The stability of FdTt is much lower at acidic pH, suggesting that electrostatic interactions are important for the high siently, resulting from rearrangement of the cubic clusters into l
Trang 1and thermal denaturation processes in solution As predicted
from the crystal structure, FdTt is extremely resistant
to perturbation The guanidine hydrochloride-induced
unfolding transition shows a midpoint at 6.5M (pH 7,
20°C), and the thermal midpoint is above boiling, at 114 °C
The stability of FdTt is much lower at acidic pH, suggesting
that electrostatic interactions are important for the high
siently, resulting from rearrangement of the cubic clusters into linear three-iron species Arange of iron–sulfur proteins has been found to accommodate these novel clusters in vitro, although no biological function has yet been assigned Keywords: ferredoxin; linear iron–sulfur cluster; protein unfolding; thermostability; Thermus thermophilus
Many proteins require the binding of cofactors to perform
their biological activity It has been demonstrated in vitro
that many proteins retain interactions with their cofactors
after polypeptide unfolding [1–6] Therefore, it is possible
that cofactors bind to their corresponding polypeptides
before or during folding in vivo Cofactors most often
stabilize the native states of the proteins with which they
interact [1–6] However, the manner in which cofactors
affect polypeptide folding and unfolding pathways remains
poorly understood Iron–sulfur ([Fe-S]) clusters represent
one of nature’s simplest, functionally versatile, and perhaps
most ancient cofactors [7] The [Fe-S] clusters, which have
2, 3 or 4 irons, are usually attached to their protein partners
by four cysteine thiol ligands [7–9] Proteins that contain
one or more [Fe-S] clusters represent a large class of
structurally and functionally diverse proteins that are
essential players in the life-sustaining processes of
respir-ation, nitrogen fixrespir-ation, and photosynthesis In these
proteins, the [Fe-S] clusters participate as agents of electron
transfer, substrate activation, catalysis, and environmental
sensing [7,10] Most [Fe-S] proteins have low reduction
potential and are known as ferredoxins Given the
struc-tural simplicity of [Fe-S] clusters and the participation of
ferredoxins in so many metabolic processes, it is somewhat
surprising that the pathways for biological formation of
[Fe-S] clusters and their incorporation into proteins are only now beginning to emerge [7]
The origin of protein thermostability is still an unsolved problem, and its understanding presents a great intellectual challenge to scientists, not to mention its potentially enormous biotechnological impact Proteins from thermo-philic organisms offer a unique opportunity to study the determinants of thermostability [11,12] Although these proteins are often very similar in sequence and structure to their mesophilic homologues (this is true also for mesophilic and thermophilic ferredoxins), they are much more resistant
to thermal denaturation and inactivation In the case of thermostable ferredoxins, it is not clear if subtle features around the [Fe-S] cluster site contribute to the additional stability or if higher stability is a result of polypeptide properties only [13] Earlier efforts to determine the origin of thermostability in monomeric proteins (most often without cofactors) have led to several hypotheses, such as stabiliza-tion by an increased number of ionic interacstabiliza-tions, an increased extent of hydrophobic-surface burial, an increased number of prolines, and smaller surface loops [12] Although evidence for these and other modes of stabiliza-tion can be found in specific examples, none applies to all or even most thermostable proteins If there are general rules for how thermophilic proteins attain their stability, then it is clear that they do not lie exclusively in individual inter-actions but may be based on properties of the whole molecule [14]
In this investigation, we focus on the seven-iron (one [4Fe-4S]2+/1+ and one [3Fe-4S]+1/° cluster) ferredoxin from Thermus thermophilus (hereafter called FdTt) T ther-mophilus is a Gram-negative aerobic bacterium found
in hot springs, thermal vents, and thermal spas It grows
at temperatures of 50–82°C, with optimum growth at 65–72°C [15] Its seven-iron ferredoxin, FdTt, is a small single-chain, single-domain protein with 78 residues The
Correspondence to P Wittung-Stafshede, Department of Chemistry,
Tulane University, 6823 St Charles Avenue, New Orleans,
LA70118, USA Fax: + 1 504 865 5596, Tel.: + 1 504 862 8943,
E-mail: pernilla@tulane.edu
Abbreviations: FdAv, ferredoxin from Azotobacter vinelandii; FdTt,
ferredoxin from Thermus thermophilus; GdnHCl, guanidine
hydro-chloride; T m , midpoint temperature of thermal unfolding transition.
(Received 2 July 2003, revised 23 September 2003,
accepted 7 October 2003)
Trang 2crystal structure of FdTt was recently solved at 1.64 A˚
resolution [16], but there has been no in vitro
characteriza-tion of the protein FdTt has a (bab)2 core structure
enveloping the two [Fe-S] clusters with an additional
C-terminal a-helix further away from the clusters (Fig 1)
According to the crystal structure of FdTt, the improved
polar and hydrophobic interactions lead to extensive
cross-linking of the secondary-structure elements (compared with
mesophilic ferredoxins), which is believed to result in high
stability It appears that most of the stabilizing features of
FdTt are found in the vicinity of the [3Fe-4S] cluster, which
is the cluster that has functional importance [16] We here
report a detailed biophysical characterization of the stability
of FdTt to thermal and chemical perturbation in solution
in vitro Interestingly, the cubic clusters in FdTt rearrange
into new, linear three-iron species on polypeptide unfolding
at high pH
Materials and methods
Materials
The chemical denaturant guanidine hydrochloride
(GdnHCl) was of highest purity All chemicals were
purchased from Sigma The FdTt protein was purified as
previously described [16]
Chemically induced unfolding
GdnHCl was used to promote FdTt unfolding at different
pH values (2.5, 4, 7, 10 and 10.5) at 20°C An FdTt
concentration of 20 lMwas used unless specified otherwise
Buffers of 25 mMconcentration were used unless otherwise
specified Samples were incubated (at 20°C) for various
lengths of time from 5 min to 120 h before spectroscopic
measurements were taken Unfolding was monitored by
far-UV CD (200–300 nm) on a JASCO-810 instrument
(1 mm cell), by visible absorption (250–700 nm) on Cary-50
and Cary-100 spectrophotometers (1 cm cell), and by tryptophan emission (300–450 nm, excitation at 280 nm)
on a Cary Eclipse instrument For each set of conditions, the transition midpoint ([GdnHCl]1/2) was obtained by direct inspection of the data Different buffers were used for experiments at different pH values: glycine/HCl was used for pH 2.5, citric acid/sodium phosphate was used for pH 4, phosphate was used for pH 7, and KCl/NaOH was used for
pH 10 and 10.5
EPR experiments were performed with liquid nitrogen at
110 K on a Bruker EMX instrument (A Tsai, University of Texas Medical School, Houston, TX, USA) using 10 mW microwave power and 9.3 GHz microwave frequency The EPR samples were 100 lMFdTt in buffer, pH 10.5 (folded protein) and 100 lM FdTt incubated for 15 min in 6.5M
GdnHCl, pH 10.5, 20°C (unfolded protein with linear [Fe-S] cluster)
Thermally induced unfolding Thermally induced unfolding of FdTt was monitored by visible absorption, fluorescence, and far-UV CD methods
To probe the reaction by cluster integrity, the absorption at
408 nm was monitored (for FdTt samples with different pH and GdnHCl conditions) as a function of temperature The signal from 20 lM FdTt was recorded on increasing the temperature at rates of 0.5, 0.25, or 0.125°C per minute (one data point collected per second; from 25°C to 95 °C)
At the end of each experiment, the temperature was decreased to 25°C, and a full absorption spectrum was taken to check for refolding The midpoint of transition (Tm) at each pH and GdnHCl concentration was deter-mined by direct inspection of the absorption vs temperature data Tm values obtained for FdTt in the presence of different GdnHCl concentrations (but the same pH and scan rate) were used to linearly extrapolate to 0MGdnHCl,
to obtain the Tmfor FdTt without denaturant at each pH (and each scan rate) Next the extrapolated T values for
Fig 1 Ribbon diagram of FdTt Protein data
bank file 1H98 The iron–sulfur clusters
(iron space-filled red, sulfur space-filled
yellow) and secondary-structure elements
(a-helices in red, b-strands in gold, random
coil in white) are highlighted.
Trang 3Chemically induced FdTt unfolding
Folded FdTt has characteristic visible absorption at 408 nm
resulting from the intact [Fe-S] clusters which disappears as
the protein unfolds (Fig 2A) FdTt has one tryptophan
at position 64, and tyrosines at positions 33, 55 and 67 in
the primary structure [16] Folded FdTt shows very little
tryptophan fluorescence because of energy transfer to the
iron–sulfur clusters As the protein unfolds, the tryptophan
emission (at 350 nm) increases dramatically because of
cluster and tryptophan separation and presumably cluster
decomposition (Fig 2B) Folded FdTt has positive CD
absorption 230 nm, arising from the tryptophan and
tyrosine contribution, and a negative CD feature at 220 nm,
characteristic of the presence of secondary structure Both
CD bands lose intensity as the protein unfolds (Fig 2C),
and the CD spectrum on unfolding resembles that of a
random-coil polypeptide Taken together, these
spectro-scopic techniques can be used to probe the unfolding
reaction of FdTt via cluster integrity (visible absorption),
cluster–tryptophan distance (emission), and secondary
structure (far-UV CD) (Fig 2)
To probe the unfolding mechanism for FdTt, visible
absorption, fluorescence, and far-UV CD probes were
monitored as a function of time after the protein had been
mixed with a high concentration of the chemical denaturant
GdnHCl (7.9M GdnHCl final concentration, 20°C)
Despite the high denaturant concentration, more than 6 h
were required for the signals to reach their endpoints at pH
7 (t1/2¼ 50 ± 10 min) At pH 2.5, the kinetics for the same
reaction were faster (t1/2¼ 10 ± 5 min) Identical (within
error) kinetic traces were observed regardless of
far-UV CD; 408-nm absorption or fluorescence signals were
used as the detection method (absorption and CD changes
shown in Fig 3) for each condition This observation
suggests that FdTt unfolding is a single process (at pH 2.5
and 7), in which polypeptide unfolding and cluster
degra-dation occur simultaneously At pH 10, however, the
kinetic process monitored by visible absorption did not
match that probed by far-UV CD (discussed below)
Next, GdnHCl titrations at 20°C were performed at
various pH values, and FdTt unfolding was probed at
different incubation times (from 2 to 48 h) using the three
spectroscopic methods The GdnHCl-induced unfolding
process is irreversible at all pH values, probably because the
clusters decompose on unfolding (in accord with complete
visible-absorption disappearance, Fig 2A) It is also
possible that cysteine oxidation takes place in the unfolded state, hampering refolding Irreversible unfolding has been reported for other ferredoxins [17–20] Because of the irreversibility of the reaction, no thermodynamic data, such
Fig 2 Visible absorption (A), tryptophan emission (B), and far-UV CD (C) of native FdTt (solid line, 0 M GdnHCl, pH 7, 20 C) and denatured FdTt (dotted line, 7.9 M GdnHCl 48 h incubation, pH 7, 20 C).
Trang 4as DGU(H2O), could be obtained for FdTt; instead, we
report unfolding-transition midpoints as a function of pH
and incubation time at 20°C (summarized in Table 1) The
transition midpoints shift to lower GdnHCl concentration
as the incubation time is increased as expected for an
irreversible reaction (Table 1)
We observe single unfolding transitions with all
spectro-scopic probes under all conditions except at high pH (see
below) The different probing methods, reporting on
different properties of FdTt, gave similar transition mid-points for FdTt samples incubated for the same amount of time and at the same pH condition (Table 1), supporting the suggestion that FdTt unfolding is a two-state process with cluster degradation occurring simultaneously with polypep-tide unfolding For example, the midpoint of the unfolding transition appeared at 6.5M GdnHCl after 48 h of incubation at pH 7 (20°C) as monitored by all three probes At pH values lower than 7, the apparent stability of FdTt decreased significantly (Fig 4); for example, at
pH 2.5, the midpoint of transition was 1.5M GdnHCl (48 h incubation, 20°C)
Transition midpoints for FdTt samples with and without
500 mMNaCl were identical (20°C, pH 7), indicating that protein stability is not affected by the presence of NaCl (data not shown) The effect of protein concentration was investigated in a separate experiment by comparing unfold-ing transitions for 10 lMand 80 lMFdTt at pH 2.5 The transition midpoints, monitored by visible absorption, was not significantly different for the two different protein concentrations In this experiment (pH 2.5, 20°C), the GdnHCl-induced midpoints were 1.7 and 1.6M GdnHCl after 2 h of incubation and 1.1 and 1.0MGdnHCl after
24 h of incubation for 80 and 10 lMFdTt, respectively Thermally induced FdTt unfolding
In buffer at pH 7, FdTt does not unfold below 100°C Therefore, thermally induced unfolding experiments were conducted in the presence of different concentrations of GdnHCl (not high enough to unfold FdTt at 20°C) Like GdnHCl-induced unfolding at 20°C, thermal unfolding of FdTt occurred in a single transition which was irreversible
In most thermal experiments, visible absorption was used
as detection probe, although some experiments were also probed by far-UV CD and fluorescence At each condition, identical Tm values were observed with the different detection probes For each scan rate studied (0.5, 0.25 and 0.13°CÆmin)1), thermal midpoints (T ) obtained at
Fig 3 FdTt unfolding kinetics (20 C), measured by visible absorption
at 408 nm, on addition of 7.9 M GdnHCl at pH 7 (solid line) and pH 2.5
(dashed line) Inset: far-UV CD detection of the same process (solid
line, pH 7; dashed line, pH 2.5).
Table 1 GdnHCl-induced unfolding midpoints (M) for FdTt at four pH
values probed by visible absorption (Abs), tryptophan emission (FL), and
far-UV CD (CD) after different incubation times (20 C) The values
have an error of ± 0.2 M *, Intermediate with new absorption bands
forms on unfolding; therefore, unfolding midpoints are not reliable by
this technique.
pH Time (h)
Unfolding midpoints (M)
7 2 No unfolding No unfolding No unfolding
Fig 4 GdnHCl concentrations at which the midpoint of unfolding transitions occur as a function of pH Incubation times 2 h (d), 8 h (j),
24 h (r), and 48 h (m).
Trang 5different concentrations of GdnHCl were extrapolated to
give a Tmvalue for each scan rate at 0MGdnHCl Next,
these Tmvalues at 0MGdnHCl for different scan rates were
extrapolated to (1/scan rate)¼ 0, to give a Tm
correspond-ing to infinite scan rate conditions The method of
extrapolation to infinite scan rate has been used before to
eliminate irreversible time-dependent steps in
protein-unfolding reactions [21] The thermal midpoints for FdTt
unfolding (at infinite scan rate) are 69°C (pH 2.5), 91 °C
(pH 4), 114°C (pH 7), and 90 °C (pH 10.5) (Fig 5) Thus,
optimum stability of FdTt to heat also occurs around
neutral pH, and the thermal stability is, like the resistance to
chemical denaturation, dramatically reduced at lower pH
Formation of linear clusters at high pH
On GdnHCl-induced unfolding of FdTt at high pH
(20°C), new visible absorption bands at 520 nm
and 610 nm appeared transiently before complete
disappearance of the visible absorption occurred (Fig 6A) The new peaks formed with rate constants that depended
on the concentration of GdnHCl (pH 10, 20°C) In 7.9M
GdnHCl, the new bands formed rapidly and became more intense (maximum intensity reached within 25 min) than at
6MGdnHCl where the formation was slower (maximum intensity reached within 80 min) Identical absorption bands at 520 nm and 610 nm have been observed transi-ently in some other ferredoxins and in beef aconitase under various conditions that perturb the protein structure [19,20,22] In those cases, it was concluded from EPR and Mo¨ssbauer studies and comparison with small model compounds [23] that the new absorption features resulted from linear three-iron clusters bound to the unfolded,
or partially unfolded, polypeptide (Fig 6A, inset) The formation of the new absorption bands for FdTt correlated with the disappearance of the far-UV CD signal, implying
Fig 5 (A) T m vs concentration of GdnHCl at pH 7 for three different
scan rates [(j) 0.5 CÆmin-1; (d) 0.25 CÆmin-1; (m) 0.13 CÆmin-1)]
and (B) T m (at infinite scan rate) as a function of pH.
Fig 6 Formation of linear species at high pH (A)Visible absorption of FdTt on addition of 7.9 M GdnHCl (pH 10.5) after 1 min (solid line),
15 min (dotted line), and 24 h (dotted-dashed line) incubation (inset: schematic drawing of a linear three-iron cluster) (B) EPR spectra (110 K, 10 mW, 9.3 GHz) of folded FdTt in pH 10.5 (thin line) and FdTt at pH 10.5 incubated for 15 min in 6.5 M GdnHCl (thick line; corresponding to dotted line in A).
Trang 6that polypeptide unfolding triggered linear-cluster
forma-tion Support for the idea that the new absorption bands
also correspond to linear three-iron clusters in FdTt comes
from EPR experiments (Fig 6B) At pH 10.5, FdTt
exhibits a typical [3Fe)4S] cluster resonance at g ¼ 2.02
and a minor contribution at g¼ 4.3 from adventitious
iron in solution On incubation in 6.5M GdnHCl for
15 min (pH 10.5, 20°C; to reach maximum intensity at
610 nm), the signal from the [3Fe)4S] center decreases
with the concomitant 15-fold increase in the g¼ 4.3
resonance and a small peak at g¼ 9.5 (features
charac-teristic of an S¼ 5/2 system in a rhombic environment)
The S¼ 5/2 signal is compatible with the presence of a
linear three-iron cluster and very similar to that reported
for purple aconitase, another seven-iron ferredoxin, and
model compounds [19,22,23] The linear [Fe–S] cluster
remained in the unfolded protein for several hours (5–10 h;
20°C, pH 10.5, 6.5–7.0MGdnHCl) before complete loss
of visible absorption occurred
Discussion
Understanding and probing protein stability at high
temperatures and extreme conditions is relevant for a
variety of biochemical and biotechnological applications
Intriguingly, the stability of a protein can be increased
by the optimization of a few interactions without large
structural modifications To further understand the
mechanisms of increased stability in bacterial ferredoxins,
we characterized chemical and thermal denaturation of
the seven-iron T thermophilus ferredoxin (FdTt) in
solution This protein was recently crystallized [16], but
in vitro solution studies have been lacking The data
presented here thus constitute the groundwork for future
experiments in which strategic FdTt mutants, designed
on the basis of the crystal structure, can be directly
compared with the biophysical behavior of the wild-type
form
Our solution study shows that wild-type FdTt is
extremely stable to heat and chemical denaturants An
explanation for this behavior, in terms of the sum of many
minor effects, is found on analyzing the crystal structure
FdTt shares 64% sequence identity with the mesophilic
ferredoxin from Azotobacter vinelandii (FdAv), which is
a protein with the same overall structure as FdTt but
significantly less stable in solution [16] Like other
mesophilic ferredoxins, FdAv has a stretch of 29 residues
at the C-terminus, which is absent from FdTt This stretch
of residues protects the [3Fe)4S] cluster in FdAv from
solvent Hence, the [3Fe)4S] cluster is more accessible to
solvent in FdTt, and shielding of this cluster from solvent
cannot be vital for FdTt function Because FdTt has a
shorter C-terminus than FdAv, it has less accessible solvent
surface area, which may aid in resistance to perturbation
[11,12] On comparing the crystal structures, polar residues
at the surface of FdTt replace topologically equivalent
negatively charged residues in FdAv Moreover, an a-helix
in FdTt, replacing a 310-helix in FdAv, is stabilized with
alanine residues [16], and the (bab)2 core of FdTt is
stabilized by additional hydrogen bonds between side
chains and the main chain, as compared with FdAv Also,
FdTt has more glycine residues than FdAv, which may
minimize conformational strain in the folded state [16] FdAv uses a cluster of glutamic and aspartic acid residues
to electrostatically interact with its physiological electron-transfer partner In FdTt, this region of the protein’s surface is less charged This difference in electrostatics is thought to increase FdTt stability by reducing unfavorable repulsions between negatively charged residues The cor-responding reduced electrostatic attraction between FdTt and its electron-transfer partners may be compensated for
by faster protein diffusion rates at the higher temperatures [16]
In addition to our biophysical study of FdTt presented here, five other thermostable seven-iron ferredoxins have been studied in vitro with respect to thermostability: from the thermostable bacteria Bacillus schlegelii [24] and Bacillus acidocaldarius [25] and the thermostable archaea Acidianus ambivalens [13,20] and Sulfolobus sp strain 7 [26] Three of these proteins (and FdTt) are stable above the boiling point of water at pH 7 Ferredoxin from
B schlegelii begins to unfold at 90°C [24], ferredoxin from B acidocaldarius is completely denatured at 88°C [25,27], ferredoxin from A ambivalens (FdA; species with zinc ion) has a thermal midpoint of 122°C (pH 7) [13], and another ferredoxin from A ambivalens (FdB; species without zinc) has a thermal midpoint of 108°C (pH 6.5) [18] The seven-iron ferredoxin from Sulfolobus sp strain 7 has a thermal midpoint of 109°C [28] FdTt, with its
114°C thermal midpoint at pH 7, is thus one of the most stable seven-iron ferredoxins (in fact, among proteins in general) investigated to date Our GdnHCl-induced unfolding data at 20°C for FdTt can only be compared with similar work on the A ambivalens ferredoxin (FdA; species with zinc) In the case of that protein, the GdnHCl-induced unfolding midpoints appear at 7.1M
(pH 7, 20°C), 2.3M (pH 2.5, 20°C), and 6.3M (pH 10,
20°C) [13] Thus both proteins have similar extreme resistance to chemical denaturants at pH 7 and higher The dramatic reduction in apparent stability at low pH for both FdTt and A ambivalens ferredoxin (using both chemical and thermal perturbation) implies that electro-static interactions contribute significantly to the proteins’ integrity at the higher pH values This occurs because, at low pH values, salt bridges are easily broken due to protonation of aspartic and glutamic acid residues (which have pKavalues around 4)
On GdnHCl-induced polypeptide unfolding at high pH,
we find that the clusters in FdTt transiently rearrange into intermediate species before complete cluster degradation occurs Clusters with the same spectroscopic features as
we found in FdTt at high pH have been shown in other studies to be linear [3Fe)4S] clusters still bound to the polypeptides (Fig 6A, inset) [19] Our spectroscopic data (absorption and EPR) support the suggestion that in FdTt also the cubic clusters rearrange into linear species
on protein unfolding under alkaline conditions The presence of a linear [3Fe)4S] cluster was first observed
in the protein bovine heart aconitase, at high pH where the protein structure was perturbed [22] Recently, the same linear cluster was discovered on in vitro unfolding of seven-iron ferredoxins from A ambivalens and S acidoc-aldarius [13,19,20] The observation of a linear cluster in FdTt and other seven-iron ferredoxins, and the recent
Trang 7observations of this cluster in [2Fe)2S] ferredoxins from
Aquifex aeolicus ([17] and unpublished data), suggest a
more general relevance of this type of linear cluster in
nature In Table 2, we summarize the known systems and
solution conditions in which linear three-iron clusters have
been observed in vitro No biological function for linear
[3Fe)4S] clusters is yet known, although reconstituted,
recombinant human cytosolic iron regulatory protein 1
has been found to contain such a cluster under
physio-logical conditions [29] We speculate that [Fe–S] cluster
rearrangements induced by protein-conformational
chan-ges may be used for regulatory purposes in vivo The
linear cluster may be a storage or transport form for iron
and sulfide in the cells ready for use in resynthesis of cubic
(functional) clusters
Summary
Examination of the crystal structure of the seven-iron
ferredoxin from T thermophilus has suggested that it
represents the minimal functional unit of this type of
protein [16] In agreement, we find FdTt to be a very stable
protein in solution in vitro: temperatures above boiling or
high denaturant concentrations and long incubation times
are necessary to perturb it From our work in solution at
different pH values, it is clear that electrostatic interactions
play a significant role in governing the high stability of
FdTt As unfolding is very slow (hours), even in high
concentrations of denaturant, there appears to be a kinetic
barrier to FdTt unfolding Slow unfolding kinetics may be a
general mechanism governing high stability of thermostable
proteins On polypeptide unfolding at high pH, linear
three-iron clusters form in FdTt Recent discoveries of the
transient appearance of such linear clusters in many
different iron–sulfur proteins imply that they may be of
biological relevance
Acknowledgements
This work was supported by the National Institute of Health
(GM5966301A2) (P.W.-S.), the Louisiana Board of Regents
(C.L.H.), and the Newcomb College Fellows Program (Tulane
University, New Orleans, Louisiana) (S.G.) We thank Ah-lim Tsai
(University of Texas Medical School, Houston) and John S Olson
(Rice University, Houston) for help with EPR experiments.
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