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monophasic unfolding curve signifying a simple two-state unfold-
ing mechanism. Clearly, use of IL as solvent affords the protein
with considerable thermodynamic stability; the onset of thermal
unfolding shifts upward to over 100 °C in water-containing IL
compared with ca. 40 °C in water. The first-derivative results also
make clear that the unfolding transition is broadened within the IL
possibly indicating a lower cooperativity for the transition relative
to that in bulk water. Noting that the equilibrium constant for
Notes and references
‡
IL was equilibrated overnight with aqueous monellin via gentle reciprocal
shaking to result in a protein-containing IL with a water content of 2.0 vol%.
Both aqueous and IL samples were centrifuged at 10 000g for 10 min prior
to experimentation.
§ The fraction of native monellin remaining at each temperature, f (T), was
determined from the expression f (T) = [F(T) 2 F ]/[F 2 F ] where F(T)
is the temperature-dependent fluorescence intensity integrated over a 4 nm
N
N
U
N
U
unfolding K(F?U) is equal to (1 2 f
N
)/f
N
, the relative free energy
slice centered about the emission maximum for the folded state, and F
N
and
F
U
are the fluorescence intensities for the folded and unfolded conforma-
stabilization as a function of temperature, DDG(T)(F?U), for
2
1
tions, respectively. Fluorescence intensities were background subtracted
and corrected for solvent thermal expansion. Temperature was controlled
using a home-built thermal stage.8
monellin in IL can be estimated: DDG(T)(F?U)/J mol ≈ 115T +
540 for 30 5 T/°C 5 80. Results from a van’t Hoff analysis reveal
that this stabilization is entropically driven. That is, the entropies of
1
¶
Emission maxima were estimated from global minima of second-
2
1
21
unfolding, DS°, are 250 and 136 J K mol for monellin in water
and IL, respectively, consistent with more rigid solvation within an
IL.
derivative spectra calculated from background subtracted experimental
emission spectra or by fitting a central 50 nm window to a Weibull
distribution; in either case, the uncertainty in estimation was 50.4 nm.
The spectral changes associated with unfolding also provide key
information about the milieu surrounding W3. Upon unfolding, the
emission contour in water is red shifted and approaches that of the
1
Several monographs and edited volumes dedicated to ionic liquids have
appeared recently: Ionic Liquids as Green Solvents: Progress and
Prospects, ed. R. D. Rogers and K. R. Seddon, ACS Symp. Ser. 856,
American Chemical Society, Washington, DC, 2003; Ionic Liquids in
Synthesis, ed. P. Wasserscheid and T. Welton, Wiley-VCH, Weinheim,
‘
naked’ Trp analog N-acetyl- -tryptophanamide (NATA) in bulk
L
water (Fig. 3). The indication here is as expected, i.e., W3 becomes
more accessible to solvent as a result of unfolding. The highly blue-
shifted emission observed for monellin in [C mpy][Tf N], how-
4 2
2
003.
ever, was completely unexpected and suggests that the state of the
protein is significantly altered within the IL. While exceptionally
short wavelength maxima are not unheard of (the known emission
maxima for tryptophan in proteins span the range from 308 to 350
nm), they are relatively uncommon and generally implicate a
complete shielding or isolation of the Trp by neighboring aromatic
residues. As a benchmark, over the entire temperature range the
emission maximum for NATA in IL lies within a narrow 336–339
nm window, irrespective of the state of hydration. This suggests a
minimal exposure of W3 to the surrounding IL solvent as well as a
tightening in protein structure which likely manifests in the higher
thermal stability. Further increases in temperature result in
additional blue shifting, possibly due to a progressive dissociation
and stripping of biological water from the local protein surface.
In summary, by monitoring the intrinsic emission from a single
Trp protein, we have been able to follow its unfolding behavior
within an IL. We believe that this is the first detailed report of
protein spectroscopy of any kind within an IL. While the improved
thermostability conferred upon proteins likely results from altera-
tion in the protein hydration level and structural compaction, the
underlying reasons are still largely speculative and may include
additional factors such as free volume contributions, ionic inter-
actions (salt bridges) and confinement effects. Nevertheless, this
remarkable stabilization against thermal inactivation suggests a
general and notable alternative to engineered or isolated thermo-
philes in high-temperature biocatalytic and biosensory applica-
tions.
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Fig. 3 Temperature-dependent emission maxima for monellin in water (-)
and in [C mpy][Tf N] equilibrated with 2.0% (v/v) water (8).¶
4 2
C h e m . C o m m u n . , 2 0 0 4 , 9 4 0 – 9 4 1
941