnetic resonance; COSY, 2D correlated spectroscopy; NOESY, 2D nuclear
Overhauser
correlated spectroscopy; enhancement.
spectroscopy;
TOCSY,
2D total
NOE, nuclear Overhauser
Table 1 Contacts between Ser28 and other residues observed in the crystal structure of barnase Other atom
Distance (A)
N
C Thr26 0 Thr26 Oyl Thr26 CB Lys27
33 33 35 33
(!
Cp Glu29 N Ala30 N Gln31
3.7 34 39
0
N Ala30 N Gln31 C’ Gln31 C Gln31 Cfi Gln31 N Ala32 c” C@
36 37 40 39 39 30 38 33
C’
Cyl Thr26 N Glue9 C Lys27
40 3.1 3.7
07’
c Lys27
40
Ser28 atom
At.oms up to 4 A apart are listed.
Other atom
Distance (A)
N
c 0 C 0 C’
Oh29 Glu29 Ala30 Ala36 Glu31
38 30 3.3 3.3 3.2
C”
0 Glu29
3.5
C
C8 Leu33 N Gly34 0 Glu29 0 Ala30
3.7 34 36 39
0
N Gly34
37
P
0 C N 0
36 37 35 33
Glu28 Gln31 Leu33 &x28
Atoms up to 4 A apart are listed.
Kellis et al., 1989), allowing for straightforward thermodynamic measurements of protein stability. As barnase contains no cysteine residues, there are no complications due to presence of disulfide bridges in the folded and unfolded state. Barnase has been used extensively in investigating the importance of specific interactions for barnase stability and in the folding pathway (Bycroft et al., 1996a; Horovitz et al., 1990; Kellis et al., 1988, 1989; Matouschek et aZ., 1989, 1996; Sali et al., 1988; Serrano & Fersht, 1989; Serrano et al., 1999). The general approach has been to make conservative site-directed mutants of the enzyme in order to disrupt, or, more rarely, add, a particular interaction and then to compare thermodynamic or kinetic properties of wild-type and mutant enzymes. Here we present a study of a surface ion pair that was introduced onto the surface of the second a-helix in barnase (residues 26 to 34) by sitedirected mutagenesis (Fig. 1). The residues mutated are Ser28 and Ala32. Since these two residues are located on the side of the helix that is fully accessible to the solvent and make no contacts with residues outside the helix (Tables 1 and 2), their substitution with charged residues was not expected to disrupt the structure of the enzyme. Ser28 was mutated to glutamic acid and Ala32 to lysine, mimicking the (i+4) Glu, Lys ion pairs from the experiments on model peptides by Marqusee & Baldwin (1987). In addition to the double mutant, the two single mutants were also made and the contribution of the interaction between Glu28 and Lys32 to stability of the protein was determined using double mutant cycles. The mutations were shown to be non-disruptive and the orientation of the mutant side-chains explored using two-dimensional ‘H n.m.r.
Protein
Electrostatic
2. Materials and Methods (a) Materials Molecular biological reagents were obtained from New England Biolabs and Boehringer-Mannheim, the radioand International, from Amersham chemicals SP-Trisacryl from IBF. Urea was highly purified AristaR grade from BDH Limited, and the buffer used in the denaturation experiments was 2-(iV-morpholino)ethanesulfonic acid (Mes) from Sigma. All other reagents were purchased from Sigma, Aldrich or BDH Limited. (b) Expression and purijication
of barnase
The vector used was pTZ18U containing the EcoRT-Hind111 fragment with the genes for barnase and bantar (Serrano et al., 1990). In this construct, the barnase gene is preceded by the promoter and signal sequence of the E. coli alkaline phosphatase gene; therefore, barnase was expressed in low-phosphate medium (Serpersu ul al., 1986). The enzyme was purified to homogeneity as described (Kellis et al., 1989; Mossakowska et al.. 1989). (v) Site-directed mutagenesis Site-directed mutants were made using the Kunkel method (Kunkel et al., 1987). The mutagenic primers used were: for Ser28-+GIu, 5’-GAGGGCTTGT(:C:TTCTT*C*TTTTGTA-3’: for Ala32+Lys. 5’.CCACCCAGCCGAGT*T*T*TTGTGCTTCTG-3’: and for Ser28-+Glu, Ala32+Lys, 5’.CAGCCGAGT*T*T*TTGTGCTTCTT*C*TTTTGTA-3’; where asterisks follow the mismatched bases. The mutants were identified by DNA sequencing. (cl) I Trua.denatura.tion Urea denaturation was observed using fluorescence intensity as the probe, with excitation at 290 nm and emission at 315 nm. Experiments were performed at 25( +@05)“C. pH 6.3. Mes acid/base buffer was used at concentrations of 7 mM, 10 mM and 50 mM. NaCl was added for high salt experiments to final concentrations of 250 mM and 556 mM. The concentration of barnase was 1 PM. The denaturation curves were analyzed as described (Kellis et al., 1988; Serrano & Fersht, 1989). The free energy of unfolding of the protein is linearly proportional to the concentration of the denaturant according to t.he following equation (Pace. 1986): A(&, = AC&,, -m
[urea].
The free energy of unfolding in water (AGH1o), the slope (m) and the half point of denaturation ([urea15c0,,) were obtained for each protein. The difference in free energy of unfolding between different forms of the protein is given by : AA(;,,2
= !!!$?t?
(lureaho:2
- [urealso~oI).
(e) Nuclear magnetic resonance Barnase was dissolved in 65 ml of 90% H,O/lO% ‘H,O to a final concentration between 4 and 6 mM. pH
781
Interactions
was adjusted to 4.5. n.m.r. spectra were acquired at 37 “C on a Bruker AM 500 spectrometer with an Aspect, 3000 computer. COSY (Aue et al., 1976; Bax & Freeman, 1981). KOESY (Jeener el al., 1979; Kumar et al.. 1980). and TOCSY (Braunschweiler & Ernst. 1983) experiments were recorded with 2048 data points in 1, and 512 t, increments, with a spectral width of 8000 Hz in both dimensions. A relaxation delay of 1.5 s was used. Suppression of the solvent signal was achieved by presaturation of the water resonance during the relaxation delay.
3. Results (a) Stability of wild-type and /mutant barnase and determination of the free energy of interaction between Glu.28 and Lys32
Table 3 lists the free energies of unfolding of the wild-type, the two single mutants and the double mutant under different conditions of ionic strength. The SE28 mutant is more stable than wild-type barnase at most ionic strengt,hs, although the difference in stability between the two proteins decreases with increasing salt concentration. ln 7 mM-MeS, the AAGu value between the wild-type and SE28 is 0.52 kcal/mol; in 10 mM-Mes it is 639 kcal/mol; in 50 mrvr-Mes, 0.23 kcal// mol; and in 50 mM-Mes with 250 mM-Nacl, 606 kcal/mol. In 50 mM-Mes, 556 mM-NaCl, the wild-type is more stable than the mutant, the AAG, value being -0.04 kcal/mol. The AK32 is less stable than the wild-type under all conditions examined; the AAG, value between the wild-type and this mutant is -919 kcal/mol in 7 mM-Mes, -018 kcal/mol in 10 mM-Mes, -914 kcal/mol in 50 mM-Mes, -622 kcal/mol in 50 mM-Mes with 250 mM-NaCl, and -024 kcal/mol in 50 mhr-Mes with 556 m&r-NaCl. The stability of the double mutant, SE28AK32, follows a pattern similar to that of the single SE28 mutant, with its AAG, value from the wild-type of 048 kcal/mol in 7 m&r-Mes, 0.33 kcal/mol in 10 m&r-Mes, 625 kcal/mol in 50 mM-Mes, 004 kcal/mol in 50 mM-Mes with 250 mM-NaCl, and -618 kcal/mol in 50 mM-Mes, 556 m&r-NaCl. Urea concentrations at the half-point of denaturation were the most accurate values measured, with the error of &@Ol M, and were therefore used to calculate the differences in free energies of denaturation. The resulting errors in AAG, were kO.03 kcal/mol. The errors in the measurement of AG,,, were much larger, at approximately 1 kcal/mol. The simplest way of estimating the magnitude of the interaction between Glu28 and Lys32 is to compare the stabilities of wild-type and the double mutant, SE28AK32. The problem with this approach is that it does not distinguish between the contribution to stability from the interaction between the two charges introduced by mutation and any contribution due to the interaction of either of these side-chains with the rest of the protein. For example, it is clear from Table 3 that the single mutation from Ser28 to Glu already stabilizes the protein under most conditions, while the single mutation from Ala32 to Lys makes barnase less
782
D. gali et al. Table 3 Free energies of unfolding for wild-type and the mutants at positions 28 and 32, at pH 6.3 Protein
Wsl
[NaCI]
(mM)
(mW
A’%,,
(kcal/mol)
m
PJ~al,O,P~l ABC, (M)
(kcal/mol)
A%, (kcal/mol)
~~28 AK32 SE28AK32
7I 7 7
0 0 0
1021 848 922 10.24
2.16 1.89 2.10 2.17
448 473 4.39 471
+052 -019 + @48
-0.15
G28 AK32 SE28AK32
10 10 10
0 0 0
944 896 8.85 8.95
1.99 1.97 1.99 190
454 4.74 445 4.71
+w39 -@18 +w33
-618
wt SE28 AK32 8E28AK32
50 50 50 50
0 0 0 0
10.25 10.14 1069 lo-63
2.23 216 2.36 2.26
459 469 453 4.70
+023 -014 +0.25
-619
G28 AK32 SE28AK32
50 50 50
250 250 250
1096 9.93 1021 993
2.01 200 2.10 I .99
497 500 486 499
+ 0.06 -0.22 + 0.04
-0.15
%28 AK32 SE28AK32
50 50 50
556 556 556
10.24 10.73 lo-42 lo-70
2.05 1-95 203 2.07
525 523 513 516
- 0.04 - 0.24 -0.18
-914
AAG, values were calculated for each ionic strength as a difference between wild-type mutant. There are small rounding errors in the last column.
stable. Both these effects have to be taken into account when measuring the interaction between Glu28 and Lys32. This problem can be overcome by using double mutant cycles (Carter et al., 1984; Serrano et al., 1990). Such cycles consist of the form of the enzyme in which neither of the partners in the interaction is present (in this case, the wild-type), the form in which both partners are present (SE28AK32) and the two forms in which only one of them is present (SE28 and AK32) (Fig. 2). The free energy of the interaction between the two residues is determined by measuring the unfolding free energies of each of the proteins in the cycle. The difference between the change in the free energy of unfolding upon mutation of the first residue when the second
X
AGE+E-Y
\
/
-
‘E’
E
AGE-E-X
AGE-x--E-XY
I Y
Y
x )
‘E’
(wt) and each
residue is present and absent is the apparent free energy of the interaction between the two residues. If the two mutations are not independent, i.e. there is an interaction between the two residues in the double mutant, the following inequalities are true (using the symbols defined in Fig. 2): A%,.,
Z A G,.,,,.,,,
(1)
A%s., ZAG,,,,,,. (2) The free energy of interaction between X and Y is given by:
By using double mutant cycles, the favorable free energy of interaction between Glu28 and Lys32 was kcaljmol in 7 m&r-Me& found to be -615 -0.18 kcal/mol in 10 mM-Mes, -0.19 kcal/mol in 50 m&r-Mes, -615 kcal/mol in 50 m&r-Mes with 250 mM-NaCl, and -0.14 kcal/mol in 50 m&i-Mes containing 556 mM-NaCl. These values are all within 0.03 kcal/mol of their average, 0.16 kcal/mol. Since the errors in AGint are approximately f604 kcal/mol, the variation of the value with ionic strength is not significant.
\/ E
AGE-Y-N-XY
Figure 2. The scheme for a double mutant cycle. X and Y are the 2 residues being introduced by mutations and AGE,,.,, AcfE.x,E.xy and AGE.y,E.xy the changes AG,+,x> in free energy of unfolding for the appropriate transitions in the cycle.
(b) Comparison of two-dimensional n.m.r. spectra of wild-type and the mutants Fingerprint regions of mutant two-dimensional n.m.r. spectra were assigned, starting with the similarities between the spectra of wild-type barnase
Protein
Electrostatic
Interactions
783
--~-____
Table 4
:
0 ‘I “f
-3.2 -3.4
Chemical shifts of backbone and side-chain protons between residues 27 and 32 in wild-typp. position 28 and 32 mutants Position
Proton
wt
KP7
NH CH VBH ( @H’ (‘?H (“H’ C6H (“H’ (“H ( “H’ (‘“NH
IO.21 398 2.11
SZS/E2S
8.6
I
i’.-
NH (‘“H (‘OH
x79 393 121
c/31
NH (‘“H (‘BH (‘#H’ CqYH (“H’
9.29
9.0
,--W.--v, 9.6 9.4
9.2
9.0 8-8 ppm
, 8.6
SH C”H ( @H (‘BH’ (‘?H (VH’ ( “H (‘6H’ (“H (“H’ C”NH
7.95 4.32 I -tiu
IO.0
I 8.4
0.2
. 8.0
7.8
Figure 3. Sequential connectivities in the second CIhelix of the mutant SE28AK32. p.p.m.. parts per million.
and the mutants. About 75% of the COSY crosspeaks corresponding to the main-chain protons in the mutant,s could be assigned by analogy with the wild-type spectra, with another 15 to 20% of assignments obtained from sequential connectivities (Wiithrich, 1986). The plots of chemical shifts of equivalent main-chain protons in the mutants and the wild-type (Fig. 4) show that the only differences occurred at the position of the mutation or at the immediately adjacent positions. The patterns of NOE connectivities between backbone protons which characterize specific secondary structure elements, i.e. d,,, d,,(i,i + 3) and daN(iri + 4) connectivities for the ol-helices (Fig. 5), and strong d,, and inter-strand NOE contacts for the o-sheets (Fig. 6), were very similar for the wild-type and the three mutants throughout the molecule, including the connectivities in the 26 to 34 cc-helix (Fig. 3). The small differences in the NOE patterns that did occur resulted from collecting data for the mutants at a lower temperature than for the wild-type barnase,
3.99 3+x)
-430
9.6 9.8
9.10 $40
7.74 4.14 IGI 2.45 SA Nl
9.2
A32/K32
SE28AK32
XA I ,46 NA 1.16 2.98 NA NA
SH (‘“H CqBH (“OH’ CYH CqYH
9.0
AK32
i?@2
E19
E 6.8 a Q
9,4
SH (‘“H (‘“H CflH’ CfYH (‘?H’
SE32
367
2.56 1.36
NA NA
-, Proton not present in side-chains; NA. proton not assigned. The wild-type (wt) data are from Psycroft rt ml. (I99Oh).
due to their changed stability. Through-bond connectivities given by TOCSY and COSY experiments were also used to determine resonances of protons in the side-chains that are found on the surface of the second u-helix in barnase. The results for the three mutants and the wild-type are shown in Table 4. Although it is not possible to interpret quantitatively changes in chemical shifts of nuclear resonances, it is known that. the chemical shifts are extremely sensitive to the local magnetic environment of the nuclei. This magnetic environment is in
784
D. gali et al. wt
and
SE28(NH1
wt
7E 4 ,o
and
SE28
(C’H)
/ 6
/
I
I
7
6
9
8 wt
chemical
wt
and
shift
IO
3
4
(p.p.m.1
wt
wt
AK32(NH)
5
chemical
and
6
shift
AK32
7
(p.p.m.)
(Can)
*
I
L
6
7 wt
wt
9
8 chemical
and
shift
SE28AK32
0
IO
3
(p.p.m.!
4
5
wt chemical
wt
(NH)
I
and
shift
SE28AK32
6
7
6
7
(p.p.m.)
(C
Ii)
I
’
I
T26
J 6
7
9
8 wt chemical
shift
IO
3
(p.p.m.1
Figure 4. Chemical shifts of amide and C” protons in the mutants plotted against The wild-type assignment is from Bycroft et al. (1990b). p.p.m., parts per million.
wt
4
5
chemical
shift
the chemical
(p&m.)
shifts
in wild-type
(wt).
785
Protein Electrostatic Interactions SE28 d.Nli, j+4)
-
dpN
-
-
-
duu
d UN
%N duu
_--
---
-
---
dPN
-
-
__30
!O
-
d UN d ou
--
-
---
---
dpN duu
-
-
--
-
-
d au
-
----
% duu d ON
-
d au
SE28AK32
AK32 d aNlbj+4)
d UN d ON
_ -
--
abN
---
d ON
d UN
____
~o”,N~FDG”*D”Lor”“K~CDNYI!KEEIO*
daNl,,j+41
-
_--
dON
d (LN
% duu d (IN
--
d.Nti,j+3) dpN
d aN(i,j+OJ
--
----
-
d.Ntisj+3)
---
~O”,N~FD~“*D”LOrVKKLPDNVlTKsE~~Kl 10
---
--
-
dsN
-
-
-
d ULI
--.
--
--
10’
dpN duu
dPN
d UN d [IN
d(IN
-
---
--
--
L~~VA~KGN~*DV”‘GKS~GGO!FSNREGKL
.o
dpu O’,,,,
dPN duu
w
60
--
-
-
--
--
-
d au
Figure 5. Sequential and secondary (Bycroft et al.. 199Ob) and the mutants.
structure
connectivities
defined by both distances and angles between magnetic nuclei. It is, therefore, assumed that, when chemical shifts of the analogous protons in the wild-type and the mutant spectra are the same, the two protons are in exactly the same environment in the two proteins. As the chemical shifts of backbone protons in the wild-type and in the three mutants are different only at the positions of the mutations and those immediately adjacent (Fig. 4), the two proteins must have largely identical structures. The fact that the NOE patterns characteristic of regular structure elements are unchanged secondary between the wild-type and the mutant (Figs 5 and 6) shows that the mutations did not perturb the turn
between main-chain
protons of t*he wild-type
(WT)
secondary structure of barnase, not even in the second a-helix, where the mutations and the changes in chemical shift occur. Chemical shifts of the assigned side-chain protons of the second a-helix are also found to be very similar between the mutants and the wild-type, except at the positions where the covalent structure has been changed (Table 4), suggesting that the environment of the side-chains was not significantly altered. Unfortunately, the overlap in the region of the twodimensional spectra containing resonances from C?, Cy, Cd and C” protons of the side-chains meant that the through-space NOE connectivities between these protons could not be assigned unambiguously
786
D. #ali WT
et al.
SE28
SE28AK32
K32
H
0
H
H
6
H
ti
6
Figure 6. NOE contacts between /?-strand main-chain protons of wild-type mutants.
MI,
barnase (Bycroft et al.. 19906) and of the
hydrogen bond; - - - - - -. NOE connectivity.
in order to find the relative side-chains.
conformation
of the
4. Discussion Stabilization energies listed in Table 3 indicate that the SE28 mutation by itself stabilizes barnase by about as much as the double mutant (SE28AK32). This may be due to the introduction of a favorable interaction between Glu28 and the a-helix dipole of the 26 to 34 a-helix. Th’_s type of interaction has been observed in barnase (Sali et al., 1988). The interaction energy between two oppositely on the barnase surface charged side-chains measured here is more than an order of magnitude
lower than that reported for buried, hydrogenbonded salt bridges (Anderson et al., 1990), but comparable to the interaction energies that have been measured for other ion pairs and salt bridges on the barnase surface. Serrano et al. (1990) examined the interaction between two oppositely charged side-chains on the first a-helix (residues 6 to 18), Asp12 and Argl6. Asp12 is present in the wild-type enzyme, while the was introduced by the ThrlS-+Arg arginine mutation. Argl6 is also present in the wild-type form of binase, the RNase from Bacillus intermedius which is highly homologous to barnase. In barnase, the TR16 mutant is more stable than the wild-type by 6.5 kcal/mol, and 633 kcal/mol of this free energy can be attributed to the interaction between
Protein Electrostatic Interactions Asp12 and Argl6 Structural evidence suggests that the two residues do not form a hydrogen-bonded salt bridge; instead, Argl6 interacts with the protein backbone and Asp12 forms salt bridge with ArgllO (Serrano et al., 1990). Studies by Horovitz et al. (1990) focused on the interactions between the C-terminal Argl 10 and two residues on the first a-helix of barnase, Asp8 and Aspl2. The side-chains of these residues are in the ideal orientation to form two salt bridges. They found that the interaction energy between Asp8 and ArgllO is 998 kcal/mol with the Aspl2-ArgllO salt and 921 kcal/mol when the bridge present Aspl2-ArgllO salt bridge is absent. Similarly, the free energy of interaction between Asp12 and ArgllO is 1.25 kcal/mol with the Asp&ArgllO salt bridge present and 0.48 when the Asp8-Argl lO salt bridge is removed by mutation. When Asp8, Asp12 and ArgllO are mutated to alanine residues, the resulting mutant is more stable than the wild-type with the two salt bridges intact. That the interaction energies between ion pairs on the protein surface measured in this laboratory are lower than the values reported for buried salt bridges may be explained by the dielectric constant in a protein core being much lower than in water or at the surface of a protein (Warshel & Russel, 1984), so that the enthalpy of interaction between two buried charges should be larger than that between two charges separated by the same distance at the surface of the molecule. In addition, as side-chains on the surface of a protein have more conformational freedom than those in the center of the protein, they lose entropy when being constrained in a salt, bridge. The difference between the results of Marqusee & Baldwin (1987), who observed a stabilizing (i&+4) Glu, Lys interaction in polypeptides largely made of alanine residues, and the work of Bradley et al. (1990), who detected no such interaction between (i,i+4) Glu and Lys in polypeptides containing a variety of residues, suggests that the two oppositely charged side-chains may only interact in the rare instance when the steric hindrance from neighboring side-chains is completely eliminated (Bradley et at., 1990). The local environment of Glu28 and Lys32 in the mutant is more similar to the situation in the synthetic polypeptide described by Bradley et al. than of that by Marqusee et al.; it is possible that a stronger interaction between the two charged sidechains may be observed if the other residues on the helix surface were all mutated to alanine residues. Our conclusion that the interaction between (Glu28 and Lys.32 is weak in the native form of the double mutant is based on the assumption that t,heir side-chains do not interact in the unfolded stat,e; this is a reasonable expectation for a pair of residues four positions apart in a random coil. The relative stability of wild-type barnase and the mutants can. however, also be explained by a model where Glu28 and Lys interact both in the unfolded and the folded state, with the interaction only slightly more favorable in the folded enzyme.
787
have no direct evidence to distinguish between these two possibilities. In either case, though, the presence of the (i,i+4) Glu, Lys ion pair does not contribute significantly to stability of the native protein relative to the denatured state.
5. Conclusion We have shown that the interaction between two oppositely charged side-chains on the surface of the second a-helix of barnase, which are ideally situated to form a surface salt bridge, contributes only approximately 615 kcal/mol to the stability of the protein. This value is an order of magnitude lower than that reported for a partially buried salt bridge (Anderson et al., 1990), but it agrees with other studies of surface electrostatic interactions in barnase (Horovitz et al., 1990; Serrano et al., 1990). We conclude that electrostatic interactions between point charges are strongly context-dependent, with interactions between single ion pairs on the protein surface generally contributing little to protein stability.
D.S. was a recipient of the Sir Hans Krebs Scholarship administered by the Biochemical Society.
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Mossakowska, D. E., Nyberg, K. $ Fersht, A. R. (1989). characterization of the recombinant Kinetic from Bacillus amyloliquefaciena ribonuclease (barnase) and investigation of key residues in catalysis by site-directed mutagenesis. Biochemistry. 28, 3843-3850. Pace, C. K’. (1986). Determination and analysis of urea and guanidinium hydrochloride denaturation curves. Methods Enzymol. 131, 26G-279. Paddon, C. J. & Hartley, R. W. (1986). Cloning, sequencing and transcription of an inactivated copy extracellular Bacillus amyloliquefaciens of ribonuclease (barnase). Gene, 40, 231-239. Perutz, M. F. (1987). Electrostatic effects in proteins. Science, 201, 1187-1191. Perutz, M. F. & Fermi, G. (1988). Stereochemistry of saltbridge formation in a-helices and b-sheets. Proteins: Struct. Fund. Genet. 4, 294-295. Perutz, M. F. & Raidt, H. (1975). Stereochemical basis of in bacterial ferredoxins and heat stability haemoglobin A2. Nature (London), 255, 256-258. Russell, A. J. & Fersht, A. R. (1987). Rational modification of enzyme catalysis by engineering surface charge. Nature (London), 328, 49&500. Sali, D., Bycroft, M. & Fersht, A. R. (1988). Stabilization of protein structure by interaction of a-helix dipole with a charged side chain. Nature (London). 335. 740-743. Serpersu, E. H., Shortle, D. & Mildvan, A. S. (1986). Kinetic and magnetic resonance studies of effects of genetic substitutions of a Ca2 +-1iganding amino acid in staphylococcal nuclease. Biochemistry, 25, 68-77. Serrano, L. & Fersht, A. R. (1989). Capping and a-helix stability. Nature (London), 342, 296-299. Serrano, L., Horovitz, A., Avron, B., Bycroft, M. & Fersht, A. R. (1990). Estimating the contribution of engineered surface electrostatic interactions to protein stability using double mutant cycles. Biochemistry, 29, 9343-9352. Warshel, A. 6 Russel, S. T. (1984). Calculations of electrostatic interactions in biological systems and in solutions. Quart. Rev. Biophys. 17, 283422. Wiithrich, K. (1986). NMR of Proteins and Nucleic Acids Wiley, Pu’ew York.
by A. Klug