9912
J. Phys. Chem. B 2010, 114, 9912–9919
Thermal Unfolding Kinetics of Ubiquitin in the Microsecond-to-Second Time Range Probed
by Tyr-59 Fluorescence
Melinda Noronha,*,† Hana Gerbelová,† Tiago Q. Faria,‡ Daniel N. Lund,§,⊥ D. Alastair Smith,§,⊥
Helena Santos,‡ and António L. Maçanita*,†
Centro de Quı́mica Estrutural, Instituto Superior Técnico, Technical UniVersity of Lisbon,
1049-001 Lisboa, Portugal, Instituto de Tecnologia Quı́mica e Biológica, UniVersidade NoVa de Lisboa, Rua
da Quinta Grande 6, Apartado 127, 2780-156 Oeiras, Portugal, and School of Physics and Astronomy,
UniVersity of Leeds, Leeds, LS2 9JT, United Kingdom
ReceiVed: May 7, 2010; ReVised Manuscript ReceiVed: June 10, 2010
Thermal folding/unfolding kinetics of wild-type ubiquitin (wt-UBQ) was studied in a wide time range, from
microseconds to seconds, by combining rapid-mixing T-jump and laser T-jump with fluorescence detection
(MTJ-F and LTJ-F, respectively) to monitor the fluorescence changes of Tyr-59 located on the 310-helix. The
kinetics occurs exclusively in the millisecond to second time range, and the decays are strictly single exponential.
From global analysis of folding and unfolding decays, the kf and ku values were determined, without use of
the equilibrium constant Ku. The activation enthalpy of folding is negative (∆Hf#(Tm) ) -10.8 kcal/mol), but
the free energy of the transition state is substantially larger than that of the unfolded state (∆Gf#(Tm) ) 7.6
kcal/mol . RTm). Thus, wt-UBQ behaves as a two-state folder, when folding is monitored by the fluorescence
of Tyr-59. The observation of kinetics on the microsecond time scale, when folding is monitored by the
disruption of hydrogen bonds between β-strands, using nonlinear infrared spectroscopy of the amide I vibrations
(LTJ-DVE) [Chung, H. S.; Tokmakoff, A. Proteins: Struct., Funct., Bioinf. 2008, 72, 474-487], seems to
result from the fact that MTJ-F monitors the effective unfolding (backbone exposure to water) of the thermally
excited protein alone, while LTJ-DVE also monitors preliminary events (hydrogen-bond breaking) and thermal
re-equilibration of the thermally excited protein.
Introduction
Kinetics of the thermal unfolding of proteins has been studied
over the past 10 years using the laser temperature jump (LTJ)
technique.1 LTJ can produce temperature jumps of 10-15 °C
while keeping constant the final temperature (Tf) for ca. 2-3
ms.1 Hence, only the so-called fast folders (peptides2-4 and small
proteins5-11) were initially studied. Later, the upper limit of 3
ms of the LTJ technique was extended to longer time scales
(10-47 ms) by taking into account the temperature time drift
after the T-jump.12
The appearance of the rapid-mixing stopped-flow technique
in 2008 (MTJ)13 provided access to both positive and negative
T-jumps, with a time resolution of a few milliseconds (dead
time ca. 4 ms) and a constant final temperature up to hours.
Therefore, by combining LTJ and MTJ, thermal unfolding/
folding kinetics can be studied for any protein from a few
microseconds to hours.
wt-Ubiquitin (wt-UBQ), a small protein of 76 amino acid
residues (8.5 kDa), has been extensively used as a model for
both chemical14-20 and thermal unfolding studies10-12,21 (Figure
1).
The resulting data have raised a number of questions
concerning the folding/unfolding kinetics of this protein and/or
of its mutants. Specifically, deviations from simple kinetics have
* To whom correspondence should be addressed. E-mail: macanita@
ist.utl.pt (A.L.M); [email protected] (M.N).
†
Technical University of Lisbon.
‡
Universidade Nova de Lisboa.
§
University of Leeds.
⊥
Present address: Avacta Group Plc, York Biocentre, Innovation Way,
York, YO10 5NY, U.K.
Figure 1. Structure of wt-ubiquitin showing the location of Tyr-59 at
the end of the 310-helix between β-strands IV and V (βIV and βV).
been observed, depending on experimental conditions17 and
detection methods.18,19 In some cases, the deviations were
consistent with the presence of intermediate states, while in other
cases the deviation consisted of a fast decay component, either
directly observed12 or indirectly deduced (burst phase).14-16
10.1021/jp104167h  2010 American Chemical Society
Published on Web 07/15/2010
Thermal Folding/Unfolding Kinetics of wt-UBQ
Intermediate states have been observed essentially in experiments involving chemical denaturation of tryptophan-mutated
UBQ.22
The fast decay component was observed with thermal (LTJDVE) denaturation of wt-UBQ by probing the disruption of
interhydrogen bonds between β-strands with nonlinear infrared
spectroscopy of the amide I vibrations in acidic conditions.11,12
The nonexponential kinetics was observed in a time range from
tenths of a microsecond to 2.7 ms, although the major change
in the probe signal occurred within 10-47 ms where the traces
could be fitted with single-exponential functions.11,12 The fast
nonexponential component accounted for a 7.7% variation of
signal intensity and was dependent on the size of the T-jump
(∆T) and final temperature (Tf). The authors attributed the fast
component to downhill unfolding of a small population of the
native protein located energetically above the transition state
immediately after the T-jump.12
Previously we studied the thermal unfolding of wt-UBQ at
pH 1.5 using time-resolved fluorescence spectroscopy (TRFS)
by following the fluorescence of its single tyrosine, Tyr-59,
located on the 310-helix.23 The purpose was to determine whether
the fluorescence of this tyrosine residue could be used as an
intrinsic probe to study the protein thermal folding/unfolding,
thus allowing the use of wt-UBQ as a model protein in our
studies on the mechanisms underlying protein stabilization by
osmolytes.24,25 The results showed that indeed the fluorescence
of Tyr-59 was an adequate probe, and, moreover, they were
consistent with the two-state model.23 However, because there
was a discrepancy in the Tm values (2.4 °C) and in the unfolding
enthalpy values (5.1 kcal/mol) determined by TRFS and DSC,
the apparent simplicity of the folding/unfolding mechanism was
not free of doubt. In this work, we combine LTJ-F and MTJ-F
to further study the thermal folding/unfolding kinetics of wtUBQ using the fluorescence of Tyr-59 in an effort to resolve
this issue.
Materials and Methods
Samples. Bovine ubiquitin was purchased from Sigma and
purified with a Mono-S column using a salt gradient as
previously described.23 N-Acetyltyrosinamide (NAYA) was
purchased from Sigma-Aldrich. Sodium acetate (Merck), sodium
chloride (Merck), acetic acid (Riedel-deHaën), and hydrochloric
acid (Riedel-deHaën) were of pro analysis grade. A final protein
concentration of 40 µM was used in all rapid-mixing T-jump
experiments. Double-distilled water was used in all samples
except for samples in D2O (Aldrich) where the pD was adjusted
using deuterium chloride 99 atom % (Aldrich).
Rapid-Mixing T-jumps (MTJ-F). Rapid-mixing T-jump
(MTJ-F) measurements were carried out using a new temperature jump accessory from BioLogic coupled to an existing
Biologic SFM-4 with a MPS-52 microprocessor unit connected
via a fiber optic cable to a xenon mercury lamp and equipped
with a circulating water bath for temperature control.13,26 The
accessory achieves temperature jumps as high as 40 °C by rapid
mixing of two solutions initially at different temperatures T1
and T2. The final temperature of the mixture (Tf) is determined
both by the initial temperatures T1 and T2 and by the volume
ratio of the two solutions. Three Peltier elements (BioLogic
TCU-250) are used to control the initial temperatures of the
two solutions and the observation cell after mixing. The heat
generated by the Peltier elements is dissipated using a water
bath circulator. Prior to the T-jump experiments for each Tf,
test shots were carried out for both unfolding and refolding to
determine the optimal conditions of the (1) appropriate voltage
J. Phys. Chem. B, Vol. 114, No. 30, 2010 9913
(V) for a sufficiently strong signal intensity without saturation,
(2) correct flow rates (Q) to ensure that the temperature is kept
constant in the region between the mixer chamber and the
observation cell (Qf ) 12 mL/s), (3) correct dilution factors to
avoid problems of aggregation while obtaining a strong enough
signal change (protein (P) to buffer (B) ratio of 1:2), and (4)
volume per shot to reduce noise (VB ) 0.146 mL, VP ) 0.073
mL, Vf ) 0.219 mL). For each Tf, the following data were
collected: T-jumps for unfolding (38 °C f Tf) and refolding
(70 °C f Tf), and shots with only UBQ and buffer both at the
same Tf, to guarantee that the three Peltier elements are
intercalibrated and to ensure that upon mixing of protein and
buffer no deviations are observed from the temperature (Tf) of
the observation cell, which if present could lead to artifacts in
kinetic curves. Sampling periods of 2.3 s were used after
checking the absence of fluorescence changes at longer times
(23 s sampling period). The folding/unfolding kinetics of wtUBQ was monitored by changes in the intrinsic fluorescence
of Tyr-59 with excitation at λ ) 275 nm, and the emission was
collected above 290 nm using a 290 nm Schott cutoff filter.
Experiments were carried out in 12.5 mM sodium acetate
buffer, and the pH was adjusted with HCl to final concentrations
of [H+] ) 33 and 100 mM (pH ) 1.5 and 1.0). Further
experiments in D2O were carried out also in 12.5 mM sodium
acetate buffer and a final concentration of [D+] ) 0.1 M (pD
) 1) using DCl.
Laser-Induced T-jumps. Laser-induced T-jumps were carried out using a home-built LTJ-F apparatus as described
elsewhere.9 The T-jumps were generated by Raman shifting the
1064 nm output (1 J per pulse) of a Q-switched Nd:YAG Laser
[Spectra-Physics (Hemel Hempstead, U.K.) Quanta Ray Lab
170] in a 1 m length stainless steel tube of methane at 30 atm
to produce an infrared pulse at 1550 nm with 10-30%
conversion efficiency. The protein sample is contained in a
quartz cell of path length 0.5 mm [Custom (Custom LC, Hellma,
U.K.)], thermostatted by a Peltier device and Peltier controller
[Marlow (Dallas) SE5010] to maintain the sample at constant
temperature. Excitation was carried out at 275 nm by using the
frequency-tripled output of a femtosecond mode-locked Ti:
sapphire laser [Coherent (Santa Clara, CA) MIRA 900/VERDI].
The mode-locked output at 37 MHz was pulse-picked to give
a pulse separation of 130 ns and a pulse width of <200 fs.
Fluorescence was selected using a cutoff filter at 290 nm and
detected using a photomultiplier tube (PMT) with a custom
dynode chain (Hamamatsu R7400U-03). The PMT output signal
was digitalized by using a digital sampling oscilloscope (LeCroy
LC584AXL) with a sampling rate of 8 GS/s. The dead time of
the experiment depends on the scattered light caused by
cavitations after each T-jump and was typically of 5-40 µs.
Calibration of the fluorescence signal was carried out using the
fluorescence signal of N-acetyltyrosinamide (NAYA) as a
function of temperature.
DSC Measurements. Differential scanning calorimetry (DSC)
measurements were carried out on a VP-DSC microcalorimeter
from Microcal equipped with 0.51 mL cells and controlled by
the VP-viewer program. Calibration of temperature and heat
flow was carried out according to the MicroCal instructions.
The samples of protein concentration 1 mg/mL were extensively
dialyzed (using a SpectraPor membrane with a molecular weight
cutoff of 3.5 kDa) at 4 °C against the buffer and further degassed
prior to the calorimetric experiments. The samples were heated
from 20 to 90 °C at a scan rate of 1 °C/min, and the calorimetric
cells were kept under an excess pressure of 28 psi during the
scan to avoid bubble formation during the experiments. Thermal
9914
J. Phys. Chem. B, Vol. 114, No. 30, 2010
Noronha et al.
Figure 2. (a) Fluorescence decay times of single-exponential decays of native wt-UBQ (squares) and double-exponential decays of unfolded
wt-UBQ (circles and triangles) and their respective (b) pre-exponential coefficients, at pH 1.5, in 12.5 mM sodium acetate. The black circles in (a)
represent the fluorescence decay times of the parent compound N-acetyltyrosinamide (NAYA) in dioxane:water mixtures.27 The pre-exponential
coefficients in (b) are equal to the mole fractions of native xN and unfolded xU protein23 and show a transition range from 38 to 70 °C. For example,
for a T-jump from 55 to 65 °C a variation in the fluorescence signal ∆F ) 66% is calculated (dashed lines; see below LTJ-F experiments).
unfolding of UBQ was carried out in 12.5 mM sodium
acetate-HCl at pH 1.0 ([H+] ) 0.1 M) and in D2O at pD 1.0
([D+] ) 0.1 M). The sample in D2O was initially dialyzed
against 12.5 mM sodium acetate, pH 1.5, lyophilized, and
dissolved in D2O adjusted to pD 1 with DCl. The thermograms
were analyzed using the DSC support software provided by
MicroCal, using as a model a two-state transition with ∆Cp.
Results
Laser and rapid-mixing T-jumps were carried out by monitoring the fluorescence signal of Tyr-59 to probe the folding/
unfolding kinetics of wt-UBQ at pH 1.5 from 5 µs-s. The
variations in the fluorescence signal from kinetic experiments
at different temperatures were cross-checked with fluorescence
data, previously obtained from equilibrium/thermodynamic
studies (Figure 2),23 to ensure that all of the kinetics was
detected.
Rapid-Mixing T-jumps. Unfolding Fu(t) (Ti ) 38 °C) and
folding Ff(t) (Ti ) 70 °C) fluorescence traces in the millisecond
time scale were determined, for final temperature values (Tf)
within the unfolding temperature range, from 45 to 65 °C. For
all final temperatures, single-exponential traces were observed
for both folding and unfolding. Moreover, the observed reciprocal decay times (kobs) had the same value for each pair of heating
and cooling jumps to the same final temperature. This is
illustrated in Figure 3, where unfolding Fu(t) and refolding Ff(t)
traces, respectively, from 38 °C (native state) and 70 °C
(denatured state) to a final temperature of Tf ) 55 °C, are fitted
with single-exponential functions with the same kobs) 16.2 s-1.
Table 1 shows the reciprocal decay times (kobs) and preexponential coefficients Fu(0), Ff(0), and F(∞) ) Fu(∞) ) Ff(∞)
obtained from global fits (see Discussion) of each pair of
unfolding-folding traces to a given Tf.
Laser T-jumps. Laser T-jumps were carried out with the
parent compound, NAYA, prior to experiments with wt-UBQ,
to calibrate the change in fluorescence intensity (F) as a function
of temperature. Figure 4a shows the fluorescence change
resulting from a T-jump from 55 to 65 °C. A sharp (within the
laser pulse) ∆F ) 7% decrease (from 1.0 to 0.93) in the
fluorescence signal (F) was observed, resultant of the decrease
of the fluorescence quantum yield of NAYA with increasing
temperature (parallel to the decrease of the lifetime; see Figure
2a) from 55 to 65 °C. After the initial steep change, the signal
remained constant.
For wt-UBQ, a similar ∆F ) 6% was observed upon a
T-jump from 55 to 65 °C (Figure 4b). However, the F at 65 °C
Figure 3. Global single-exponential fits (red and blue lines) of the
fluorescence changes of wt-UBQ on unfolding (black circles) and
refolding (gray circles) T-jumps, from Ti ) 38 °C to Tf ) 55 °C
and Ti ) 70 °C to Tf ) 55 °C, give an observable rate constant, kobs
) 16.2 s-1 (λexc ) 275 nm; λem > 290 nm). Fu,f(0) are the preexponential coefficients at t ) 0 for the unfolding and refolding
traces; fN and fU are the fluorescence signal of native and unfolded
protein, respectively.
(no T-jump) is much less than that observed after the T-jump,
which indicates that practically all the unfolding kinetics of wtUBQ is on a time scale greater than 1.8 ms. Similar results
were obtained upon a T-jump from 50 to 60 °C (data not shown).
Discussion
Kinetic Analysis. The foregoing data shows that the thermal
unfolding/folding kinetics of wt-UBQ at pH 1.5 is adequately
described with the two-state model represented in scheme 1
below, where ku and kf are the rate constants of unfolding and
folding and N and U the native and unfolded states, respectively.
ku
N {\} U
scheme 1
kf
The time evolution of the native N(t) and unfolded U(t)
conformations obeys eqs 1 and 2, which express the well-known
result that the decay functions, N(t) and U(t), are sums of a
Thermal Folding/Unfolding Kinetics of wt-UBQ
J. Phys. Chem. B, Vol. 114, No. 30, 2010 9915
TABLE 1: Rate Constant (kobs) λ2), Fluorescence Intensity
Signals (in V) at t ) 0 of the Unfolding Fu(0) and Folding
Ff(0) Fluorescence Traces, and Fluorescence Intensity Signal
(in V) at t ) ∞, F(∞)
T (°C)
kobs (s-1)
Fu(0)
Ff(0)
F(∞)
45
47
49
51
53
55
57
58
59
62
63
65
11.0
11.3
11.9
12.1
12.7
16.2
19.2
27.7
31.3
52.6
69.1
110.2
1.02
0.99
0.94
0.91
0.95
0.74
0.72
0.57
0.64
0.54
0.70
0.35
0.61
0.52
0.49
0.36
0.34
0.31
0.23
0.25
0.20
0.19
0.14
0.15
0.99
0.93
0.86
0.76
0.65
0.52
0.41
0.36
0.32
0.21
0.19
0.14
constant term with an exponential term with rate constant kobs
)λ2 ) ku + kf (see Supporting Information for details).
[]
][ ]
[
kf ku
1
N
(t) ) (1/λ2)
-λ2t
k
-k
U
u
u e
[]
[
][ ]
kf -kf
1
N
(t) ) (1/λ2)
ku kf e-λ2t
U
(for unfolding)
(1)
(for folding)
(2)
The determination of ku and kf is usually carried out by
coupling the value of kobs ) ku + kf, obtained from either a
folding or unfolding trace, to that of the equilibrium constant
Ku ) ku/kf, determined from DSC or any other suitable method.23
However, the availability of both folding and unfolding traces,
from MTJ-F experiments, provides an alternative, more robust
methodology to extract the values of ku and kf from the kinetic
data alone, using the information contained the pre-exponential
coefficients (the values of N(0), U(0), and N(∞) ) U(∞)).
In order to profit from this information, the experimental
signal (UV absorption, fluorescence, etc.) has to be converted
into concentrations (or mole fractions) of the native and unfolded
protein. The experimental signal, in our case the fluorescence
signal F(t), is the summed fluorescence signals of N and U,
which are in turn proportional to their respective concentrations,
N(t) and U(t),23 (eq 3, where the coefficients fN and fU are the
fluorescence quantum yields of N and U, respectively, apart
from a common instrumental constant).
F(t) ) fNN(t) + fUU(t)
(3)
Ff(t) ) [fNkf + fUku + (fUkf - fNkf) exp(-λ2t)]/(ku + kf)
(5)
Therefore, the unfolding Fu(t) and folding Ff(t) traces contain
all the information required for the evaluation of ku and kf,
without using the equilibrium constant obtained from independent experiments, i.e, two equations (eqs 6 and 7) for the two
unknowns (kf and ku), with all data, fN, fU, Fu,f(∞), and kobs, taken
from the decays (see Figure 3).
kf + ku ) kobs
(6)
fNkf + fUku ) Fu,f(∞)/kobs
(7)
The global analysis of each pair of folding and unfolding
traces, subjected to the foregoing constrains (eqs 6 and 7),
besides being more robust, provides the possibility of comparing
the equilibrium constant derived from kinetic data Kukin to that
independently obtained from equilibrium measurements KuTRFS,
and thus verifying the internal consistency of all (equilibrium
and kinetic) data. An additional and useful advantage of this
comparison is the possibility of checking the presence/absence
of fast decay components (on the submillisecond time scale)
from the disagreement/agreement of equilibrium and kinetic
data.
Table 2 shows the values of kf and ku derived from the global
fit of eqs 6 and 7 to each pair of unfolding Fu(t) and folding
Ff(t) experimental traces (fits in Figure 3) and compares the
values of the equilibrium constant calculated from the kinetic
data (Kukin ) ku/kf) to those obtained from equilibrium studies
(KuTRFS ) xU/xN) using time-resolved fluorescence spectroscopy
(Figure 2b).23 The values of Kukin and KuTRFS are in reasonable
agreement within experimental error.
Temperature Dependence of the Rate Constants. Arrhenius
plots of kf and ku (Figure 5) show that ku is strongly activated
(Eau) 49 ( 1 kcal mol-1, A0u ) 8.3 ( 7 × 1033 s-1), whereas
kf is slightly negatively activated (Eaf ) -6 ( 1 kcal mol-1,
A0f ) 4.3 ( 2 × 10-4 s-1).
The non-Arrhenius behavior of kf (negative Eaf) has been
previously reported for several other proteins.9,12,13,28-32 Because
the negative Eaf could result from a positive ∆Hf# coupled to a
sufficiently negative activation heat capacity of folding (∆CPf#),
as reported for CI2 (∆CPf# ) -0.31 kcal mol-1 K-1),28 we have
analyzedthetemperaturedependenceofkf withtheGibbs-Helmholtz
equation (eq 8), by globally fitting both folding and unfolding
rate constants with Eyring’s equation, eq 9, subject to the
constrain ∆CPu# - ∆CPf# ) ∆CPTRFS ) 0.89 kcal mol-1 K-1 from
equilibrium (TRFS) experiments.23
(
#
#
#
#
∆Gu/f
) ∆Hu/f
(Tm) + ∆CPu/f
(T - Tm) - T ∆Su/f
(Tm) +
( ))
#
ln
∆CPu/f
Combination of eqs 1-3 leads to eqs 4 and 5 (see also
Supporting Information), which show that the values of the
unfolding Fu(t) and folding Ff(t) traces at t ) 0 are equal to fN
and fU, respectively, and the traces take a common value of
(fNkf + fUku)/kobs at t ) ∞ (see Figure 3).
Fu(t) ) [fNkf + fUku + (fNku - fUku) exp(-λ2t)]/(ku + kf)
(4)
ku/f )
(
#
∆Gu/f
k BT
〈κ〉 exp h
RT
)
T
Tm
(8)
(9)
Acceptable fits (Figure 6a) could be obtained only with
negative ∆Hf#(Tm) values. Positive ∆Hf# values always induced
large errors in the temperature range below the Tm.
9916
J. Phys. Chem. B, Vol. 114, No. 30, 2010
Noronha et al.
Figure 4. (a) Normalized fluorescence signal (F) of NAYA at 65 °C (gray) and the laser T-jump from Ti ) 55 °C to Tf ) 65 °C (black); (b)
fluorescence signal of wt-UBQ, pH 1.5, at 65 °C (gray), and the laser T-jump from Ti ) 55 °C to Tf ) 65 °C (black) shows a negligible decrease
in the fluorescence signal up to 1.8 ms.
TABLE 2: Rate Constants of Unfolding (ku) and Folding
(kf) Calculated from Global Analysis of Folding and
Unfolding Traces, and Equilibrium Constants Calculated
from Kinetic (Kukin ) ku/kf) and from Equilibrium TRFS
(KuTRFS) Data
T (°C)
ku (s-1)
kf (s-1)
Kkin
u
KTRFS
u
45
47
49
51
53
55
57
58
59
62
63
65
1.1
1.4
2.5
3.7
5.1
8.2
12.3
20.3
24.0
46.5
63.3
105
9.9
9.9
9.5
8.6
7.6
8.0
6.9
7.4
7.2
6.1
5.8
5.6
0.11
0.14
0.26
0.43
0.67
1.03
1.78
2.74
3.33
7.62
10.9
18.8
0.09
0.14
0.24
0.39
0.66
1.06
1.90
2.45
3.28
7.67
10.1
17.9
As expected, inclusion of solvent friction using Kramer’s
equation (eq 10)13,33 provides an even more negative value for
∆Hf# (Table 3).
( ) (
ku/f ) ν
ηTm
η
#
∆Gu/f
exp RT
)
(10)
Interestingly, our data for wt-UBQ folding kinetics is very
close to that obtained by Torrent et al.13 for RNase A using
MTJ-F (Table 3, third row).
A negative ∆Hf#(Tm) value means that the enthalpy decrease
in the transition from the unfolded to the transition state
(resulting from the intraprotein hydrophobic forces) is larger
Figure 5. Arrhenius plots of rate constant of folding kf (black) and
unfolding ku (gray) of wt-UBQ at pH 1.5 using the fluorescence of
Tyr-59 showing that ku is strongly activated whereas kf is slightly
negatively activated.
than the increase in enthalpy that is required for reaching the
transition state through rotations of the protein-backbone bonds
in the presence of solvent friction. This situation has been
predicted from theory.34-38 In this case, the folding free energy
barrier ∆Gf#(Tm), if existent, is exclusively entropic.
The value of ∆Gf#(Tm), obtained from the data in the second
row of Table 3 and eq 10, is 7.6 kcal/mol, ca. 1 order of
magnitude larger than RTm. Thus, within the limitations arising
from the application of eq 10, there is a sufficiently high free
energy barrier separating the native and unfolded states so that
transition-state theory can be applied.
Summarizing, the foregoing results show that thermal unfolding of wt-UBQ at pH 1.5 probed by Tyr-59, from both
equilibrium (TRFS and DSC) and kinetic experiments, follows
a two-state model, occurring in the millisecond time scale, with
no kinetics observed in the µs-2 ms time range.
Comparison with Literature Data. The kinetics of thermal
unfolding/folding of wt-UBQ has been previously studied
in D2O at pD 1.0, using LTJ with infrared (IR) or differential
vibrational echo spectroscopy (DVE) detection to probe the
temperature-induced changes in the amide I vibrational mode
of β-strands I-V.11,12,21 Comparison of the data in ref 12with
ours shows qualitative agreement on the millisecond time
scale; namely, “single-exponential kinetics” was observed,
indicating the absence of intermediate states. However, there
are a number of discrepancies, namely, (1) observation of
kinetics on the microsecond time scale vs our observation
of its absence, and (2) different values of kf and ku leading
to (3) different Tm values (64 °C, in D2O at pD 112,21 vs 54.6
°C in H2O, at pH 1.523).
Because the discrepancies in the Tm and rate constants could
arise from the different experimental conditions of the two
studies, we have carried out rapid-mixing T-jumps at pH 1 ([H+]
) 0.1 M) and at pD 1 ([D+] ) 0.1 M in D2O). The folding and
unfolding decays were qualitatively similar to those obtained
in H2O at pH 1.5; namely, they could be globally fitted with
single-exponential functions, and there was no indication of
kinetics in the microsecond time range.39 The resulting values
of kf and ku are plotted in Figure 7, together with those obtained
in ref 12. Our values measured in H2O at pH 1.5 are also shown
for comparison.
Decreasing the pH from 1.5 to 1 stabilizes wt-UBQ by
increasing only kf (Figure 7a, red squares) with no change in ku
(Figure 7b, red squares). The consequent stabilization (ca. 3 °C
in the Tm, from 57.2 °C at pH 1.523 to 60.9 °C at pH 1.0) is in
apparent contrast with the reported destabilization of wt-UBQ
with decreasing pH.40 The larger kf (and unchanged ku) likely
results from the screening of the intraprotein electrostatic
repulsion (positive charges at pH 1) at higher [Cl-] (ionic
Thermal Folding/Unfolding Kinetics of wt-UBQ
J. Phys. Chem. B, Vol. 114, No. 30, 2010 9917
Figure 6. Fitting of kf values calculated using Erying transtion-state theory (open circles) to the experimental (a) kf and (b) ku (black squares) of
UBQ at pH 1.5, imposing ∆CPu# - ∆CPf# ) ∆CPTRFS ) 890.0 cal mol-1 K-1 (obtained from TRFS) and at Tm ) 54.6 °C. The values of ∆S# are
affected by the pre-exponential factor of Eyring’s equation (kBT/h ) 6.8 × 1012 s-1) and should be regarded just as fitting parameters.
TABLE 3: Values of the Activation Parameters Obtained from Fitting Eqs 9-10 to kf and ku for Wild-Type Ubiquitin
# a
(wt-UBQ) at pH 1.5, Using Eyring’s Transition-State Theory and Kramers Model Corrected for ∆Cpu/f
wt-UBQ (Eyring)
wt-UBQ (Kramers)
RNase A (Kramers)
∆Hf#(Tm),
kcal mol-1
∆Sf#(Tm)b,
kcal mol-1 K-1
∆Cpf#, kcal mol-1
K-1
∆H#u(Tm),
kcal mol-1
∆S#u(Tm)b,
kcal mol-1 K-1
∆Cpu#,
kcal mol-1 K-1
-5.6
-10.8
-10.5
-0.072
-0.056
-0.064
-0.113
-0.093
49.6
44.9
105.2
0.097
0.114
0.284
0.757
0.797
a
The third row shows data obtained by Torrent et al.13for wild-type ribonuclease A (RNase A) at pH 5.0 for comparison of the folding
parameters. b The values of ∆S# are affected by the pre-exponential factors of either eq 9 or 10.
Figure 7. Comparison of the rate constants of (a) folding and (b) unfolding obtained by probing the 310-helix (black; pH 1.5), (red; pH 1), (blue;
pD 1), and the β-strands III-V (gray, pD 1; Chung and Tokmakoff) of wt-UBQ.
strength) in the denatured state. Actually, it has been reported
that addition of 0.5 M NaCl to wt-UBQ at pH 2 increases the
Tm by 22.5 °C.41
Changing from H2O at 0.1 M HCl to D2O at 0.1 M DCl
induces an additional increase in kf (Figure 7b, blue squares)
and a decrease in ku (Figure 7a, blue squares), consistent with
a further increase in the Tm (Tm ) 62.7 °C from DSC). In terms
of activation energies, it is interesting to note that kf becomes
even more negatively activated (Eaf ) -15 ( 1 kcal mol-1)
whereas ku has a similar activation energy as in H2O (Eau) 50
( 2 kcal mol-1, Table 4).
Comparison of our data with that presented by Chung and
Tokmakoff in the same solvent conditions (Table 4, third
and fourth row, respectively) shows that the differences
persist: Our values for kf are smaller (Figure 7a), although
the discrepancies result from very small differences in the
values of Ea,f and Af (Table 4, 3rd and fourth row), and both
Eu,f and Au are substantially different, although the ku values
themselves are not much different (Figure 7b). Moreover,
no indication of kinetics on the microsecond time scale was
found.
Probe-Dependent Kinetics. There are three possible explanations for the observed differences. The first is that the
differences result from accumulated intrinsic errors of the three
experimental techniques used to derive the data (DSC, MTJ-F,
LTJ-F, and LTJ-DVE). However, this could account for
differences in rate constants and activation energies, but not
for the presence vs absence of kinetics on the microsecond time
scale.
The second is that the 310-helix and β-strands III-V simply
unfold differently from each other. As Tyr-59 is located on the
310-helix of the C-terminal of wt-UBQ, while data from Chung
and Tokmakoff probe β-strands III-V, the foregoing differences
in ku could suggest that unfolding of the β-strands of wt-UBQ
at pD 1 begins at a lower temperature and ends at a higher
temperature than the 310-helix unfolding; i.e., at low temperatures
9918
J. Phys. Chem. B, Vol. 114, No. 30, 2010
Noronha et al.
TABLE 4: Values of Activation Energies and Pre-exponential Coefficients of Unfolding (Ea,u, Au), and Folding (Ea,f, Af) of
wt-UBQ, from Arrhenius Plots of kf and ku Measured under Different Experimental Conditions and Different Probes
experiment in
H2O,
H2O,
D2O,
D2O,
a
pH
pH
pD
pD
1.5
1.0
1.0
1.0a
probed by
310-helix
310-helix
310-helix
β-strands
Ea,u, kcal mol-1
49
48
50
21
Ea,f, kcal mol-1
-6
-9
-14.9
-14.7
Au, s-1
8 × 10
4 × 1030
5 × 1032
1.2 × 1015
33
Af, s-1
Tm, °C
-4
4.3 × 10
1.0 × 10-5
5.9 × 10-9
5.8 × 10-9
57.2b
60.9b
62.7b
64.0
Values calculated with data from ref 12. b From DSC measurements.
the 310-helix region would be more stable than the β-strands,
whereas at higher temperatures the inverse would be true. The
310-helix and the R-helix of wt-UBQ have been reported as
recognition sites through noncovalent protein-protein interactions,42 and their higher stability at temperatures below the Tm
may have functional importance. Although interesting, this
possibility is odd because the 310-helix is located between
β-strands IV and V (see Figure 1) that do unfold.
The third, more plausible, explanation for the difference in
rate constants on the millisecond time scale and the observation
of kinetics by LTJ-DVE versus the absence of kinetics by MTJ-F
on the microsecond time scale results from the fact that the
two kinetic techniques probe different phenomena at the
molecular level. The MTJ-F probes the exposure of tyrosine to
water, while the LTJ-DVE probes the breaking of interstrand
hydrogen bonds. It is, therefore, intuitive that for unfolding,
the incipient hydrogen-bond breaking should be immediately
detected by LTJ-DVE, while observation of a fluorescence signal
change (by MTJ-F) requires further displacements and backbone
rotations leading to water exposure of Tyr-59. Consequently,
MTJ-F monitors the unfolding of the thermally excited protein
alone, while LTJ-DVE also monitors the preparation and thermal
re-equilibration of the thermally excited protein. This explains
the observation of microsecond kinetics by LTJ-DVE and not
by MTJ-F.
Furthermore, the resulting greater complexity of the LTJDVE signal and the low upper limit of its temporal range make
an accurate characterization of the longest decay time difficult,
due to mixing with the shorter decay times (or stretched
exponential). This difficulty, possibly leading to larger kobs
values, coupled to the preliminary nature of our data in D2O
may explain the differences observed in the rate constants. A
global analysis of the MTJ-F, LTJ-F, and LTJ-DVE experimental data would certainly provide a more accurate basis for
modeling the folding/unfolding pathway of wt-UBQ.
Conclusions
The thermal unfolding of wt-UBQ at pH 1.5, followed by
the fluorescence of its single tyrosine residue Tyr-59, from both
equilibrium and kinetic studies, shows that wt-UBQ behaves
as a two-state folder on the millisecond time scale. No kinetics
was observed in the microsecond time region, and no evidence
for intermediate states was found.
Arrhenius plots for kf and ku at pH 1.5 show that kf is slightly
negatively activated (Eaf ) -6 ( 1 kcal mol-1), whereas ku is
highly activated, Eau ) 49 ( 1 kcal mol-1). Analysis of the
temperature dependence of kf with the Gibbs-Helmholtz
equation results in negative values for both ∆H#f and∆Cpf#. Thus,
the negative value of ∆Cpf# alone cannot explain the observation
of a decreasing kf in the entire transition temperature range (from
38 to 70 °C).
Comparison of kf and ku values at two different pH values
shows an increase only in kf at pH 1 as compared to pH 1.5
(due to Cl--induced acceleration of folding), whereas compari-
son of kf and ku in H2O and D2O shows that D2O has a stabilizing
effect brought about by both an increase in kf and a decrease in
k u.
Comparison of data from LTJ-DVE (ref 12)with our results
from MTJ-F in the same solvent conditions, at pD 1 in D2O,
revealed differences in the nature of the kinetic traces and in
the kf and ku values. We propose that the differences result from
the fact that MTJ-F monitors the actual unfolding (backbone
exposure to water) of the thermally excited protein alone, while
LTJ-DVE also monitors the preliminary events (hydrogen-bond
breaking) and thermal re-equilibration of the thermally excited
protein.
Acknowledgment. The work was supported by the Fundaçãopara a Ciência e a Tecnologia (FCT), Portugal, Projects
POCI/QUI/56585/04 and POCI/BIA-PRO/57263/04, and by the
European Commission, 5th Framework Programme contract
QLK3-CT-2000-00640. M.N. and T.F. acknowledge the FCT
for postdoc grants SRFH/BPD/27128/2006 and SRFH/BPD/
44428/2008.
Supporting Information Available: This information is
available free of charge via the Internet at http://pubs.acs.org.
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