What are the environmental challenges faced by marine organisms?

1. What are the environmental challenges faced by marine organisms?

2. 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): • Increase the rate of metabolic flux. • Decrease the efficiency of metabolic flux.

3. Temperature: Heat production/conservation 2H+ 4H+ 4H+ H+ Brown Adipose Tissue in Mammals Oxygen consumption is normally tightly coupledto ATP production. Uncoupling proteins (UCP)uncouples this process to generate heat

4. Muscle is a good tissue for heat generation ATP ADP + Pi Myosin ATPase (contraction) ADP + Pi ADP + Pi SR 2K+ 3Na+ Ca2+ 3 Major ATP sinks: Na+-K+ ATPase Myosin ATPase SR Ca2+ ATPase ATP Na+/K+ ATPase (membrane potential) ATP SR-Ca2+ ATPase (relaxation)

5. 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

6. 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.

7. 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).

8. 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

9. 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).

10. Temperature: Effects on Membranes

11. Cholesterol Major membrane components Phosphotidylcholine (PC) Phosphotidylethanolamine (PE) PC PE

12. Membranes are stabilized by the hydrophobic interaction and van der Waals forces.

13. 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.

14. 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. PE PC Increased cholesterol also helps stabilize membranes – important in warm adaptation/acclimation

15. 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)

16. High T, protein damage, Q10<1 Arrhenius plot of the effect of temperature on reaction rate. Arrhenius plot is linear over the physiological range. Physiological T range Log rate Low T, elevated energy barriers, Q10>2 1/T 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.

17. 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.

18. 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)

19. 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.

20. 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?

21. 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).

22. 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

23. 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.

24. 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 N-terminal of adjoining subunit. These traits promote flexibility in LDH.

25. 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.

26. No free E Vmax V0 Km Km [S] • Binding affinity is described by the Km. Low Km = high substrate affinity, High Km = low substrate affinity.

27. 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.

28. 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: • Be structurally stable enough to competently bind substrates, but flexible enough to facilitate conformational change. • Be able to rapidly catalyze reactions. • Be able to recognize and bind substrates at physiological concentrations. • These enzyme properties co-evolve!!

29. 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? • Increased charged amino acids. • Increased bulky, hydrophobic residues. • Also, thermoprotectant molecules and macromolecular crowding may enhance stability.

30. Fixing temperature-induced damage: Proteins that are denatured must be refolded properly. • Heat-shock proteins (HSP) play a role in refolding.

31. Cool Cool Warm Very warm (Tomanek and Somero, 1999)