Transcript Temperature: Effects on Proteins

What are the environmental
challenges faced by marine
organisms?
Temperature: Body T terms
Ectotherms: body temperature varies in concert with the temperature of the
surrounding environment.
Endotherms: regulate the amount of heat produced catabolically, either regionally
or globally.
Homeotherms: maintain a stable body T through behavior, insulation, coloration,
circulatory regulation, and the regulation of cellular heat production. e.g.,
homeothermic endotherm: regulate metabolism to maintain constant body T.
Heterotherms: can temporally or regionally regulate endothermic heat production.
e.g., regional heterothermic endotherm: use metabolic processes to heat a
region of the body. e.g., temporal heterothermic endotherm: body temperature
varies over time.
2 Strategies for endothermy, based on the fact that no process is 100% efficient
(e.g., 38% of energy from glucose oxidation is stored as ATP):
1) Increase the rate of metabolic flux.
2) Decrease the efficiency of metabolic flux.
Temperature: Heat production/conservation
Brown Adipose
Tissue in
Mammals
Oxygen
consumption is
normally
tightly coupled
to ATP
production.
Uncoupling
proteins (UCP)
uncouples this
process to
generate heat
4H+
4H+
2H+
H+
Muscle is a good tissue
for heat generation
ATP ADP + Pi
Myosin ATPase
(contraction)
SR
3 Major ATP sinks:
Na+-K+ ATPase
Myosin ATPase
SR Ca2+ ATPase
ADP
+ Pi
Ca2+
ADP
+ Pi
2K+
3Na+
ATP
ATP
SR-Ca2+ ATPase
(relaxation)
Na+/K+ ATPase
(membrane potential)
•Internalized red muscle of tuna (top left), and
the heat exchanger (top right) and heater organ
cell of the blue marlin (bottom).
•Skipjack tuna has heat exchanger below
vertebral column.
Block et al. 1991
Proposed mechanism of excitation-thermogenic coupling in cranial
heater organs of fishes. Ca+2 futile cycling, ATP hydrolysis and the
electron transport system all produce heat. From Block, 1991.
Temperature: Protection against freezing
•Why is freezing bad for organisms?
Colligative properties: fishes elevate glycerol and TMAO
concentrations in the cold (including seasonal variations).
•This depresses the freezing point.
Non-colligative properties: Antifreeze proteins (AFP) and
antifreeze glycoproteins (AFGP). First found in antarctic
Nototheniid fishes by DeVries (1971). Since found in many taxa
including plants, fungi and bacteria.
•AFPs and AFGPs depress the freezing point below the
thermodynamic melting point. Often called thermal hysteresis
proteins (THPs).
Convergent evolution of AFPs and
AFGPs in fishes. 4 AFPs and 1 AFGP
that are known do not follow
evolutionary lineages in fish.
Other properties of THPs:
•THPs integrate in the ice.
•THPs make ice crystals grow quickly
and in a needle-like conformation.
•THPs limit ice recrystallization.
•They accomplish these goals by
binding to ice crystals.
Fletcher et al. 2001
Mechanism of THP action. Normal ice crystal growth occurs with a low
radius of curvature (top left). When THPs bind the ice, the available
surfaces for crystal growth have a high radius of curvature (top right). In
insects, an ice-nucleating protein (PIN) aggregates several THPs to
enhance the antifreeze function (Hochachka and Somero, 2002).
Temperature: Effects on Membranes
Major membrane components
O
H2C
O
C
O
HC
O
C
O
H2C
O
P
O
CH 2
CH 2
N+(CH 3)3
O-
Phosphotidylcholine (PC)
O
H2C
O
C
O
HC
O
C
O
H2C
O
P
CH 3
CH-CH 3
O
CH 2
CH 2
N+H3
(CH 2)3
-
O
CH-CH 3
Phosphotidylethanolamine (PE)
CH 3
CH 3
PC
PE
HO
Cholesterol
Membranes are stabilized by the hydrophobic interaction and
van der Waals forces.
Membrane “phase” and “static order” (fluidity/viscosity)
must be conserved across temperatures
Homeophasic acclimation/adaptation: Conservation of phase at different
temperatures. Phosphatidylcholine (PC) is cylindrical, while
phosphatidylethanolamine (PE) is conical (more unsaturated). The PC:PE ratio is
higher in more warm adapted species.
PE
PC
Effects of acclimation temperature on
PC:PE ratio in membranes from rainbow
trout gills. PC has a more cylindrical
conformation than PE. Note that the
acclimation response includes a rapid
change in PC:PE but it does not reach a
constant level over this time course (other
compensations invoked). Hazel and
Carpenter, 1985.
Adaptation temperature versus the
degree of unsaturated acyl chains in
vertebrate synaptic membranes. The
open circles are PE and the filled
circles are PC. Note decreased
PC:PE ratio in cold adapted species.
Logue et al. 2000.
Increased cholesterol also helps stabilize membranes – important in
warm adaptation/acclimation
•Homeoviscous adaptation in
brain synaptic membranes:
maintenance of membrane
fluidity at different
temperatures.
•DPH is a probe that
intercalates in membranes.
•High DPH anisotropy
indicates low fluidity.
•Note effect of temperature
(top) and homeoviscous
adaptation at the adaptation
temperature (below). This
mode of adapation is
complete within the
horizontal lines.
(Logue et al. 2000)
Temperature: Effects on Proteins
• Temperature effects on metabolism encapsulated by Q10.
• Q10 = (k1/k2)10/(t1-t2)
• For many processes (e.g., rates of respiration, enzyme
activities), Q10  2, when the temperature effects are measured
within an organism’s physiological range.
Arrhenius plot of the effect
of temperature on reaction
rate. Arrhenius plot is linear
over the physiological
range.
Log rate
High T, protein damage, Q10<1
Physiological T range
Low T, elevated energy
barriers, Q10>2
1/T
•Temperature is a measure of the level of kinetic energy most frequently
occupied by molecules in the system. Higher kinetic energies lead to
higher chemical reactivities.
•However, if body T is 298 K, a 10° change is only a 10/298 (3%)
change in the average kinetic energy of the system. So how is Q10  2?
•Arrhenius - examine not only most common energy state (temperature),
but also the high energy states that exceed the activation energy.
Distribution of kinetic energies.
Adaptations/Acclimations to temperature include
modifications of protein quality and quantity
• Organisms in the cold must “speed up”
metabolism to compensate for slowing effects
of cold.
• Often found that at a common temperature,
cold-adapted or cold acclimated species have
higher biological rates.
• How might this be accomplished?
• Enzyme activity: units of Units! (moles/min)
Temperature compensation of metabolism may involve changes in
enzyme quantity (here is an example in a cold-acclimated species)
Striped bass Morone saxatilis red muscle cell acclimated to 25° C
(left) and to 5° C (right). From Egginton and Sidell (1989). Note
increase in mitochondria and lipid droplets in cold acclimated cell.
Thus, cold acclimation usually involves increases in the number of
enzymes, not in their quality.
Temperature compensation may involve changes in protein quality
(here is an example for temperature adaptation)
Fields and Somero, 1998
A4-LDH catalytic rates for differently thermally adapted vertebrates.
At a common temperature, cold adapted enzymes perform “better”
than warm adapted enzymes. kcat = rate of catalysis/active site =
Vmax/[E]. Why isn’t a very fast catalytic rate selected for in warm
adapted enzymes?
Temperature compensation may involve changes in protein quality and
quantity (here is an example for temperature adaptation)
Temperature compensation of LDH and
CS activity (kcat x [enzyme]) in brain of
Antarctic and tropical adapted fish.
Measurements were made at 10° C and
extrapolated to the habitat temperature (0
or 25 C). Numbers on plot represent the
activity at habitat temperature. Although
temperature compensation occurs (higher
activities at a common temperature in the
cold adapted species), there is still a 2-fold
difference in activity at the habitat
temperatures; temperature compensation is
incomplete.
Importantly, the higher activities at a
common temperature are proportional to
the change in kcat in the cold adapted
species. Thus, cold adapted enzymes are
“better”, not more abundant. (Kawall et
al. 2001).
How do enzymes adjust kcat values during cold adaptation?
•You can only increase a process rate by increasing the rates of the
slow steps in that process.
Lactate dehydrogenase (LDH): Model for temperature adaptation
O
H3C
C
OH
O
+ NADH + H+
C
H3C
CH
O
O-
O-
pyruvate
+ NAD+
C
lactate
Conformational changes in
LDH during catalysis and
the LDH active site. Note
that the -helix “doors”
above clamp shut when
substrates are in the active
site. Also note essential
His-193 and Arg-171.
3D structure of LDH. The catalytic loop is highly conserved, and
along with the H helix and the 1G-2G, form highly mobile
“doors” that swing open to allow substrates to get to the active site
and close to lock substrates in the appropriate position for catalysis.
Hypervariable loop is “hinge” for the 1G-2G “door”. In cold adapted species
increased glycine residues, and more hydrophilic, so it interacts less well with Nterminal of adjoining subunit. These traits promote flexibility in LDH.
Why isn’t kcat maximized for all enzymes?
•There is a trade-off between kcat and binding affinity (Km).
•Enzymes “breath”, and a population of enzymes occupies an
ensemble of conformations, only some of which can bind substrates.
•Binding affinity is described by the Km.
No free E
Vmax
V0
Km Km
[S]
Low Km = high substrate affinity, High Km = low substrate affinity.
Km of pyruvate for LDH as a function of
temperature at different experimental
temperatures. Dark bars are normal
body temperature. Cold adapted species
are to the left, warm adapted to the right.
Note that at a common temperature, Km
values are higher in the more flexible
enzymes from cold adapted species.
Also note that Km increases with T as
expected, but its value is conserved at
normal body T across species.
Km of species at 10°C (open triangles)
and at the upper limit of body
temperature (filled circles). Points to the
left are cold adapted species and to the
right are warm adapted species. Note
that when adjusted for natural body
temperature, Km is remarkably
consistent.
How much change in a protein
is required to make it thermally
adapted? Here are Kms for
congeners in the genus
Sphyraena that are adapted to
different climates. Only 1
amino acid is different between
S. idiastes and S. lucasana, and
S. argentea differs from the first
two by only 4 amino acids.
In summary, enzymes must:
1. Be structurally stable enough to competently bind substrates, but flexible
enough to facilitate conformational change.
2. Be able to rapidly catalyze reactions.
3. Be able to recognize and bind substrates at physiological concentrations.
These enzyme properties co-evolve!!
Global protein stability is often independent of kinetic properties.
•Proteins are only marginally stable.
•Protein stability also adapts to different thermal regimes.
T at which 50% of
secondary structure is
lost in eye lens proteins
of differently thermally
adapted vertebrates
(McFall-Ngai and
Horwitz, 1990).
How do proteins become more thermally stable?
1. Increased charged amino acids.
2. Increased bulky, hydrophobic residues.
Also, thermoprotectant molecules and macromolecular crowding may enhance
stability.
Fixing temperature-induced damage: Proteins that are denatured must
be refolded properly.
•Heat-shock proteins (HSP) play a role in refolding.
Cool
Cool
Warm
Very warm
(Tomanek and
Somero, 1999)