Peter A. Fields

Review: Protein function at thermal extremes: balancing stability and flexibility.

No organism can survive across the entire temperature range found in the biosphere, and a given species can rarely support active metabolism across more than a few tens of degrees C. Nevertheless, life can be maintained at surprisingly extreme temperatures, from below -50 to over 110 degrees C. That proteins, which are assembled with the same 20 amino acids in all species, can function well at both extremes of this range illustrates the plasticity available in the construction of these macromolecules. In studying proteins from extremophiles, researchers have found no new amino acids, covalent modifications or structural motifs that explain the ability of these molecules to function in such harsh environments. Rather, subtle redistributions of the same intramolecular interactions required for protein stabilization at moderate temperatures are sufficient to maintain structural integrity at hot or cold extremes. The key to protein function, whether in polar seas or hot springs, is the maintenance of an appropriate balance between molecular stability on the one hand and structural flexibility on the other. Stability is needed to ensure the appropriate geometry for ligand binding, as well as to avoid denaturation, while flexibility is necessary to allow catalysis at a metabolically appropriate rate. Comparisons of homologous proteins from organisms spanning a wide range of thermal habitats show that adaptive mutations, as well as stabilizing solutes, maintain a balance between these two attributes, regardless of the temperature at which the protein functions.

Temperature sensitivities of cytosolic malate dehydrogenases from native and invasive species of marine mussels (genus Mytilus): sequence-function linkages and correlations with biogeographic distribution.

SUMMARY The blue mussel Mytilus galloprovincialis, a native of the Mediterranean Sea, has invaded the west coast of North America in the past century, displacing the native blue mussel, Mytilus trossulus, from most of its former habitats in central and southern California. The invasive success of M. galloprovincialis is conjectured to be due, in part, to physiological adaptations that enable it to outperform M. trossulus at high temperatures. We have examined the structure and function of the enzyme cytosolic malate dehydrogenase (cMDH) from these species, as well as from the more distantly related ribbed mussel, Mytilus californianus, to characterize the effects of temperature on kinetic properties thought to exhibit thermal adaptation. The M. trossulus cMDH ortholog differs from the other cMDHs in a direction consistent with cold adaptation, as evidenced by a higher and more temperature-sensitive Michaelis-Menten constant for the cofactor NADH (KmNADH). This difference results from minor changes in sequence: the M. trossulus ortholog differs from the M. galloprovincialis ortholog by only two substitutions in the 334 amino acid monomer, and the M. californianus and M. trossulus orthologs differ by five substitutions. In each case, only one of these substitutions is non-conservative. To test the effects of individual substitutions on kinetic properties, we used site-directed mutagenesis to create recombinant cMDHs. Recombinant wild-type M. trossulus cMDH (rWT) has high KmNADH compared with mutants incorporating the non-conservative substitutions found in M. californianus and M. galloprovincialis - V114H and V114N, respectively - demonstrating that these mutations are responsible for the differences found in substrate affinity. Turnover number (kcat) is also higher in rWT compared with the two mutants, consistent with cold adaptation in the M. trossulus ortholog. Conversely, rWT and V114H appear more thermostable than V114N. Based on a comparison of KmNADH and kcat values among the orthologs, we propose that immersion temperatures are of greater selective importance in adapting kinetic properties than the more extreme temperatures that occur during emersion. The relative warm adaptation of M. galloprovincialis cMDH may be one of a suite of physiological characters that enhance the competitive ability of this invasive species in warm habitats.

Proteomic responses of blue mussel (Mytilus) congeners to temperature acclimation

SUMMARY The ability to acclimate to variable environmental conditions affects the biogeographic range of species, their success at colonizing new habitats, and their likelihood of surviving rapid anthropogenic climate change. Here we compared responses to temperature acclimation (4 weeks at 7, 13 and 19°C) in gill tissue of the warm-adapted intertidal blue mussel Mytilus galloprovincialis, an invasive species in the northeastern Pacific, and the cold-adapted M. trossulus, the native congener in the region, to better understand the physiological differences underlying the ongoing competition. Using two-dimensional gel electrophoresis and tandem mass spectrometry, we showed that warm acclimation caused changes in cytoskeletal composition and proteins of energy metabolism in both species, consistent with increasing rates of filtration and respiration due to increased ciliary activity. During cold acclimation, changes in cytoskeletal proteins were accompanied by increasing abundances of oxidative stress proteins and molecular chaperones, possibly because of the increased production of aldehydes as indicated by the upregulation of aldehyde dehydrogenase. The cold-adapted M. trossulus showed increased abundances of molecular chaperones at 19°C, but M. galloprovincialis did not, suggesting that the two species differ in their long-term upper thermal limits. In contrast, the warm-adapted M. galloprovincialis showed a stronger response to cold acclimation than M. trossulus, including changes in abundance in more proteins and differing protein expression profiles between 7 and 13°C, a pattern absent in M. trossulus. In general, increasing levels of oxidative stress proteins inversely correlate with modifications in Krebs cycle and electron transport chain proteins, indicating a trade-off between oxidative stress resistance and energy production. Overall, our results help explain why M. galloprovincialis has replaced M. trossulus in southern California over the last century, but also suggest that M. trossulus may maintain a competitive advantage at colder temperatures. Anthropogenic global warming may reinforce the advantage M. galloprovincialis has over M. trossulus in the warmer parts of the latter’s historical range.

Sublethal Exposure to UV Radiation Affects Respiration Rates of the Freshwater Cladoceran Daphnia catawba

Abstract We examined the effects of UV radiation (UVR) on metabolic rates of the freshwater cladoceran Daphnia catawba. We exposed D. catawba to UVB for 12 h in a lamp phototron at levels of 2.08 and 4.16 kJ m−2 both with and without concomitant exposure to UVA and visible photorepair radiation (PRR). We also included a group that received PRR only and a dark control group. Respiration rates were measured for 6 h following exposure. Respiration rates increased by 31.8% relative to the dark control at the lowest level of UVB stress (2.08 kJ m−2 UVB with PRR), whereas respiration was inhibited by 70.3% at the highest stress level (4.16 kJ m−2 UVB without PRR). Survival rates in the group that received PRR only and the group exposed to 2.08 kJ m−2 and PRR were not significantly different from that in the control group; however, the survival rate was reduced for all other UVR exposures. We hypothesize that enhanced respiration rates reflect energetic costs related to repair of cellular components damaged by sublethal levels of UVR. Increases in respiration rate of the magnitude we found in our experiment could significantly reduce energetic reserves available for growth and reproduction, especially in cases where these costs are incurred repeatedly during a series of days with high levels of UVR.

Adaptations of protein structure and function to temperature: there is more than one way to 'skin a cat'.

ABSTRACT Sensitivity to temperature helps determine the success of organisms in all habitats, and is caused by the susceptibility of biochemical processes, including enzyme function, to temperature change. A series of studies using two structurally and catalytically related enzymes, A4-lactate dehydrogenase (A4-LDH) and cytosolic malate dehydrogenase (cMDH) have been especially valuable in determining the functional attributes of enzymes most sensitive to temperature, and identifying amino acid substitutions that lead to changes in those attributes. The results of these efforts indicate that ligand binding affinity and catalytic rate are key targets during temperature adaptation: ligand affinity decreases during cold adaptation to allow more rapid catalysis. Structural changes causing these functional shifts often comprise only a single amino acid substitution in an enzyme subunit containing approximately 330 residues; they occur on the surface of the protein in or near regions of the enzyme that move during catalysis, but not in the active site; and they decrease stability in cold-adapted orthologs by altering intra-molecular hydrogen bonding patterns or interactions with the solvent. Despite these structure–function insights, we currently are unable to predict a priori how a particular substitution alters enzyme function in relation to temperature. A predictive ability of this nature might allow a proteome-wide survey of adaptation to temperature and reveal what fraction of the proteome may need to adapt to temperature changes of the order predicted by global warming models. Approaches employing algorithms that calculate changes in protein stability in response to a mutation have the potential to help predict temperature adaptation in enzymes; however, using examples of temperature-adaptive mutations in A4-LDH and cMDH, we find that the algorithms we tested currently lack the sensitivity to detect the small changes in flexibility that are central to enzyme adaptation to temperature. Summary: Studies over the past 40 years have shown that one to a few amino acid substitutions are sufficient to alter enzyme function in temperature-adaptive ways, but that these substitutions can occur in a variety of locations throughout the protein.

Cold Adaptation and Stenothermy in Antarctic Notothenioid Fishes: What Has Been Gained and What Has Been Lost?

Antarctic notothenioid fishes inhabit the coldest and most thermally stable waters in the ocean. During their evolutionary histories, these fishes have adapted to the threats posed by potentially freezing temperatures as well as the decelerating effects of reduced temperatures on rates of physiological processes. Cold adaptation of numerous biochemical systems has been a major feature of the evolutionary development of these species [1, 2, 3, 4].

Changes in protein expression in the salt marsh mussel Geukensia demissa: evidence for a shift from anaerobic to aerobic metabolism during prolonged aerial exposure

During aerial exposure (emersion), most sessile intertidal invertebrates experience cellular stress caused by hypoxia, and the amount and types of hypoxia-induced stress will differ as exposure time increases, likely leading to altered metabolic responses. We examined proteomic responses to increasing emersion times and decreasing recovery (immersion) times in the mussel Geukensia demissa, which occurs in salt marshes along the east coast of North America. Individuals are found above mean tide level, and can be emersed for over 18 h during spring tides. We acclimated mussels to full immersion at 15°C for 4 weeks, and compared changes in gill protein expression between groups of mussels that were continually immersed (control), were emersed for 6 h and immersed during recovery for 18 h (6E/18R), were emersed for 12 h and recovered for 12 h (12E/12R), or were emersed for 18 h with a 6 h recovery (18E/6R). We found clear differences in protein expression patterns among the treatments. Proteins associated with anaerobic fermentation increased in abundance in 6E/18R but not in 12E/12R or 18E/6R. Increases in oxidative stress proteins were most apparent in 12E/12R, and in 18E/6R changes in cytoskeletal protein expression predominated. We conclude that G. demissa alters its strategy for coping with emersion stress over time, relying on anaerobic metabolism for short- to medium-duration exposure, but switching to an air-gaping strategy for long-term exposure, which reduces hypoxia stress but may cause structural damage to gill tissue.

Phylogenetic Relationships and Biochemical Properties of the Duplicated Cytosolic and Mitochondrial Isoforms of Malate Dehydrogenase from a Teleost Fish, Sphyraena idiastes

Abstract. Unlike birds and mammals, teleost fish express two paralogous isoforms (paralogues) of cytosolic malate dehydrogenase (cMDH; EC 1.1.1.37; NAD+: malate oxidoreductase) whose evolutionary relationships to the single cMDH of tetrapods are unknown. We sequenced complementary DNAs for both cMDHs and the mitochondrial isoform (mMDH) of the fish Sphyraena idiastes (south temperate barracuda) and compared the sequences, kinetic properties, and thermal stabilities of the three isoforms with those of mammalian orthologues. Both fish cMDHs comprise 333 residues and have subunit masses of approximately 36 kDa. One cytosolic isoform, cMDH-S, was significantly more heat-stable than either the other cMDH (cMDH-L) or mMDH. In contradiction to the generally accepted model of vertebrate cMDH evolution, our phylogenetic analysis indicates that the duplication of the fish cytosolic paralogues occurred after the divergence of the lineages leading to teleosts and tetrapods. cMDH-L and cMDH-S differed in optimal concentrations of substrates and cofactors and apparent Michaelis–Menten constants, suggesting that the two paralogues may play distinct physiological roles. Differences in intrinsic thermal stability among MDH paralogues may reflect different degrees of stabilization in vivo by extrinsic stabilizers, notably protein concentration in the case of mMDH. Thermal stabilities of porcine mMDH and cMDH-L, but not cMDH-S, were significantly increased when denaturation was measured at a high protein (bovine serum albumin; BSA) concentration, but the BSA-induced stabilization reduced the catalytic activity.

Ultraviolet Radiation and Daphnia Respiration in Context: The Facts

In their letter, Lucia Fidhiany and Klaus Winckler (1) raise several concerns with our recent article (2) in Photochemistry and Photobiology. Several of the comments questioned our treatment of the literature, and others challenged our experimental design and results. We strongly disagree with these concerns for the reasons we outline below and remain confident in the conclusions of our study. We do not agree with Fidhiany and Winckler’s assertion that an extensive review of the last 80–200 years of literature on respiratory responses to various types of stressors is necessary to provide proper context for the research results we report. In the introduction to our paper we chose to cite several recent studies in which other authors used oximetry to document metabolic responses of freshwater fish to sublethal UV radiation (UVR) stress. In the first paragraph of the introduction, we state ‘‘In addition to studies that have shown behavioral changes associated with UVR exposure, several recent studies have documented a metabolic response to UV exposure (3– 4). However, the nature of the metabolic response to UVR is inconsistent among studies. Respiration rates of juvenile rainbow trout increase with UV exposure (3), whereas maximum routine respiration rates and metabolic scope of vendace and whitefish larvae decrease in response to UVR (4).’’ These specific examples were chosen for two reasons: (1) the experimental protocols of these studies are parallel to our own because they involve manipulation of UVB radiation and (2) they reinforced the point that respiratory responses to sublethal doses of UVR are inconsistent. Fidhiany and Winckler also cautioned against ‘‘using second-level literature for citation.’’ In our paper, 17 of 20 citations are from the primary literature. The three citations from the secondary literature included two chapters from highly regarded edited books (5–6) to support broad statements in the introduction about general effects of UVR on aquatic organisms and a reference to the World Meteorological Organization report (7) on the international UV index scale. Fidhiany and Winckler’s assertions that our paper lacked an adequate treatment of previous studies on UVR effects on Daphnia and was missing appropriate references about effects of environmental stress on general Daphnia metabolism are unfounded. In the introduction and discussion, we included references to previous studies on Daphnia responses to UVR, including both laboratory and field studies (8–14). In addition, we make a clear statement in the introduction that our work is an extension of a larger literature on UVR and Daphnia by stating ‘‘Most research has focused on the effects of UVR on the survival of D. catawba and, consequently, UVR exposure levels have been relatively high (e.g. .26 kJ m 2 UVR) (11). In this study, we extend these previous studies by exposing D. catawba to lower levels of UVR and monitoring respiration and survival rates.’’ The list of references that Fidhiany and Winckler cite in their letter includes a wealth of excellent information about Daphnia; however, we do not believe that specific reference needs be made to these papers in order to correctly interpret our results. In particular, none of the listed papers address the effects of sublethal levels of UVR exposure on Daphnia metabolism. Furthermore, some of the papers have only a peripheral relationship to our work. For example, the work by Scott et al. (15) on effects of UVB irradiated food on survival and fecundity of Daphnia pulex is not directly relevant to our work because it was the Daphnia, not their food source, that were differentially irradiated in our experiment. Similarly, we did not consider the role of hemoglobin in our discussion because previous studies indicate that lacustrine Daphnia species do not synthesize hemoglobin under well-oxygenated conditions such as those in our oligotrophic study site and our experiment (16,17). We recognize the valuable contributions of Fidhiany and Winckler to the field of UVR research, in particular their previous work on sublethal effects of UVA on the metabolism of the convict cichlid fish (18,19). However, we disagree strongly with their statement that we have ignored the effects of UVA on Daphnia respiration. Our experimental design explicitly included a photorepair radiation treatment (referred to as PRR) to examine effects of UVA and photosynthetically available radiation (PAR) on Daphnia respiration. We found that respiration rates in the PRR treatment did not differ from the dark control, indicating that neither UVA nor PAR was driving the significant metabolic responses that we observed in other treatments in our experiment. The first paragraph of our discussion addresses the lack of a UVA effect in our results in detail. In addition to the behavioral mechanisms that we review, it is possible that UVA could affect Daphnia respiration through other pathways such as UVA-induced DNA damage or oxidative stress (20,21). However, our results are not consistent with these hypothesized UVA effects. Rather, the

Rapid proteomic responses to a near-lethal heat stress in the salt marsh mussel Geukensia demissa.

ABSTRACT Acute heat stress perturbs cellular function on a variety of levels, leading to protein dysfunction and aggregation, oxidative stress and loss of metabolic homeostasis. If these challenges are not overcome quickly, the stressed organism can die. To better understand the earliest tissue-level responses to heat stress, we examined the proteomic response of gill from Geukensia demissa, an extremely eurythermal mussel from the temperate intertidal zone of eastern North America. We exposed 15°C-acclimated individuals to an acute near-lethal heat stress (45°C) for 1 h, and collected gill samples from 0 to 24 h of recovery. The changes in protein expression we found reveal a coordinated physiological response to acute heat stress: proteins associated with apoptotic processes were increased in abundance during the stress itself (i.e. at 0 h of recovery), while protein chaperones and foldases increased in abundance soon after (3 h). The greatest number of proteins changed abundance at 6 h; these included oxidative stress proteins and enzymes of energy metabolism. Proteins associated with the cytoskeleton and extracellular matrix also changed in abundance starting at 6 h, providing evidence of cell proliferation, migration and tissue remodeling. By 12 h, the response to acute heat stress was diminishing, with fewer stress and structural proteins changing in abundance. Finally, the proteins with altered abundances identified at 24 h suggest a return to the pre-stress anabolic state. Summary: After exposure to an acute and near-lethal heat stress, gill from the mussel Geukensia demissa responds immediately with a coordinated time course of changes in protein abundance.