Figure 1. The protein folding funnel for a fast-folding helical
protein. Copyright Elsevier Cell Press 1996.
While the difference in the Gibbs free energy, ΔG, between the initial
and the final thermodynamic equilibrium states of the
unfolding/refolding mechanisms has been investigated from both a
theoretical and experimental points of view,5 the
study of intermediate nonequilibrium states is also important as these
states may influence the folding process during biosynthesis and may
help understand misfolding, which is the cause of some
diseases.6 Thermodynamically, the folding process
might occur at constant temperature (the physiological environment is
assumed to be a thermal bath) but stochastic nonequilibrium intermediate
states may present thermal fluctuations that may be related to energy
(heat) exchange between the protein and the physiological
environment.7 Therefore, the measurement of those
temperature fluctuations/heat exchange is important to better understand
the thermodynamics of those intermediate nonequilibrium states and its
influence during the (mis)folding process. Besides the folding process,
real time temperature measurements would also be appealing for the study
of fluctuations due to thermal noise in proteins that work as motors
(rotary and linear engines) and shuttles.8 These
proteins generally use chemical reactions such as ATP hydrolysis as a
fuel to generate mechanical work. Taking into account the first law of
thermodynamics, part of the energy received by the protein is converted
into useful work and the remaining energy is lost as heat. When proteins
help transporting ions/molecules through cell membrane, for example, the
work is considered thermodynamically favorable when the transport is
performed from a region of high ion/molecule concentration to a region
of low ion/molecule concentration. In this case, ΔG is associated to the
chemical potential difference and the reversible work needed to perform
the transport of ions/molecules across the membrane.9As a transport phenomenon, it may also be viewed as a nonequilibrium
thermodynamical process in which heat conduction may lead to gradients
of temperature and entropy production with time. Thus, the direct
measurement of those temperature gradients may be relevant information
to assess and perhaps optimize the efficiency of the energy cycle of the
molecular motor.
Protein dynamics: theoretical and experimental analyses
Protein dynamics has been investigated by a number of theoretical
methodologies and experimental techniques. From a theoretical point of
view, computational and modeling have been using to analyze energy
change in protein folding using statistical mechanics and nonequilibrium
thermodynamics.10,11 From the experimental point of
view, X-ray crystallography, optical spectroscopy, and nuclear magnetic
resonance have been commonly used.12 All these
experimental techniques are employed in ensembles, i.e., the information
retrieved is based on the collective behavior (average) of the objects
of study, and therefore individual behavior associated to stochastic
nonequilibrium fluctuations are not detectable. Single molecule
experiments have been focused on fluorescence correlation spectroscopy
to study protein folding as a diffusive process on a free-energy
surface13 and optical tweezer/atomic force microscopy
to establish mechanical forces to stretch (unfold) the macromolecule and
observe the dynamics of the refolding process.14Concerning proteins working as motors and shuttles, experimental
techniques such as magnetic tweezers and electrorotation have been used
to estimate the thermodynamic efficiency under some constraint
conditions such as quasi static limit.15 All the
single molecule experiments mentioned here were based on an external
stimulus to induce some mechanical motion (folding, rotation) in order
to study the dynamics of the system. However, biological systems under
external stimulation and without external stimulation may undergo
different metabolisms. Nothing has been tried concerning real time
temperature measurements on individual proteins spontaneously
folding/performing a mechanical work. As mentioned earlier in the text,
valuable information about misfolding during biosynthesis and poor
engine efficiency, both possibly associated to stochastic
thermodynamical nonequilibrium states, may be hypothetically retrieved
by measurements of thermal fluctuations and, consequently, estimates of
energy related thermodynamic variables. Actually, the study of
temperature fluctuations in nonequilibrium thermodynamical phenomena can
be more widespread, not restricted only to proteins but it can be
extended to other types of biomacromolecules, such as for example DNA.
Thus, the potential for using nanothermometers for probing
nonequilibrium phenomena in biochemical reactions involving
macromolecules is substantial.
Luminescence thermometry in a single protein: the experimental
limitations and challenges
Optical temperature sensing using a single luminescent object linked to
a biomacromolecule presents many challenges. For accurate real time
temperature monitoring of biomacromolecules, the luminescence
nanothermometer has to be in contact with the biomacromolecule while the
temperature is estimated through some remote detection system that
records some change of the luminescence profile with the temperature on
the object of study. Imaging and sensing of biological ensemble systems
have been accomplished using different classes of luminophores, such as
semiconductor quantum dots, lanthanide doped nanocrystals, and
fluorescent proteins.16 Inorganic compounds have
better photostability, i.e., they do not undergo photodegradation, a
characteristic of organic compounds, and therefore they are more
convenient for single molecule studies. Due to its lower toxicity level,
lanthanide doped nanocrystals are generally more appropriate than
semiconductor quantum dots, which contains elements such as Cd, Se and
S. However, due to its larger size and constitution, inorganic
nanoparticles-biomacromolecules conjugates are much more complicated to
fabricate than fluorescent proteins-biomacromolecules
conjugates.16 Thermometry using a single fluorescent
protein is complicated by the fact that besides photodegradation due to
long exposure to light excitation, organic molecules also show blinking,
a characteristic of electronic population of dark states during
relaxation. Lanthanide doped nanoparticles, on the other hand, do not
present blinking because a single nanoparticle has many luminophores,
the lanthanide ions. Recent imaging and spectral analysis of the
luminescence profile of individual lanthanide doped nanoparticles are
available in literature.17,18 Figure 2 shows the
luminescence spectral profile of a NaYF4 nanoparticle
doped lanthanides (Yb3+ and Er3+).
The signal was recorded with a scanning confocal microscopy
set-up.17 Observe that the relative intensity of the
two emission bands at 520-535 nm and 540-560 nm, which are radiative
relaxations from thermally coupled electronic states4S3/2 and2H11/2 of Er3+,
changes with the temperature in a way that optical temperature sensing
is feasible.