# Molecular Processes in Biological Thermosensation

Authors: I. Digel; P. Kayser; G. M. Artmann
Journal: Journal of Biophysics
 (2008)

---

## Abstract

Since thermal gradients are almost everywhere, thermosensation could represent one of the oldest sensory transduction processes that evolved in organisms. There are many examples of temperature changes affecting the physiology of living cells. Almost all classes of biological macromolecules in a cell (nucleic acids, lipids, proteins) can present a target of the temperature-related stimuli. This review discusses some features of different classes of temperature-sensing molecules as well as molecular and biological processes that involve thermosensation. Biochemical, structural, and thermodynamic approaches are applied in the paper to organize the existing knowledge on molecular mechanisms of thermosensation. Special attention is paid to the fact that thermosensitive function cannot be assigned to any particular functional group or spatial structure but is rather of universal nature. For instance, the complex of thermodynamic, structural, and functional features of hemoglobin family proteins suggests their possible accessory role as “molecular thermometers”.


---

## Body

## 1. Introduction

Temperature changes are one of the main stresses experienced by organisms from bacteria to plants and animals and therefore temperature is one of the environmental cues under constant vigilance in living cells. Several problems arise from exposing a cell to a sudden change in temperature [1]: firstly, membrane fluidity changes, that affect many membrane-associated vital
functions. Secondly, nucleic acid topology will be affected causing shifts in
processes such as transcription and translation. Finally, the protein function
is affected both from structural and catalytic points.Hence, living cells need devices for sensing
environmental temperature changes in order to adapt their biochemical processes
accordingly. A successful adaptive response
to temperature changes cannot be performed by corresponding changes in the rate
and equilibrium of enzymatic reactions only. Such a mechanism of adaptive reaction
is too unspecific and uncontrollable. To cope with temperature variation, living
organisms need sensing temperature alterations and translating this sensory
event into a pragmatic gene response.While such regulatory cascades may ultimately be
complicated, it appears that they contain primary sensor machinery at the top
of the cascade. The functional core of such machinery is usually that of a
temperature-induced conformational or physicochemical change in the central constituents
of the cell. Hence, a specific sensory transduction mechanism is needed,
including, as a key element, a molecular sensor, transforming physical
parameter (temperature) into a biologically significant signal (change in
membrane permeability, specific inhibition/stimulation of gene expression,
etc.). In a sense,
a living organism can use structural alterations in its biomolecules as the
primary thermometers or thermostats. Thus, sensory
transduction is a complex biological process aimed at integrating and decoding
physical and chemical stimuli performed by primary sensory molecular devices.
Furthermore, sensory perception of potentially harmful stimuli functions as a
warning mechanism to avert potential tissue/organ damage.Among temperature-controlled processes in living
organisms, most well-known are the expression of heat-shock and cold-shock
genes [2]. Relocation of a culture of Escherichia coli adapted
to an optimal growth to a sudden temperature increase, or decrease, by some 10–15°C results in
adaptive shock responses. Such responses involve a remodeling of bacterial gene
expression, aimed at adjusting bacterial cell physiology to the new
environmental demands [3, 4]. The response of prokaryotic and eukaryotic systems
to heat-shock stress has been investigated widely in a large number of
organisms and model cell systems. Notably, all organisms from prokaryotes to
plants and higher eukaryotes respond to cold and heat shock in a comparatively
similar manner. The general response of cells to temperature stress (cold or
heat) is the elite and rapid overexpression of small groups of proteins, the
so-called CSPs (cold-shock proteins) or HSPs (heat shock proteins),
respectively, but the initial launching mechanism is different in both cases.In bacteria, the heat response generally invokes some
20 heat-shock proteins, whose functions are primarily to help deal with, and
alleviate, the cellular stress imposed by heat [5]. Many of these proteins participate in reconstituting
and stabilizing protein structures and in removing misfolded ones. The
expression of this special chaperone system, which includes the proteins DnaK, DnaJ, and GrpE is
activated by the presence of misfolded, temperature-denatured proteins. Thus,
one could implicate the binding of partially unfolded proteins by chaperones as
the thermosensoric event regulating expression of heat-shock proteins, where
the primary sensory element is constituted by some easily denaturing proteins.
This, in turn, demonstrates that even bacteria can practically utilize destructive
changes in protein conformation as a means for temperature sensing.In case of cold shock, the primary sensing
event is more obscure. Various reports have now shown
that when in vitro cultivation temperature is lowered, the rigidity of
the cell membrane is increased which results in compromised membrane-associated
cellular functions. Furthermore, cold stress dramatically hinders
membrane-bound enzymes, slows down diffusion rates, and induces cluster
formation of integral membranous proteins [6].In mammalian cells the five known mechanisms by which
cold-shock-induced changes occur in gene expression are: (i) a general
reduction in transcription and translation, (ii) inhibition of RNA degradation,
(iii) increased transcription of specific target genes via elements in the
promoter region of such genes, (iv) alternative pre-mRNA splicing, and (v) via
the presence of cold-shock specific IRESs (internal ribosome entry segments) in
mRNAs that result in the preferential and enhanced translation of such mRNAs
upon cold shock [7].It has been pointed out that cold stress exposes cells
to two major stresses: those relating to changes in temperature and those
related to changes in dissolved oxygen concentration at decreased temperature,
and it is therefore necessary to consider potential responses to each, either
independently or as part of a coordinated response. Separating the relative
effects of temperature and oxygen as a result of decreased temperature is
difficult and has not been extensively addressed to date. Both changes in
dissolved oxygen and temperature reduction result in similar changes in
cultured mammalian cells [7].The shock response systems discussed above belong to
ultimate mechanisms aimed to survival under extreme temperature conditions. However,
the ability to express certain factors can be affected by reasonably small
temperature changes. Less drastic changes in temperature may not induce shock
responses, but can be sufficient to modulate the expression of virulence genes,
for example inShigellae [8] and Yersiniae [9]. While one might be surprised that organisms built on
such minimalist approaches as bacteria respond to temperature changes, the
consequence of these observations is that even bacteria actually sense
temperature shifts in order to control gene expression accordingly. Investigators
have now been studying the moderate temperature sensation in a variety of
organisms for at least several decades or more. Recently, a number of reports
have shown that exposing yeast or mammalian cells to sub-physiological
temperatures (<30°C or <37°C, resp.) invokes
a coordinated cellular response involving modulation of transcription,
translation, metabolism, the cell cycle and the cell cytoskeleton [7, 10–13]. Nevertheless very little is known about the
molecular mechanisms that govern initial response on small thermal stimuli,
particularly the primary sensory transduction mechanisms.Below, we have tried to uncover some aspects of the
molecular basis of temperature sensing by biological molecular thermometers, to
summarize some known aspects of primary components of temperature signal transduction
and to show possible thermosensitive role of even “common” molecules such as
hemoglobin.


## 2. Temperature-Sensing Biomolecules

In
addition to specificity and sensitivity, the pragmatic thermoresponse should be
one that is reversible and controlled. Such
complexity of thermosensing and thermoregulation may reflect the demands to
handle and fine-tune responses to an important environmental factor in a
dynamic fashion. However, ultimately, it seems that basic and uncomplicated
biochemical processes are used as primary sensors and, for that purpose changes
in the nucleic acid, protein or membrane physicochemical state appear highly
suitable. Bellow we make a short overview of temperature-sensing properties of
most important groups of biological macromolecules.


### 2.1. Membrane Lipids

While the information available is somewhat
scant, the picture emerging shows that cells can use signals generated through
changes in nucleic acid or protein conformation, or changes in membrane lipid
behavior, as sensory devices. The physical state of
membranes does change in response to temperature shifts in phase-transition
manner [14], but the temperature-induced changes in real
biological membranes are not sharp because many kinds of fatty acids present,
having different characteristic temperature points of phase transition. Thus,
it would not be surprising if cells (even those of bacteria) could utilize, changes
in membrane fluidity as a thermometer device, assisted by protein helpers,
playing a role of switchers, “sharpening” the temperature response. Microorganisms
counteract the propensity for membranes to rigidify at lower temperature by
adapting to the conditions in order to maintain a more-or-less constant degree
of membrane fluidity (homeoviscous adaptation). The cyanobacterium Synecocystis responds to decreased temperature by increasing the cisunsaturation
of membrane-lipid fatty acids through expressing acyl-lipid desaturases [15–17]. Lipid
unsaturation would then restore membrane fluidity at the lower temperature. In B.
subtilis, this lipid modification is initiated through the activity of a
so-called two-component regulatory system consisting of the DesK and DesR
proteins [15]. Prokaryotic two-component regulatory systems usually
consist of protein pairs, a sensor kinase and a regulatory protein [18].It appears that it is a combination of membrane
physical state and protein conformation that is able to sense temperature and
to translate this sensing event into proper gene expression. However, sensing of temperature
through alteration in nucleic acid conformation could be
more efficient temperature-mediated mechanism of gene expression.


### 2.2. RNA

Messenger RNAs apart from carrying their coding
information for protein generation are also rapidly emerging as regulators of
expression of the encoded message. With unique chemical and structural
properties, sensory RNAs perform vital regulatory roles in gene expression by
detecting changes in the cellular environment through interactions with small
ligands [19, 20] and proteins [21, 22].Regulatory RNA elements, “riboswitches,” have been
reported recently, responding to intracellular signals by conformational
changes. Riboswitches are
conceptually divided into two parts: anaptamer and an expression platform. The aptamer directly binds the small molecule, and
the expression platform undergoes structural changes in response to the changes
in the aptamer. The expression platform is what regulates gene expression. Riboswitches
demonstrate that naturally occurring RNA can specifically response on versatile
physical and chemical stimuli, a capability that many previously believed was
the domain of proteins or artificially constructed RNAs called aptamers [23].Theoretically, RNA molecules have a strong
potential as temperature sensors, in that they can form pronounced secondary
and tertiary structures [24], and through their ability to form
intermolecular RNA : RNA hybrids [25]. Both of these processes greatly depend on the
formation of complementary base pairing, and consequently one would anticipate
these to be dependent on environmental temperature.RNA thermometers operate at the post-transcriptional
level to sense selectively the temperature and transduce a signal to the
translation machinery via a conformational change. They have usually a highly
structured 5’-end that shields the ribosome binding site at physiological
temperatures [1, 26–29]. Changes in temperature are
manifested by the liberation of the Shine-Dalgarno (SD)
sequence, thereby facilitating ribosome binding and translation initiation.


### 2.3. DNA

It is known that both in prokaryotic and eukaryotic cells, the geometry and
tension of DNA are highly dynamic and correspond to its functional activity. In
the bacterial cell, chromosome and plasmid DNA is contained in a “twisted”
superhelical conformation [30, 31],
where the degree of superhelicity varies in response to changes in the ambient
temperature. In many examples, the expression of many genes is dependent on DNA
conformation, and temperature-dependent gene regulation is mastered through
changes in DNA supercoiling [3, 32, 33].Seemingly, the temperature-induced conformational changes in DNA are mainly
controlled through the presence of  “nucleotid-associated” proteins, of which H-NS is the best
characterized [30, 34]. In E.
coli, creating and maintaining conformational structures in the DNA
molecule are mainly regulated through the balance of two opposing topoisomerase
activities, mainly those of topoisomerases II and I [35, 36].Examples of pure DNA-related temperature sensitivity are rare if ever
reported. In most cases, genomic thermosensitivity appears to be a result of
certain interplay among DNA, RNA, and proteins. Some bacteria carry a DNA-plasmid which shows a
controlled constant plasmid copy number at one temperature and a much higher or
totally uncontrolled copy number at a different temperature. The high-copy number phenotype of pLO88 plasmid maintained inEscherichia coli (HB101) is observed only at elevated temperatures,
(above 37°C), and is due to the precise position of a Tn5 insertion in DNA, but
the exact mechanism remains obscure [37].All abovementioned examples of membrane- and nucleic acid-based temperature
sensitivity apparently include proteins as a key regulatory component. Therefore,
from the point of view of molecular temperature sensation, protein-based molecular
“thermometers” represent an extremely interesting group.


### 2.4. Proteins

Many sensory pathways in living organisms use
structural changes in proteins as a primary perceptive event, activating
further signaling cascades. IfE. coli is exposed to an oxidative
substance such as hydrogen peroxide, it responds by the activation of a
transcriptional regulator protein OxyR [38]. Activation of OxyR is achieved through the
formation of a disulphide bound within the protein, upon which OxyR induces the
expression of a set of genes adapting the bacterial cell to oxidative stress.
This illustrates how it is possible both to sense and respond to an
abrupt change in a specific environmental factor in a simple, yet elegant mode.One would expect the organisms and cells to be
similarly elegant when sensing temperature shifts. Indeed, a striking example
is the temperature-controlled switching of the flagellar rotary motor ofE.
coli between the two rotational states, clockwise (CW) and counterclockwise
(CCW) [39]. The molecular mechanism for switching remains
unknown, but seems to be connected to the response regulator CheY-P. Two
possible models of CheY-P action explain shifting the difference in
free energy between CW and CCW states in terms of (i) conformation-related
differential binding [40, 41] and (ii) thermodynamic changes in dissociation
constants [42].Further studies on the thermosensory transducing
system inE. coli revealed that two
major chemoreceptors, Tar and Tsr, which detect aspartate and serine,
respectively, also function as thermoreceptors, as well as Trg and Tap receptors [43]. Interestingly, in spite of different specificity and
sensitivity, amino acid sequences of all four chemoreceptors have a significant
homology. These are transmembrane proteins with two functional domains in their
role as chemoreceptors; one is a ligand-binding domain located in the periplasm
and the other is a signaling domain located in the cytoplasm. Thus, it is
suggested that a temperature change induces a conformational change in these
two receptors and that this conformational change triggers the signaling for
thermoresponse. In the simplest model of thermoreception by these receptors,
two conformational states of these receptors are assumed: a low-temperature
state and a high-temperature state [44]. The swimming pattern of the Trg- and Tap-containing
cells was determined simply by the temperature of the medium, indicating that
these cells under nonadaptive conditions sense
the absolute temperature as the thermal stimulus, and not the relative change in temperature.The understanding of proteins temperature-related sensory transductions in
terms of their underlying molecular mechanism is fast-advancing thanks to the
discovery and functional characterization of the transient receptor potential
(TRP) channels. This protein family, first identified inDrosophila, is at the forefront of our sensory stem, responding to
both physical and chemical stimuli and, thus, having diverse functions [45, 46].The superfamily of TRP
channels currently comprises nearly 30 mammalian members grouped into six related families: TRPC, TRPV, TRPP, TRPM, TRPN, and
mucolipins. In higher organisms, TRPV
channels are important polymodal integrators of noxious stimuli mediating
thermosensation and nociception. The transient
receptor potential channel vanilloid receptor subunit 1 (TRPV1) is widely
recognized as a molecular integrator of physical and chemical stimuli in the
peripheral nociceptor terminals [11, 47].A subset of these channels, the thermo-TRPs, is
activated by distinct physiological temperatures. Six thermo-TRP channels, which are all
characterized by their unusually high-temperature sensitivity (Q10>10), have been cloned: TRPV(1)–(4) are heat-activated
[48–50], whereas TRPM8 [50, 51] and TRPA1 [52] are activated by cold. With a Q10 of about 26 for TRPV1 [53] and approx. 24 for TRPM8 [54, 55], they far surpass the temperature dependence of the
gating processes characterized by other ion channels (Q10>3) [53]. In spite of the great advances made, the molecular
basis for regulation by temperature remains unknown because of the lack of
structural information. More detailed consideration of
protein dynamics and thermodynamics can bring us closer to understanding of
universal principles of thermal sensation.


## 2.1. Membrane Lipids

While the information available is somewhat
scant, the picture emerging shows that cells can use signals generated through
changes in nucleic acid or protein conformation, or changes in membrane lipid
behavior, as sensory devices. The physical state of
membranes does change in response to temperature shifts in phase-transition
manner [14], but the temperature-induced changes in real
biological membranes are not sharp because many kinds of fatty acids present,
having different characteristic temperature points of phase transition. Thus,
it would not be surprising if cells (even those of bacteria) could utilize, changes
in membrane fluidity as a thermometer device, assisted by protein helpers,
playing a role of switchers, “sharpening” the temperature response. Microorganisms
counteract the propensity for membranes to rigidify at lower temperature by
adapting to the conditions in order to maintain a more-or-less constant degree
of membrane fluidity (homeoviscous adaptation). The cyanobacterium Synecocystis responds to decreased temperature by increasing the cisunsaturation
of membrane-lipid fatty acids through expressing acyl-lipid desaturases [15–17]. Lipid
unsaturation would then restore membrane fluidity at the lower temperature. In B.
subtilis, this lipid modification is initiated through the activity of a
so-called two-component regulatory system consisting of the DesK and DesR
proteins [15]. Prokaryotic two-component regulatory systems usually
consist of protein pairs, a sensor kinase and a regulatory protein [18].It appears that it is a combination of membrane
physical state and protein conformation that is able to sense temperature and
to translate this sensing event into proper gene expression. However, sensing of temperature
through alteration in nucleic acid conformation could be
more efficient temperature-mediated mechanism of gene expression.


## 2.2. RNA

Messenger RNAs apart from carrying their coding
information for protein generation are also rapidly emerging as regulators of
expression of the encoded message. With unique chemical and structural
properties, sensory RNAs perform vital regulatory roles in gene expression by
detecting changes in the cellular environment through interactions with small
ligands [19, 20] and proteins [21, 22].Regulatory RNA elements, “riboswitches,” have been
reported recently, responding to intracellular signals by conformational
changes. Riboswitches are
conceptually divided into two parts: anaptamer and an expression platform. The aptamer directly binds the small molecule, and
the expression platform undergoes structural changes in response to the changes
in the aptamer. The expression platform is what regulates gene expression. Riboswitches
demonstrate that naturally occurring RNA can specifically response on versatile
physical and chemical stimuli, a capability that many previously believed was
the domain of proteins or artificially constructed RNAs called aptamers [23].Theoretically, RNA molecules have a strong
potential as temperature sensors, in that they can form pronounced secondary
and tertiary structures [24], and through their ability to form
intermolecular RNA : RNA hybrids [25]. Both of these processes greatly depend on the
formation of complementary base pairing, and consequently one would anticipate
these to be dependent on environmental temperature.RNA thermometers operate at the post-transcriptional
level to sense selectively the temperature and transduce a signal to the
translation machinery via a conformational change. They have usually a highly
structured 5’-end that shields the ribosome binding site at physiological
temperatures [1, 26–29]. Changes in temperature are
manifested by the liberation of the Shine-Dalgarno (SD)
sequence, thereby facilitating ribosome binding and translation initiation.


## 2.3. DNA

It is known that both in prokaryotic and eukaryotic cells, the geometry and
tension of DNA are highly dynamic and correspond to its functional activity. In
the bacterial cell, chromosome and plasmid DNA is contained in a “twisted”
superhelical conformation [30, 31],
where the degree of superhelicity varies in response to changes in the ambient
temperature. In many examples, the expression of many genes is dependent on DNA
conformation, and temperature-dependent gene regulation is mastered through
changes in DNA supercoiling [3, 32, 33].Seemingly, the temperature-induced conformational changes in DNA are mainly
controlled through the presence of  “nucleotid-associated” proteins, of which H-NS is the best
characterized [30, 34]. In E.
coli, creating and maintaining conformational structures in the DNA
molecule are mainly regulated through the balance of two opposing topoisomerase
activities, mainly those of topoisomerases II and I [35, 36].Examples of pure DNA-related temperature sensitivity are rare if ever
reported. In most cases, genomic thermosensitivity appears to be a result of
certain interplay among DNA, RNA, and proteins. Some bacteria carry a DNA-plasmid which shows a
controlled constant plasmid copy number at one temperature and a much higher or
totally uncontrolled copy number at a different temperature. The high-copy number phenotype of pLO88 plasmid maintained inEscherichia coli (HB101) is observed only at elevated temperatures,
(above 37°C), and is due to the precise position of a Tn5 insertion in DNA, but
the exact mechanism remains obscure [37].All abovementioned examples of membrane- and nucleic acid-based temperature
sensitivity apparently include proteins as a key regulatory component. Therefore,
from the point of view of molecular temperature sensation, protein-based molecular
“thermometers” represent an extremely interesting group.


## 2.4. Proteins

Many sensory pathways in living organisms use
structural changes in proteins as a primary perceptive event, activating
further signaling cascades. IfE. coli is exposed to an oxidative
substance such as hydrogen peroxide, it responds by the activation of a
transcriptional regulator protein OxyR [38]. Activation of OxyR is achieved through the
formation of a disulphide bound within the protein, upon which OxyR induces the
expression of a set of genes adapting the bacterial cell to oxidative stress.
This illustrates how it is possible both to sense and respond to an
abrupt change in a specific environmental factor in a simple, yet elegant mode.One would expect the organisms and cells to be
similarly elegant when sensing temperature shifts. Indeed, a striking example
is the temperature-controlled switching of the flagellar rotary motor ofE.
coli between the two rotational states, clockwise (CW) and counterclockwise
(CCW) [39]. The molecular mechanism for switching remains
unknown, but seems to be connected to the response regulator CheY-P. Two
possible models of CheY-P action explain shifting the difference in
free energy between CW and CCW states in terms of (i) conformation-related
differential binding [40, 41] and (ii) thermodynamic changes in dissociation
constants [42].Further studies on the thermosensory transducing
system inE. coli revealed that two
major chemoreceptors, Tar and Tsr, which detect aspartate and serine,
respectively, also function as thermoreceptors, as well as Trg and Tap receptors [43]. Interestingly, in spite of different specificity and
sensitivity, amino acid sequences of all four chemoreceptors have a significant
homology. These are transmembrane proteins with two functional domains in their
role as chemoreceptors; one is a ligand-binding domain located in the periplasm
and the other is a signaling domain located in the cytoplasm. Thus, it is
suggested that a temperature change induces a conformational change in these
two receptors and that this conformational change triggers the signaling for
thermoresponse. In the simplest model of thermoreception by these receptors,
two conformational states of these receptors are assumed: a low-temperature
state and a high-temperature state [44]. The swimming pattern of the Trg- and Tap-containing
cells was determined simply by the temperature of the medium, indicating that
these cells under nonadaptive conditions sense
the absolute temperature as the thermal stimulus, and not the relative change in temperature.The understanding of proteins temperature-related sensory transductions in
terms of their underlying molecular mechanism is fast-advancing thanks to the
discovery and functional characterization of the transient receptor potential
(TRP) channels. This protein family, first identified inDrosophila, is at the forefront of our sensory stem, responding to
both physical and chemical stimuli and, thus, having diverse functions [45, 46].The superfamily of TRP
channels currently comprises nearly 30 mammalian members grouped into six related families: TRPC, TRPV, TRPP, TRPM, TRPN, and
mucolipins. In higher organisms, TRPV
channels are important polymodal integrators of noxious stimuli mediating
thermosensation and nociception. The transient
receptor potential channel vanilloid receptor subunit 1 (TRPV1) is widely
recognized as a molecular integrator of physical and chemical stimuli in the
peripheral nociceptor terminals [11, 47].A subset of these channels, the thermo-TRPs, is
activated by distinct physiological temperatures. Six thermo-TRP channels, which are all
characterized by their unusually high-temperature sensitivity (Q10>10), have been cloned: TRPV(1)–(4) are heat-activated
[48–50], whereas TRPM8 [50, 51] and TRPA1 [52] are activated by cold. With a Q10 of about 26 for TRPV1 [53] and approx. 24 for TRPM8 [54, 55], they far surpass the temperature dependence of the
gating processes characterized by other ion channels (Q10>3) [53]. In spite of the great advances made, the molecular
basis for regulation by temperature remains unknown because of the lack of
structural information. More detailed consideration of
protein dynamics and thermodynamics can bring us closer to understanding of
universal principles of thermal sensation.


## 3. Biophysical Aspects of Protein-Aided Thermosensation

It appears from the
above mentioned examples of protein participation in temperature sensing events
that sudden conformational changes, “structural transitions” play essential
role on the primary conversion of physical stimulus
into biologically relevant signal.Phase transitions and critical phenomena
continue to be the subject of intensive experimental and theoretical
investigation. In this context, systems consisting primarily of well
characterized proteins and water can serve as particularly valuable objects of
study. The importance of studies of specific phase transitions in protein/water
solutions derives also from their physiological relevance to the supramolecular
organization of normal tissues and to certain pathological states. For example,
such phase transitions play an important role in the deformation of the
erythrocyte in sickle-cell disease [21, 56] and in the cryoprecipitation of immunoglobulins
in cryoglobulinemia and rheumatoid arthritis [57].Discussions about
protein stability and temperature-induced structural transitions are usually
limited to the stability of the native state against denaturation. Yet the
native state may include different functionally relevant conformations
characterized by different Gibbs energies and therefore different stabilities
(e.g., the R and T states of hemoglobin). Even when the native state does not
undergo a conformational change, it is still characterized by the occurrence of
a large number of local unfolding events that give rise to many substates.
Thus, the native state itself needs to be considered as a statistical ensemble
of conformations rather than unique entity. These distinctions are very
important from the functional point of view since different conformations are
usually characterized by different functional properties.The stabilizing
contributions that arise from the hydrophobic effect and hydrogen bonding are
largely offset by the destabilizing configurational entropy. The hydrophobic
effect is strongly temperature-dependent, and is considerably weaker and
perhaps even destabilizing at low temperatures than at elevated temperatures.
The contribution of various interactions for a “typical” protein is reported in
many works [58–62]. Apparently, the transition from stabilizing to
destabilizing conditions is achieved by relatively small changes in the
environment. These can be changes in temperature, pH, and addition of
substrates or stabilizing cosolvents. While the exact contribution of different
interactions to the stability of globular proteins remains a question, our
understanding seems to be refined enough to allow for the reasonable prediction
of the overall folding thermodynamics [61, 62]. Important to mention that both the enthalpy
end entropy changes are not constant but increasing functions of temperature,
and that the Gibbs energy stabilization of a protein can be written as follows:
(1)ΔG=ΔH(TR)+ΔCp(T−TR)−TΔS(TR)+ΔCpln(T/TR),
where TR is
a convenient reference temperature. ΔCp is the heat capacity change, and ΔH(TR) and ΔS(TR) are the enthalpy and entropy
values at that temperature. The temperature dependency of ΔH and ΔS is an important issue because it transforms
the Gibbs energy function from a linear into a parabolic function of
temperature.For large values ofΔCp
, the Gibbs Energy crosses zero point twice—temperature (heat denaturation) and one at low temperature (cold denaturation).
The native state is thermodynamically stable between those two temperatures and
ΔG exhibits a maximum at the temperature at
which ΔS=0. The peculiar shape of the Gibbs energy
function of a protein does not permit a unique definition of protein stability.
For example, having a higher denaturation temperature does not necessarily
imply that a protein will be more stable at room temperature. Within the context of
the structural parameterization of the energetics, the Gibbs energy of protein stabilization
is approximated by
(2)ΔG=ΔGgen+ΔGion+ΔGtr+ΔGother,
where ΔGgen contains the contributions
typically associated with the formation of secondary and tertiary structure
(van der Waals interactions, hydrogen bonding, hydration, and conformational
entropy), ΔGion the electrostatic and
ionization effects, and ΔGtr the contribution of the change
in translational degrees of freedom existing in oligomeric proteins. The term ΔGother includes interactions unique
to specific proteins that cannot be classified in a general way (e.g.,
prosthetic groups, metals, and ligands) and must be treated on a case-by-case
basis.Nilius and coworkers have recently applied a simple
thermodynamic formalism to describe the shifts in voltage dependence due to
changes in temperature [63, 64], where the probability of the opening of a protein channel is given as a
function of temperature, the gating charge, Faraday’s constant, and the free-energy
difference between open and closed states of the channel.At biological
temperatures, some proteins alternate between well-defined, distinct
conformations. In order for two conformational states to be distinct, there
must be a free-energy barrier separating them. The notions involved to get from
one state to another are usually much more complex than the oscillation of
atoms and groups about their average positions. In proteins, because most of
the forces that stabilize the native state are noncovalent, there is enough
thermal energy at physiological temperature for weak interactions to break and
reform frequently. Thus a protein molecule is more flexible than a molecule in
which only covalent forces dictate the structure.To further understand the nature of dynamic
transitions in proteins, it is particularly important to characterize solvent effects.
Solvent can in principle affect protein dynamics by modifying the effective
potential surface of the protein and/or by frictional damping. Changes in the
structure and internal dynamics of proteins as a function of solvent conditions
at physiological temperatures have been found by using several experimental
techniques [65]. It is clear from the works of Zaccai and others that
solvent affects protein dynamics at physiological temperatures [66–68]. They
reported that in the absence of minimal hydration, proteins do not function at
all. Therefore, a solvent dependence of the dynamic transition might be
expected. Indeed, measurements on CO binding to myoglobin indicate that dynamic
behavior of the protein is correlated with a glass transition in the
surrounding solvent [69], and a recent molecular dynamics analysis of hydrated
myoglobin also indicates a major solvent role in protein dynamic transition behavior
[70].From the point of view
of structural biophysics, thermosensation is a special sort of mechanosensation and
therefore many theoretical models and considerations developed for protein
mechanosensors are also applicable for thermosensors. The difference between
mechanosensitive channels and thermosensitive molecules is only the size and
the organization of “pushing” agents—a lot of noncoordinated
events (thermal stimuli) versus a net stretch (mechanical stimuli). Interestingly,
many members of thermosensing TRPV family are known osmo- and mechanosensors. Because mechanical stimuli are everywhere,
mechanosensation could represent one of the oldest sensory transduction
processes that evolved in living organisms. Similar to thermal sensors, what exactly
makes these channels respond to membrane tension is unclear. The answer will
not be simple, because not thermal and mechanosensors are very diverse [71, 72]. However, there are interesting parallels in
structural composition of different classes of known temperature-sensory
proteins.


## 4. Structural Features of Protein Molecular Thermometers

Despite significant evolutionary distances and
apparent differences of primary structure all temperature-sensitive proteins
known so far display some remarkable similarities in their tertiary/quaternary
structure. The ability of a big protein TlpA responsible inSalmonella typhimurium for temperature
regulation of transcription resides in its structural design. Two-thirds of the
C-terminal portion of TlpA is contained in an alpha-helical-coiled-coil
structure that constitutes an oligomerization domain. As the temperature
increases, the proportion of DNA-binding oligomers decreases, leading to a
derepression of the target gene. At moderate temperatures, the concentration of
TlpA increases, shifting the balance to the formation of DNA-binding oligomers
and, in part, restoring the repression potential of TlpA. Thus, TlpA undergoes
a reversible conformational shift in response to temperature alteration,
leading to an alteration in the oligomeric structure and subsequently in the
regulatory capacity of TlpA [44].The sensory capacity is contained in the coiled-coil
structure of TlpA, which illustrates the means of sensing temperature through
changes in protein conformation. The coiled-coil structure is a versatile and a
rather flexible motif in mediating protein: protein interactions. In
vertebrates, the thermosensitive elements of transcriptional mechanism
typically contain coiled-coil folding motifs, such as those in leucine zipper
family.TRPV channel subunits in turn have a common topology of
six transmembrane segments (S1–S6) with a pore
region between the fifth and sixth segments, and cytoplasmic N- and C-termini. In both TRPV1 and TRPM8, modulation of channel
gating behavior by temperature arises from the C-terminal structure that
follows the S6 inner helix [51]. Partial
deletions performed in the C-terminal domain of TRPV1 result in functional
channels with attenuated heat sensitivity, and truncation of the whole TRPV1
C-terminal domain completely hindered channel expression [53]. Interestingly, in TRPM8 channels, binding of phosphatidylinositol bisphosphate (PIP2) leads to channel activation
[73]. The
proximal C-terminal TRP domain is conserved in TRPM8 and appears to serve as a
PIP2 site [74]. These observations, and the fact that the key
question regarding  what makes
thermo-TRPs temperature sensitive remained unanswered, suggests building
C-terminal chimeras between different members of TRPV family as a further step
in structural approach [11].In thermo-TRP channels, it has been proposed that the
structural rearrangement leads to a change in tension on the helical linker
connecting the C-terminal domains with S6 segment. This tension on the linker
provides the energy necessary to move the S6 inner helix to the open
conformation [54, 55]. Another possibility could be
that temperature affects the interaction between a particular portion of the
proximal C-terminal and some other region of the channel, probably an
intracellular loop. Finally, it may be that independent arrangements induced by
temperature on C-terminal domains directly promote gate opening [53].Bernd Nilius'
group in their study on the voltage dependence of TRP channel gating by
temperature pointed
out that the small gating charge of TRP channels compared to that of classical
voltage-gated channels could lie at the basis of the large shifts of their
voltage-dependent activation curves, and may be essential for their gating
versatility [63, 64]. Thus, small changes of the free energy of activation of these channels can
result in large shifts of their voltage-dependent activation curves, and
concomitant gating of these channels.In membrane, TRP channels form tetramers of identical
subunits [47]. The crystal structure of mechanosensitive/thermosensitive
membrane proteins reveals that the channel folds as a homoheptamer that has a
large, cytoplasmic region. Recently obtained data indicate that the modular
nature of the structures involved in activation processes allow different
stimuli (voltage, temperature, and agonists) to promote thermo-TRP channel
opening by different interrelated mechanisms as has been suggested in the form
of allosteric interaction [54, 55, 75].The very interesting aspect resides in the observation
that bacterial proteins H–NS and StpA may form hetero-oligomers exactly the same way as TRPV thermosensory channels
of higher animals sometimes do [30, 54]. In this context, it is important to note that the temperature-sensitive
H–NS function is
also associated with oligomerization and that the H–NS
oligomerization domain most evidently relies on the formation of coiled-coil
oligomers [31, 69].The molecular dynamics and organization of the temperature-sensing proteins
signaling complexes are still elusive, although fast-advancing progress in this
arena is uncovering the molecular identity of these elements. A series of
papers published by Artmann and coworkers revealed intriguing temperature-related
structural transitions phenomena in hemoglobins (Hb) and myoglobins of
different species [58, 76, 77]. The reported nonlinearity in hemoglobin temperature behavior seems to be
connected to physiological body temperature of the given species and therefore might
surprisingly reflect the role of Hb as a molecular thermometer [78].


## 5. Novel Classes of Molecular Thermometers: Hemoglobin and CO

Proteins of the hemoglobin (Hb) family, also referred
to as the myoglobin (Mb) or globin family are gas-binding heme proteins found
in all domains of life. Hbs have evolved slightly different structures and
functions, but both the predominantly helical structure and certain aminoacids
are well conserved (1). Distinct-but-related classes of Hbs are widespread in
Bacteria, Archaea, and Eucarya. Although the
physiological functions of vertebrate Hbs known so far are the transport of
molecular O2 and have a role in nitric oxide (NO) metabolism, those of
nonvertebrate Hbs are much more diverse. In addition to O2 transport and
storage, they include facilitation of O2 diffusion, reactions with sulfide and
its transport, complex and as yet incompletely elucidated roles in NO
regulation and metabolism, maintenance of acid-base balance, O2 scavenging, O2
sensing, oxidase and peroxidase activities, the latter related to
detoxification, vitellogenin-like function and roles as light-shading pigments
and regulators of the buoyancy of aquatic insects [79].The reported [58, 76, 77] temperature
effects on hemoglobin hydration and aggregation may reflect an unknown,
possibly atavistic, yet expectable function of Hb in keeping homeostasis.The suggested by Zerlin et al. [78] ability of mammalian hemoglobins to
thermoadaptation finds support in many studies made for thermophilic organisms. A gene encoding a protein
homologous to Hb was identified in Aquifex aeolicus, a
hydrogen-oxidizing obligate chemolithoautotroph that grows at temperatures of
>95°C under microaerobic conditions. A. aeolicus thermoglobin, AaTgb,
is monomeric, resistant to thermal and chemical denaturation, pentacoordinate in
the ferrous deoxygenated state, and oxygen-avid. Key strongly, although not
strictly, conserved positions are preserved in the AaTgb sequence. Proline
occupies the C2 position, initiating the start of the C helix. Although
histidine occupies the distal E7 position in most plant and animal Hbs, this
residue is commonly adaptively replaced by glutamine in many invertebrate and
bacterial Hbs. Similarly large thermal variations are also encountered by Hb-containing
prokaryotes like the cyanobacterium Nostoc that extends from tropical to
polar terrestrial environments [79]. Most of
thermophilic hemoglobins discovered so far may be described as basic
ones. The aminoacid sequence is compact, without additional residues or domains
at either terminus beyond the A and H helices of the canonical fold. This basic
fold may even be fused to other domains or duplicated and fused onto itself to
yield Hbs with multiple copies of the globin domain.The equilibrium constants for dimer-tetramer
association of Hb have been determined as a linear function of temperature from
kinetic studies of the forward and reverse rate constants [60]. It is worthy to note that these studies have
been performed at temperatures below 30°C and therefore do not correspond to
physiological conditions. The thermodynamic parameters calculated for Hb are
consistent with an increased role of hydrophobic interactions within the dimer-dimer contact region, or a decreased
role of hydrogen bonds and ion pair interactions.Thermodynamic experiments by Frauenfelder,
Petsko, and Tsernoglu [80] showed that myoglobin can assume a large
number of slightly different structures, conformational substates, separated by
energy barriers. Evidence for multiple potential energy minima also comes from
molecular dynamics simulations made for myoglobin. The complex of
thermodynamic, structural, and functional features of hemoglobin family
proteins supports the hypothesis of their possible secondary role as
temperature-sensing molecules. For homeothermic organisms (birds and mammals) such multiple protein-mediated temperature
control could be of special importance, supporting its strengthening during
evolution.



---

*Source: 602870-2008-05-12.xml*