Pyroccocus furiosus rubredoxin (PFRD), like most studied hyperthermophilic proteins, does not undergo reversible folding. The irreversibility of folding is thought to involve PFRD’s iron-binding site. Here we report a PFRD variant (PFRD-XC4) whose iron binding site was redesigned to eliminate iron binding using a computational design algorithm. PFRD-XC4 folds without iron and exhibits reversible folding with a melting temperature of 82 °C, a thermodynamic stability of 3.2 kcal mol^(-1) at 1 °C, and NMR chemical shifts similar to that of the wild-type protein. This variant should provide a tractable model system for studying the thermodynamic origins of protein hyperthermostability

Mayo, Stephen L.

Strop, Pavel

English

Caltech Authors - Main

Rubredoxin Variant Folds without Iron
Pavel Strop† and Stephen L. Mayo*,‡
Contribution from Biochemistry Option and Howard Hughes Medical Institute and DiVision of Biology,
California Institute of Technology, Mail Code 147-75, Pasadena, California 91125
ReceiVed October 1, 1998
Abstract: Pyroccocus furiosus rubredoxin (PFRD), like most studied hyperthermophilic proteins, does not
undergo reversible folding. The irreversibility of folding is thought to involve PFRD’s iron-binding site. Here
we report a PFRD variant (PFRD-XC4) whose iron binding site was redesigned to eliminate iron binding
using a computational design algorithm. PFRD-XC4 folds without iron and exhibits reversible folding with a
melting temperature of 82 °C, a thermodynamic stability of 3.2 kcal mol-1 at 1 °C, and NMR chemical shifts
similar to that of the wild-type protein. This variant should provide a tractable model system for studying the
thermodynamic origins of protein hyperthermostability.
Introduction
Hyperthermophilic proteins have attracted considerable at-
tention because they can serve as model systems for the
determination of factors responsible for protein stability. These
include an optimized use of structure elements such as hydro-
phobic interactions, packing forces, hydrogen bonds, and salt
bridges.1-5 In most cases, however, the usual methods for
determining thermodynamic stability cannot be used for hyper-
thermophilic proteins. The measurement of protein stability
requires reversible folding, but in almost all studied hyperther-
mophilic proteins, the denaturation is not reversible.6-8 In the
few hyperthermophilic proteins that exhibit reversible folding,
there are other elements that complicate the determination of
the free energy of folding. These proteins either are dimeric9,10
or do not fit a two-state denaturation model.10 Irreversible
denaturation has been addressed by hydrogen exchange studies11
and by molecular dynamics simulations,12 but neither of these
methods provides an accurate measure of free energy.
Rubredoxins are small (6 kDa) iron-sulfur proteins with
the active site arranged around an iron tetrahedrally coordinated
to four cysteinate sulfurs (Figure 1a). Thirteen rubredoxins have
† Biochemistry Option.
‡ Howard Hughes Medical Institute and Division of Biology. E-mail:
steve@mayo.caltech.edu.
(1) Lee, B.; Vasmatzis, G. Curr. Opin. Biotechnol. 1997, 8, 423-428.
(2) Straume, M., Murphy, K. P., Freire, E., Adams, M. W. W., Kelly,
R. M., Eds. Thermodynamic Strategies for Protein DesignsIncreased
Temperature Stability; American Chemical Society: Washington, DC, 1992;
Vol. 498, pp 122-135.
(3) Rees, D. C.; Adams, M. W. W. Structure 1995, 3, 251-254.
(4) Eidsness, M. K.; Richie, K. A.; Burden, A. E.; Kurtz, J., D. M.; Scott,
R. A. Biochemistry 1997, 36, 10406-10413.
(5) Jiang, X.; Bishop, E. J.; Farid, R. S. J. Am. Chem. Soc. 1997, 119,
838-839.
(6) Cavagnero, S.; Zhou, Z.; Adams, M.; Chan, S. Biochemistry 1998,
37, 3377-3385.
(7) Klump, H. H.; W, A. M. W.; Robb, F. T. Pure Appl. Chem. 1994,
66, 485-489.
(8) Wampler, E. J.; Neuhaus, B. E. J. Protein Chem. 1997, 16, 721-
732.
(9) Li, W.; Grayling, R. A.; Sandman, K.; Edmonson, S.; Shriver, J.
W.; Reeve, J. N. Biochemistry 1998, 37, 10563-10572.
(10) MacBeath, G.; Kast, P.; Hilvert, D. Biochemistry 1998, 37, 10062-
10073.
(11) Hiller, R.; Zhou, Z.; Adams, M.; Englander, W. Proc. Natl. Acad.
Sci. 1997, 11329-11332.
(12) Bradley, A. E.; Stewart, E. D.; Adams, W. M.; Wampler, J. E.
Protein Sci. 1993, 650-665.
VOLUME 121, NUMBER 11
MARCH 24, 1999
© Copyright 1999 by the
American Chemical Society
10.1021/ja9834780 CCC: $18.00 © 1999 American Chemical Society
Published on Web 03/05/1999
been sequenced, and all conserve the four cysteine ligands
(residues 5, 8, 38, and 41 in the hyperthermophilic Pyroccocus
furiosus rubredoxin (PFRD) and residues 6, 9, 39, and 42 in
the mesophilic DesulfoVibrio Vulgaris rubredoxin (DVRD)).
Since several rubredoxin structures are available from the
Brookhaven Protein Data Bank at resolutions of 0.95-1.8
Å,13-19 these proteins are valuable models for the investigation
of the origins of thermostability.
It is believed that the iron binding site in rubredoxin is the
cause of irreversible unfolding.20 Here we report a Pyroccocus
furiosus rubredoxin variant (PFRD-XC4) which was designed
to eliminate the iron-binding site using a computational design
algorithm. PFRD-XC4 is able to fold in the absence of iron
and undergoes reversible denaturation. The variant provides an
excellent opportunity for systematic exploration of the factors
determining protein thermostability.
Experimental Section
Computational Modeling. In the calculation using a mesophilic
structure, the four cysteine residues that form rubredoxin’s iron binding
site were optimized in the absence of iron coordination. Two of the
cysteines were classified as core residues (Cys6 and Cys39) and two as
boundary residues (Cys9 and Cys42). Residues were classified into core,
surface, and boundary groups as previously described.21 Seven amino
acid types (Ala, Val, Leu, Ile, Phe, Tyr, and Trp) were considered at
the two core positions, and 16 amino acid types (Ala, Val, Leu, Ile,
(13) Bernstein, F. C.; Koetzle, T. F.; Williams, G. J. B.; Meyer Jr, E. F.;
Brice, M. D.; Rodgers, J. R.; Kennard, O.; Shimanouchi, T.; Tasumi, M. J.
Mol. Biol. 1977, 112, 535-542.
(14) Adman, E. T.; Sieker, L. C.; H, J. L. J. Mol. Biol. 1991, 337-352.
(15) Dauter, Z.; Sieker, L.; Wilson, K. Acta Crystallogr. 1992, 42-59.
(16) Day, M.; Hsu, B.; Joshua-tor, L.; Park, J.; Zhou, Z.; Adams, M.;
Rees, D. Protein Sci. 1992, 1494-1507.
(17) Frey, M.; Sieker, L. C.; Payan, F.; Haser, R.; Bruschi, M.; Pepe,
G.; LeGall, J. J. Mol. Biol. 1987, 525-541.
(18) Sieker, L. C.; Stenkamp, R. E.; Jensen, L. H.; Prickril, B.; LeGall,
J. FEBS 1986, 73-76.
(19) Watenpaugh, K. D.; Sieker, L. C.; Jensen, L. H. J. Mol. Biol. 1979,
509-522.
(20) Cavagnero, S.; Debe, D.; Zhou, Z.; Adams, M.; Chan, S. Biochem-
istry 1998, 3369-3376. (21) Dahiyat, B. I.; Mayo, S. L. Science 1997, 278, 82-87.
Figure 1. (a) Ribbon diagram of wild-type mesophilic DesulfoVibrio
Vulgaris rubredoxin showing the four cysteine residues that coordinate
the iron. (b) Ribbon diagram showing the computed side chain
orientations of the four mutations. Structure figures were generated
using MOLMOL.41
Figure 2. Circular dichroism measurements of wild-type Pyroccocus
furiosus rubredoxin (PFRD) and the PFRD-XC4 variant. (a) Wavelength
scans of PFRD-XC4 at 1 °C (circles), 99 °C (diamonds), and refolded
at 1 °C (squares). PFRD is also shown at 1 °C for comparison
(triangles). (b) Thermal unfolding curve for PFRD-XC4 monitored by
CD at 225 nm. (c) Guanidinium chloride denaturation curve for PFRD-
XC4 at 1 °C.
2342 J. Am. Chem. Soc., Vol. 121, No. 11, 1999 Strop and Mayo
Phe, Tyr, Trp, Ser, Thr, Asp, Asn, Glu, Gln, His, Lys, and Arg) were
considered at the two boundary positions. Both amino acid identities
and side chain conformations were determined by the algorithm for
the four optimized residues. Neighboring residues 7, 10, 40, and 43
were fixed in identity, but their conformations were allowed to change
in the calculation. All other residues as well as the backbone were held
fixed. Computational details, potential functions, and parameters for
van der Waals, solvation, and hydrogen bonding are described in our
previous work.21-26
Mutagenesis and Protein Purification. The hyperthermophilic
mutant PFRD-XC4 was constructed by inverse PCR27 using a synthetic
Pyroccocus furiosus rubredoxin gene in plasmid pt7-7.4 The mutations
for PFRD-XC4 are C5L, C8T, C38A, and C41T. A synthetic gene based
on the sequence of the DesulfoVibrio Vulgaris rubredoxin was con-
structed for the mesophilic variant (DVRD-XC4) by recursive PCR28
and cloned into pt7-7. The mutations for DVRD-XC4 are C6L, C9T,
C39A, and C42T. Both mutants were verified by sequencing. Recom-
binant proteins were expressed by IPTG induction in BL21(DE3) hosts
(Invitrogen) as described29 and isolated using a freeze/thaw method.30
Purification was accomplished by reverse-phase high-performance liquid
chromatography using first a linear 1%/min followed by a 0.07%/min
acetonitrile/water gradient containing 0.1% TFA. Molecular weights
were verified by mass spectrometry.(22) Dahiyat, B. I.; Mayo, S. L. Protein Sci. 1996, 5, 895-903.(23) Dahiyat, B. I.; Sarisky, C. A.; Mayo, S. L. J. Mol. Biol. 1997, 273,
789-796.
(24) Dahiyat, B. I.; Mayo, S. L. Proc. Natl. Acad. Sci. U.S.A. 1997, 94,
10172-10177.
(25) Dahiyat, B. I.; Gordon, D. B.; Mayo, S. L. Protein Sci. 1997, 6,
1333-1337.
(26) Malakauskas, S. M.; Mayo, S. L. Nat. Struct. Biol. 1998, 5, 470-
475.
(27) Hemsley, A.; Arnheim, N.; Toney, M. D.; Cortopassi, G.; Galas,
D. J. Nucleic Acids Res. 1989, 17, 6545-6551.
(28) Prodromou, C.; Pearl, L. H. Protein Eng. 1992, 5, 827-829.
(29) Alexander, P.; Fahnestock, S.; Lee, T.; Orban, J.; Bryan, P.
Biochemistry 1992, 31, 3597-3603.
(30) Johnson, B. H.; Hecht, M. H. Bio/Technology 1994, 12, 1357-
1360.
Table 1. 1H NMR Chemical Shifts for PFRD-XC4, pH 6.3, 25 °Ca
residue HN HR Hâ others
1 Ala
2 Lys 8.68 5.45 1.62 HE, 2.92
3 Trp 9.39 4.95 2.74 HD1, 6.87; HE1, 9.73; HE3, 6.61; HZ2, 7.58; HZ3, 6.94; HH2, 7.21
4 Val 9.97 5.57 HG1, 0.78; HG2, 0.93
5 Leu 8.85 4.35 0.16, 1.91 HG, 1.21; HD1, 0.53; HD2, 0.70
6 Lys 9.09 3.79 HG1, 1.41; HG2, 1.57; HE1, 2.86; HE2, 2.95; HD, 1.71
7 Ile 8.25 4.09 1.88
8 Thr 6.92 4.73 4.47 HG2, 1.19
9 Gly 8.72 3.47, 4.24
10 Tyr 7.59 4.27 2.70, 3.30 HD, 7.33; HE, 7.20
11 Ile 7.34 4.97 1.49 HG2, 0.71; HD1, 0.80; HG1, 1.79
12 Tyr 9.53 4.64 2.92, 3.17 HD, 7.10; HE, 6.43
13 Asp 8.78 4.92 2.26, 2.86
14 Glu 8.26 3.86 1.94, 2.11 HG, 3.03
15 Asp 8.03 4.27
16 Ala 7.2 4.35 1.49
17 Gly 8.12 3.59, 3.98
18 Asp 8.32 5.17 3.01
19 Pro
20 Asp 9.09 4.5 2.75
21 Asn 7.73 5.19 2.69, 3.16
22 Gly 7.83 3.86, 4.15
23 Ile 7.67 4.38 1.94 HG2, 0.91; HD1, 0.82; HG1, 1.34
24 Ser 8.72 4.61 3.74, 3.90
25 Pro
26 Gly 8.22 3.74, 4.25
27 Thr 7.3 4.01 3.94 HG1, 6.35; HG2, 1.04
28 Lys 8.92 4.01 1.50, 2.12 HD, 1.69; HE, 3.09
29 Phe 10.06 3.29 HZ, 6.52; HD, 5.70; HE, 6.12
30 Glu 9.34 3.72 1.92, 2.08 HG1, 2.21; HG2, 2.32
31 Glu 7.41 4.09 2.06, 2.20
32 Leu 6.86 3.88 0.34, 0.88 HG, 0.70; HD2, -0.14
33 Pro
34 Asp 8.76 4.25 2.59, 2.69
35 Asp 8.23 4.53 2.58, 2.87
36 Trp 7.7 4.15 2.97 HD1, 7.19; HE1, 10.41; HE3, 6.84; HZ2, 7.49; HZ3, 6.25; HH2, 7.00
37 Val 6.72 4.32 HG1, 1.33
38 Ala 8.49 1.28
39 Pro 4.13 1.70, 2.27 HD1, 2.76; HD2, 3.48; HG, 1.90
40 Ile
41 Thr
42 Gly 7.62 3.64, 3.86
43 Ala 8.88 4.19
44 Pro
45 Lys 6.92 2.19, 2.39 HG1, 1.00; HG2, 1.18; HD, 1.70
46 Ser 8.12 4.38 4.03, 4.19
47 Glu 8.04 4.48 1.98, 2.32
48 Phe 8.16 4.94, 3.76
49 Glu 9.34 4.93
50 Lys 8.55 3.2 1.12, 1.28 HG1, 0.07; HG2, 0.22; HD, 0.65; HE, 2.68
51 Leu 8.68 4.3 1.29, 1.48 HD, 0.83
52 Glu 8.1 4.35 1.84 HG1, 2.07; HG2, 2.19
53 Asp 8.03 4.34 2.49, 2.65
a Chemical shifts were referenced to H2O (4.77 ppm at 25 °C).
Rubredoxin Variant Folds without Iron J. Am. Chem. Soc., Vol. 121, No. 11, 1999 2343
CD Analysis. CD data were collected on an Aviv 62DS spectrometer
equipped with a thermoelectric unit and using a 1 mm path length cell.
Protein samples were 40 íM in 50 mM sodium phosphate buffer at
pH 6.3. Concentrations were determined by UV spectrophotometry.
Thermal melts were monitored at 225 nm. Data were collected every
2 °C with an equilibration time of 2 min and an averaging time of 10
s. Tm was determined by fitting the melting curves to a two-state model
as described.31 Guanidinium chloride denaturations were performed at
1 °C. ¢G’s, m values, and error estimates were obtained by fitting the
denaturation data to a two-state transition as described.32
NMR Studies. NMR data were collected on a Varian Unity Plus
600 MHz spectrometer equipped with a Nalorac inverse probe with a
self-shielded z gradient. NMR samples were prepared in 90/10 H2O/
D2O or 99.9% D2O with 200 mM NaCl and 25 mM acetate-d3, pH
6.3. The sample concentration was approximately 1.5 mM. Sequential
assignment of resonances was achieved by the standard homonuclear
method.33 Two-dimensional DQF-COSY,34 TOCSY,35 and NOESY36
spectra were acquired at 25 °C. The TOCSY spectrum was recorded
with a 80 ms mixing time using a clean-MLEV17 mixing sequence.
The NOESY spectrum was acquired with a 150 ms mixing time. Water
suppression was accomplished either with presaturation during the
relaxation delay or with pulsed field gradients.37 Spectra were processed
with VNMR (Varian Associates) and were assigned with ANSIG.38
3JHNHA values were derived from NOESY cross-peak fine structure using
the INFIT module of XEASY.39
Results and Discussion
Backbone coordinates for the mesophilic rubredoxin structure
used in the calculations were obtained from Brookhaven Protein
Data Bank entry 8rxn.13,15 A radius scale factor of 0.9 for the
van der Waals interactions24 resulted in two threonine residues
at the boundary positions and two alanine residues at the core
positions. To obtain an alternate sequence that provides greater
core volume, selection for the core positions in the calculation
was repeated with the radius scale factor decreased to 0.7 and
boundary positions 9 and 42 fixed as threonines. The amino
acid sequence selected by the algorithm under these conditions
contains the following mutations: C6L, C9T, C39A, and C42T
(Figure 1b). The equivalent mutations for the hyperthermophilic
variant, PFRD-XC4, are C5L, C8T, C38A, and C41T.
The far-ultraviolet circular dichroism (CD) spectrum of
PFRD-XC4 is nearly identical to that of the wild-type protein
at 1 °C (Figure 2a). Wavelength scans performed at 1 °C, 99
°C, and then again at 1 °C demonstrate reversible folding (Figure
2a). Thermal denaturation of the variant, monitored by CD at
225 nm, shows a cooperative unfolding transition with a melting
temperature (Tm) of 82 °C (Figure 2b). Based on the estimated
Tm for the wild-type hyperthermophilic rubredoxin,11 PFRD-
XC4 is destabilized by about 80 °C. The mesophilic variant,
(31) Minor, D. L.; Kim, P. S. Nature 1994, 371, 264-267.
(32) Santoro, M. M.; Bolen, D. W. Biochemistry 1988, 27, 8063-8068.
(33) Wu¨thrich, K. NMR of Proteins and Nucleic Acids; John Wiley and
Sons: New York, 1986.
(34) Piantini, U.; Sorensen, O. W.; Ernst, R. R. J. Am. Chem. Soc. 1982,
104, 6800-6801.
(35) Bax, A.; Davis, D. G. J. Magn. Reson. 1986, 65, 355-360.
(36) Jeener, J.; Meier, B. H.; Bachmann, P.; Ernst, R. R. J. Chem. Phys.
1979, 71, 4546-4553.
(37) Piotto, M.; Saudek, V.; Sklenar, V. J. Biomol. NMR 1992, 2, 661-
665.
(38) Kraulis, P. J. J. Magn. Reson. 1989, 24, 627-633.
(39) Bartels, C.; Xia, T. H.; Billeter, M.; Guntert, P.; Wuthrich, K. J.
Biol. NMR 1995, 5, 1-10.
Figure 3. (a) NMR assignments summary and NOE connectivities of PFRD-XC4. Bars represent unambiguous connectivities with bar thickness
indexed to the intensity of the resonance. Coupling constants with 3JHNHA > 8.0 Hz are shown as open squares, and 3JHNHA < 4.0 Hz are shown as
black squares. Secondary structural elements of PFRD are given at the bottom of the figure for comparison.42 (b) Chemical shift difference between
PFRD and PFRX-XC4 for amide nitrogen (black lines) and R hydrogens (shaded line). Proline residues 19, 25, 33, 39, and 44 not shown. Residues
40 and 41 also not shown due to a missing sequential assignments. Mutations are at positions 5, 8, 38, and 41. Residues 7, 9, 10, and 48 are in the
close proximity of the mutated site.
2344 J. Am. Chem. Soc., Vol. 121, No. 11, 1999 Strop and Mayo
DVRD-XC4, was also expressed and found to be unfolded at 1
°C. Chemical denaturation of PFRD-XC4 at 1 °C using
guanidinium chloride (GdmCl), monitored by CD at 225 nm,
yielded a free energy of unfolding (¢Gu) of 3.2 ( 0.1 kcal mol-1
and a m value of 2.0 kcal mol-1 M-1 (Figure 2c).
Sequential assignments were obtained for 90% of backbone
hydrogens (Table 1). On the basis of the chemical shifts for the
amide and R hydrogens, the PFRD-XC4 variant adopts a fold
similar to that of PFRD.40 Chemical shift differences between
PFRD and PFRD-XC4 are shown in Figure 3b. The chemical
shifts of PFRD-XC4 residues that are distal from the site of
mutation do not deviate significantly from those of PFRD,
suggesting that these residues are in the same chemical
environment as in PFRD. The chemical shifts of residues 7, 9,
10, and 48 significantly diverge from the corresponding PFRD
chemical shifts. These residues are expected to have different
chemical shifts because they are in close proximity to the
mutated site. Lysine 45 does not make a direct contact with the
mutated site but makes contact with residues 10 and 48.
Examination of NOE sequential connections (Figure 3a) reveals
strong HN-HA cross-peaks present in the â-sheet regions
(residues 1-6, 10-14, and 49) and HN-HN cross-peaks in
the turn regions (residues 6-10, 18-22, 29-32, and 46-47).
Large coupling constants (3JHNHA > 8.0 Hz) are also observed
for most of the â-sheet residues (Figure 3a). These observations
provide additional evidence that PFRD-XC4 adopts a fold
similar to that of PFRD.
Given that PFRD-XC4 adopts a fold similar to that of PFRD
and undergoes reversible unfolding, it should provide a tractable
model system for determination of protein hyperthermostability.
PFRD-XC4 should be amenable to mutagenesis studies aimed
at addressing the contribution of salt bridges to protein stability
using methods such as differential scanning calorimetry and
chemical denaturation.
Conclusion
We have successfully eliminated the iron center in hyper-
thermophilic rubredoxin and created a small protein that displays
reversible folding. The PFRD-XC4 variant has a melting
temperature of 82 °C, a thermodynamic stability of 3.2 kcal
mol-1 at 1 °C, and NMR chemical shifts that are similar to
those of the wild-type protein. This variant should provide a
tractable model system for studying the thermodynamic origins
of protein hyperthermostability.
Acknowledgment. We thank M. K. Eidness for the wild-
type Pyroccocus furiosus rubredoxin gene used in this study,
S. M. Malakauskas, S. Ross, and C. Sarisky for technical
assistance and discussions, and B. I. Dahiyat for discussions.
The work was supported by the Howard Hughes Medical
Institute (S.L.M.) and a NIH training grant (P.S.).
JA9834780
(40) Blake, P.; Park, J.; Zhou, Z.; Hare, D.; Adams, W. W. M.; Summers,
M. Protein Sci. 1992, 1508-1521.
(41) Koradi, R.; Billeter, M.; Wu¨thrich, K. J. Mol. Graph. 1996, 14, 51.
(42) Blake, P.; Park, J.; Bryant, F.; Aono, S.; Magnuson, J.; Eccleston,
E.; Howard, J.; Summers, M.; Adams, M. Biochemistry 1991, 30, 5-10895.
Rubredoxin Variant Folds without Iron J. Am. Chem. Soc., Vol. 121, No. 11, 1999 2345


Rubredoxin Variant Folds without Iron

Rubredoxin Variant Folds without Iron
Pavel Strop† and Stephen L. Mayo*,‡
Contribution from Biochemistry Option and Howard Hughes Medical Institute and DiVision of Biology,
California Institute of Technology, Mail Code 147-75, Pasadena, California 91125
ReceiVed October 1, 1998
Abstract: Pyroccocus furiosusrubredoxin (PFRD), like most studied hyperthermophilic proteins, does not
undergo reversible folding. The irreversibility of folding is thought to involve PFRD’s iron-binding site. Here
we report a PFRD variant (PFRD-XC4) whose iron binding site was redesigned to eliminate iron binding
using a computational design algorithm. PFRD-XC4 folds without iron and exhibits reversible folding with a
melting temperature of 82°C, a thermodynamic stability of 3.2 kcal mol-1 at 1°C, and NMR chemical shifts
similar to that of the wild-type protein. This variant should provide a tractable model system for studying the
thermodynamic origins of protein hyperthermostability.
Introduction
Hyperthermophilic proteins have attracted considerable at-
tention because they can serve as model systems for the
determination of factors responsible for protein stability. These
include an optimized use of structure elements such as hydro-
phobic interactions, packing forces, hydrogen bonds, and salt
bridges.1-5 In most cases, however, the usual methods for
determining thermodynamic stability cannot be used for hyper-
thermophilic proteins. The measurement of protein stability
requires reversible folding, but in almost all studied hyperther-
mophilic proteins, the denaturation is not reversible.6-8 In the
few hyperthermophilic proteins that exhibit reversible folding,
there are other elements that complicate the determination of
the free energy of folding. These proteins either are dimeric9,10
or do not fit a two-state denaturation model.10 Irreversible
denaturation has been addressed by hydrogen exchange studies11
and by molecular dynamics simulations,12 but neither of these
methods provides an accurate measure of free energy.
Rubredoxins are small (∼6 kDa) iron-sulfur proteins with
the active site arranged around an iron tetrahedrally coordinated
to four cysteinate sulfurs (Figure 1a). Thirteen rubredoxins have
† Biochemistry Option.
‡ Howard Hughes Medical Institute and Division of Biology. E-mail:
steve@mayo.caltech.edu.
(1) Lee, B.; Vasmatzis, G.Curr. Opin. Biotechnol.1997, 8, 423-428.
(2) Straume, M., Murphy, K. P., Freire, E., Adams, M. W. W., Kelly,
R. M., Eds. Thermodynamic Strategies for Protein DesignI creased
Temperature Stability; American Chemical Society: Washington, DC, 1992;
Vol. 498, pp 122-135.
(3) Rees, D. C.; Adams, M. W. W.Structure1995, 3, 251-254.
(4) Eidsness, M. K.; Richie, K. A.; Burden, A. E.; Kurtz, J., D. M.; Scott,
R. A. Biochemistry1997, 36, 10406-10413.
(5) Jiang, X.; Bishop, E. J.; Farid, R. S.J. Am. Chem. Soc.1997, 119,
838-839.
(6) Cavagnero, S.; Zhou, Z.; Adams, M.; Chan, S.Biochemistry1998,
37, 3377-3385.
(7) Klump, H. H.; W, A. M. W.; Robb, F. T.Pure Appl. Chem.1994,
66, 485-489.
(8) Wampler, E. J.; Neuhaus, B. E.J Protein Chem.1997, 16, 721-
732.
(9) Li, W.; Grayling, R. A.; Sandman, K.; Edmonson, S.; Shriver, J.
W.; Reeve, J. N.Biochemistry1998, 37, 10563-10572.
(10) MacBeath, G.; Kast, P.; Hilvert, D.Biochemistry1998, 37, 10062-
10073.
(11) Hiller, R.; Zhou, Z.; Adams, M.; Englander, W.Proc. Natl. Acad.
Sci.1997, 11329-11332.
(12) Bradley, A. E.; Stewart, E. D.; Adams, W. M.; Wampler, J. E.
Protein Sci.1993, 650-665.
VOLUME 121, NUMBER 11
MARCH 24, 1999
© Copyright 1999 by the
American Chemical Society
10.1021/ja9834780 CCC: $18.00 © 1999 American Chemical Society
Published on Web 03/05/1999
been sequenced, and all conserve the four cysteine ligands
(residues 5, 8, 38, and 41 in the hyperthermophilicPyroccocus
furiosus rubredoxin (PFRD) and residues 6, 9, 39, and 42 in
the mesophilicDesulfoVibrio Vulgaris rubredoxin (DVRD)).
Since several rubredoxin structures are available from the
Brookhaven Protein Data Bank at resolutions of 0.95-1.8
Å,13-19 these proteins are valuable models for the investigation
of the origins of thermostability.
It is believed that the iron binding site in rubredoxin is the
cause of irreversible unfolding.20 Here we report aPyroccocus
furiosusrubredoxin variant (PFRD-XC4) which was designed
to eliminate the iron-binding site using a computational design
algorithm. PFRD-XC4 is able to fold in the absence of iron
and undergoes reversible denaturation. The variant provides an
excellent opportunity for systematic exploration of the factors
determining protein thermostability.
Experimental Section
Computational Modeling. In the calculation using a mesophilic
structure, the four cysteine residues that form rubredoxin’s iron binding
site were optimized in the absence of iron coordination. Two of the
cysteines were classified as core residues (Cys6 and Cys39) and two as
boundary residues (Cys9 and Cys42). Residues were classified into core,
surface, and boundary groups as previously described.21 Seven amino
acid types (Ala, Val, Leu, Ile, Phe, Tyr, and Trp) were considered at
the two core positions, and 16 amino acid types (Ala, Val, Leu, Ile,
(13) Bernstein, F. C.; Koetzle, T. F.; Williams, G. J. B.; Meyer Jr, E. F.;
Brice, M. D.; Rodgers, J. R.; Kennard, O.; Shimanouchi, T.; Tasumi, M.J.
Mol. Biol. 1977, 112, 535-542.
(14) Adman, E. T.; Sieker, L. C.; H, J. L.J Mol. Biol. 1991, 337-352.
(15) Dauter, Z.; Sieker, L.; Wilson, K.Acta Crystallogr. 1992, 42-59.
(16) Day, M.; Hsu, B.; Joshua-tor, L.; Park, J.; Zhou, Z.; Adams, M.;
Rees, D.Protein Sci.1992, 1494-1507.
(17) Frey, M.; Sieker, L. C.; Payan, F.; Haser, R.; Bruschi, M.; Pepe,
G.; LeGall, J.J. Mol. Biol. 1987, 525-541.
(18) Sieker, L. C.; Stenkamp, R. E.; Jensen, L. H.; Prickril, B.; LeGall,
J. FEBS1986, 73-76.
(19) Watenpaugh, K. D.; Sieker, L. C.; Jensen, L. H.J. Mol. Biol.1979,
509-522.
(20) Cavagnero, S.; Debe, D.; Zhou, Z.; Adams, M.; Chan, S.Biochem-
istry 1998, 3369-3376. (21) Dahiyat, B. I.; Mayo, S. L.Science1997, 278, 82-87.
Figure 1. (a) Ribbon diagram of wild-type mesophilicDesulfoVibrio
Vulgaris rubredoxin showing the four cysteine residues that coordinate
the iron. (b) Ribbon diagram showing the computed side chain
orientations of the four mutations. Structure figures were generated
using MOLMOL.41
Figure 2. Circular dichroism measurements of wild-typePyroccocus
furiosusrubredoxin (PFRD) and the PFRD-XC4 variant. (a) Wavelength
scans of PFRD-XC4 at 1°C (circles), 99°C (diamonds), and refolded
at 1 °C (squares). PFRD is also shown at 1°C for comparison
(triangles). (b) Thermal unfolding curve for PFRD-XC4 monitored by
CD at 225 nm. (c) Guanidinium chloride denaturation curve for PFRD-
XC4 at 1°C.
2342 J. Am. Chem. Soc., Vol. 121, No. 11, 1999 Strop and Mayo
Phe, Tyr, Trp, Ser, Thr, Asp, Asn, Glu, Gln, His, Lys, and Arg) were
considered at the two boundary positions. Both amino acid identities
and side chain conformations were determined by the algorithm for
the four optimized residues. Neighboring residues 7, 10, 40, and 43
were fixed in identity, but their conformations were allowed to change
in the calculation. All other residues as well as the backbone were held
fixed. Computational details, potential functions, and parameters for
van der Waals, solvation, and hydrogen bonding are described in our
previous work.21-26
Mutagenesis and Protein Purification. The hyperthermophilic
mutant PFRD-XC4 was constructed by inverse PCR27 using a synthetic
Pyroccocus furiosusrubredoxin gene in plasmid pt7-7.4 The mutations
for PFRD-XC4 are C5L, C8T, C38A, and C41T. A synthetic gene based
on the sequence of theDesulfoVibrio Vulgaris rubredoxin was con-
structed for the mesophilic variant (DVRD-XC4) by recursive PCR28
and cloned into pt7-7. The mutations for DVRD-XC4 are C6L, C9T,
C39A, and C42T. Both mutants were verified by sequencing. Recom-
binant proteins were expressed by IPTG induction in BL21(DE3) hosts
(Invitrogen) as described29 and isolated using a freeze/thaw method.30
Purification was accomplished by reverse-phase high-performance liquid
chromatography using first a linear 1%/min followed by a 0.07%/min
acetonitrile/water gradient containing 0.1% TFA. Molecular weights
were verified by mass spectrometry.(22) Dahiyat, B. I.; Mayo, S. L.Protein Sci.1996, 5, 895-903.
(23) Dahiyat, B. I.; Sarisky, C. A.; Mayo, S. L.J Mol. Biol.1997, 273,
789-796.
(24) Dahiyat, B. I.; Mayo, S. L.Proc. Natl. Acad. Sci. U.S.A.1997, 94,
10172-10177.
(25) Dahiyat, B. I.; Gordon, D. B.; Mayo, S. L.Protein Sci.1997, 6,
1333-1337.
(26) Malakauskas, S. M.; Mayo, S. L.Nat. Struct. Biol.1998, 5, 470-
475.
(27) Hemsley, A.; Arnheim, N.; Toney, M. D.; Cortopassi, G.; Galas,
D. J. Nucleic Acids Res.1989, 17, 6545-6551.
(28) Prodromou, C.; Pearl, L. H.Protein Eng.1992, 5, 827-829.
(29) Alexander, P.; Fahnestock, S.; Lee, T.; Orban, J.; Bryan, P.
Biochemistry1992, 31, 3597-3603.
(30) Johnson, B. H.; Hecht, M. H.Bio/Technology1994, 12, 1357-
1360.
Table 1. 1H NMR Chemical Shifts for PFRD-XC4, pH 6.3, 25°Ca
residue HN HR Hâ others
1 Ala
2 Lys 8.68 5.45 1.62 HE, 2.92
3 Trp 9.39 4.95 2.74 HD1, 6.87; HE1, 9.73; HE3, 6.61; HZ2, 7.58; HZ3, 6.94; HH2, 7.21
4 Val 9.97 5.57 HG1, 0.78; HG2, 0.93
5 Leu 8.85 4.35 0.16, 1.91 HG, 1.21; HD1, 0.53; HD2, 0.70
6 Lys 9.09 3.79 HG1, 1.41; HG2, 1.57; HE1, 2.86; HE2, 2.95; HD, 1.71
7 Ile 8.25 4.09 1.88
8 Thr 6.92 4.73 4.47 HG2, 1.19
9 Gly 8.72 3.47, 4.24
10 Tyr 7.59 4.27 2.70, 3.30 HD, 7.33; HE, 7.20
11 Ile 7.34 4.97 1.49 HG2, 0.71; HD1, 0.80; HG1, 1.79
12 Tyr 9.53 4.64 2.92, 3.17 HD, 7.10; HE, 6.43
13 Asp 8.78 4.92 2.26, 2.86
14 Glu 8.26 3.86 1.94, 2.11 HG, 3.03
15 Asp 8.03 4.27
16 Ala 7.2 4.35 1.49
17 Gly 8.12 3.59, 3.98
18 Asp 8.32 5.17 3.01
19 Pro
20 Asp 9.09 4.5 2.75
21 Asn 7.73 5.19 2.69, 3.16
22 Gly 7.83 3.86, 4.15
23 Ile 7.67 4.38 1.94 HG2, 0.91; HD1, 0.82; HG1, 1.34
24 Ser 8.72 4.61 3.74, 3.90
25 Pro
26 Gly 8.22 3.74, 4.25
27 Thr 7.3 4.01 3.94 HG1, 6.35; HG2, 1.04
28 Lys 8.92 4.01 1.50, 2.12 HD, 1.69; HE, 3.09
29 Phe 10.06 3.29 HZ, 6.52; HD, 5.70; HE, 6.12
30 Glu 9.34 3.72 1.92, 2.08 HG1, 2.21; HG2, 2.32
31 Glu 7.41 4.09 2.06, 2.20
32 Leu 6.86 3.88 0.34, 0.88 HG, 0.70; HD2,-0.14
33 Pro
34 Asp 8.76 4.25 2.59, 2.69
35 Asp 8.23 4.53 2.58, 2.87
36 Trp 7.7 4.15 2.97 HD1, 7.19; HE1, 10.41; HE3, 6.84; HZ2, 7.49; HZ3, 6.25; HH2, 7.00
37 Val 6.72 4.32 HG1, 1.33
38 Ala 8.49 1.28
39 Pro 4.13 1.70, 2.27 HD1, 2.76; HD2, 3.48; HG, 1.90
40 Ile
41 Thr
42 Gly 7.62 3.64, 3.86
43 Ala 8.88 4.19
44 Pro
45 Lys 6.92 2.19, 2.39 HG1, 1.00; HG2, 1.18; HD, 1.70
46 Ser 8.12 4.38 4.03, 4.19
47 Glu 8.04 4.48 1.98, 2.32
48 Phe 8.16 4.94, 3.76
49 Glu 9.34 4.93
50 Lys 8.55 3.2 1.12, 1.28 HG1, 0.07; HG2, 0.22; HD, 0.65; HE, 2.68
51 Leu 8.68 4.3 1.29, 1.48 HD, 0.83
52 Glu 8.1 4.35 1.84 HG1, 2.07; HG2, 2.19
53 Asp 8.03 4.34 2.49, 2.65
a Chemical shifts were referenced to H2O (4.77 ppm at 25°C).
Rubredoxin Variant Folds without Iron J. Am. Chem. Soc., Vol. 121, No. 11, 19992343
CD Analysis.CD data were collected on an Aviv 62DS spectrometer
equipped with a thermoelectric unit and using a 1 mmpath length cell.
Protein samples were 40µM in 50 mM sodium phosphate buffer at
pH 6.3. Concentrations were determined by UV spectrophotometry.
Thermal melts were monitored at 225 nm. Data were collected every
2 °C with an equilibration time of 2 min and an averaging time of 10
s.Tm was determined by fitting the melting curves to a two-state model
as described.31 Guanidinium chloride denaturations were performed at
1 °C. ∆G’s, m values, and error estimates were obtained by fitting the
denaturation data to a two-state transition as described.32
NMR Studies. NMR data were collected on a Varian Unity Plus
600 MHz spectrometer equipped with a Nalorac inverse probe with a
self-shieldedz gradient. NMR samples were prepared in 90/10 H2O/
D2O or 99.9% D2O with 200 mM NaCl and 25 mM acetate-d3, pH
6.3. The sample concentration was approximately 1.5 mM. Sequential
assignment of resonances was achieved by the standard homonuclear
method.33 Two-dimensional DQF-COSY,34 TOCSY,35 and NOESY36
spectra were acquired at 25°C. The TOCSY spectrum was recorded
with a 80 ms mixing time using a clean-MLEV17 mixing sequence.
The NOESY spectrum was acquired with a 150 ms mixing time. Water
suppression was accomplished either with presaturation during the
relaxation delay or with pulsed field gradients.37 Spectra were processed
with VNMR (Varian Associates) and were assigned with ANSIG.38
3JHNHA values were derived from NOESY cross-peak fine structure using
the INFIT module of XEASY.39
Results and Discussion
Backbone coordinates for the mesophilic rubredoxin structure
used in the calculations were obtained from Brookhaven Protein
Data Bank entry 8rxn.13,15 A radius scale factor of 0.9 for the
van der Waals interactions24 resulted in two threonine residues
at the boundary positions and two alanine residues at the core
positions. To obtain an alternate sequence that provides greater
core volume, selection for the core positions in the calculation
was repeated with the radius scale factor decreased to 0.7 and
boundary positions 9 and 42 fixed as threonines. The amino
acid sequence selected by the algorithm under these conditions
contains the following mutations: C6L, C9T, C39A, and C42T
(Figure 1b). The equivalent mutations for the hyperthermophilic
variant, PFRD-XC4, are C5L, C8T, C38A, and C41T.
The far-ultraviolet circular dichroism (CD) spectrum of
PFRD-XC4 is nearly identical to that of the wild-type protein
at 1 °C (Figure 2a). Wavelength scans performed at 1°C, 99
°C, and then again at 1°C demonstrate reversible folding (Figure
2a). Thermal denaturation of the variant, monitored by CD at
225 nm, shows a cooperative unfolding transition with a melting
temperature (Tm) of 82 °C (Figure 2b). Based on the estimated
Tm for the wild-type hyperthermophilic rubredoxin,11 PFRD-
XC4 is destabilized by about 80°C. The mesophilic variant,
(31) Minor, D. L.; Kim, P. S.Nature1994, 371, 264-267.
(32) Santoro, M. M.; Bolen, D. W.Biochemistry1988, 27, 8063-8068.
(33) Wüthrich, K. NMR of Proteins and Nucleic Acids; John Wiley and
Sons: New York, 1986.
(34) Piantini, U.; Sorensen, O. W.; Ernst, R. R.J. Am. Chem. Soc.1982,
104, 6800-6801.
(35) Bax, A.; Davis, D. G.J. Magn. Reson.1986, 65, 355-360.
(36) Jeener, J.; Meier, B. H.; Bachmann, P.; Ernst, R. R.J. Chem. Phys.
1979, 71, 4546-4553.
(37) Piotto, M.; Saudek, V.; Sklenar, V.J Biomol. NMR1992, 2, 661-
665.
(38) Kraulis, P. J.J. Magn. Reson.1989, 24, 627-633.
(39) Bartels, C.; Xia, T. H.; Billeter, M.; Guntert, P.; Wuthrich, K.J.
Biol. NMR1995, 5, 1-10.
Figure 3. (a) NMR assignments summary and NOE connectivities of PFRD-XC4. Bars represent unambiguous connectivities with bar thickness
indexed to the intensity of the resonance. Coupling constants with3JHNHA > 8.0 Hz are shown as open squares, and3JHNHA < 4.0 Hz are shown as
black squares. Secondary structural elements of PFRD are given at the bottom of the figure for comparison.42 (b) Chemical shift difference between
PFRD and PFRX-XC4 for amide nitrogen (black lines) andR hydrogens (shaded line). Proline residues 19, 25, 33, 39, and 44 not shown. Residues
40 and 41 also not shown due to a missing sequential assignments. Mutations are at positions 5, 8, 38, and 41. Residues 7, 9, 10, and 48 are in the
close proximity of the mutated site.
2344 J. Am. Chem. Soc., Vol. 121, No. 11, 1999 Strop and Mayo
DVRD-XC4, was also expressed and found to be unfolded at 1
°C. Chemical denaturation of PFRD-XC4 at 1°C using
guanidinium chloride (GdmCl), monitored by CD at 225 nm,
yielded a free energy of unfolding (∆Gu) of 3.2( 0.1 kcal mol-1
and am value of 2.0 kcal mol-1 M-1 (Figure 2c).
Sequential assignments were obtained for 90% of backbone
hydrogens (Table 1). On the basis of the chemical shifts for the
amide andR hydrogens, the PFRD-XC4 variant adopts a fold
similar to that of PFRD.40 Chemical shift differences between
PFRD and PFRD-XC4 are shown in Figure 3b. The chemical
shifts of PFRD-XC4 residues that are distal from the site of
mutation do not deviate significantly from those of PFRD,
suggesting that these residues are in the same chemical
environment as in PFRD. The chemical shifts of residues 7, 9,
10, and 48 significantly diverge from the corresponding PFRD
chemical shifts. These residues are expected to have different
chemical shifts because they are in close proximity to the
mutated site. Lysine 45 does not make a direct contact with the
mutated site but makes contact with residues 10 and 48.
Examination of NOE sequential connections (Figure 3a) reveals
strong HN-HA cross-peaks present in theâ-sheet regions
(residues 1-6, 10-14, and 49) and HN-HN cross-peaks in
the turn regions (residues 6-10, 18-22, 29-32, and 46-47).
Large coupling constants (3JHNHA > 8.0 Hz) are also observed
for most of theâ-sheet residues (Figure 3a). These observations
provide additional evidence that PFRD-XC4 adopts a fold
similar to that of PFRD.
Given that PFRD-XC4 adopts a fold similar to that of PFRD
and undergoes reversible unfolding, it should provide a tractable
model system for determination of protein hyperthermostability.
PFRD-XC4 should be amenable to mutagenesis studies aimed
at addressing the contribution of salt bridges to protein stability
using methods such as differential scanning calorimetry and
chemical denaturation.
Conclusion
We have successfully eliminated the iron center in hyper-
thermophilic rubredoxin and created a small protein that displays
reversible folding. The PFRD-XC4 variant has a melting
temperature of 82°C, a thermodynamic stability of 3.2 kcal
mol-1 at 1 °C, and NMR chemical shifts that are similar to
those of the wild-type protein. This variant should provide a
tractable model system for studying the thermodynamic origins
of protein hyperthermostability.
Acknowledgment. We thank M. K. Eidness for the wild-
type Pyroccocus furiosusrubredoxin gene used in this study,
S. M. Malakauskas, S. Ross, and C. Sarisky for technical
assistance and discussions, and B. I. Dahiyat for discussions.
The work was supported by the Howard Hughes Medical
Institute (S.L.M.) and a NIH training grant (P.S.).
JA9834780
(40) Blake, P.; Park, J.; Zhou, Z.; Hare, D.; Adams, W. W. M.; Summers,
M. Protein Sci.1992, 1508-1521.
(41) Koradi, R.; Billeter, M.; Wu¨thrich, K. J. Mol. Graph.1996, 14, 51.
(42) Blake, P.; Park, J.; Bryant, F.; Aono, S.; Magnuson, J.; Eccleston,
E.; Howard, J.; Summers, M.; Adams, M.Biochemistry1991, 30, 5-10895.
Rubredoxin Variant Folds without Iron J. Am. Chem. Soc., Vol. 121, No. 11, 19992345


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Rubredoxin Variant Folds without Iron

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