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Their Roles in Acclimatizing to Fluctuating Temperatures in the Rocky Intertidal Environment Jacob R

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Their Roles in Acclimatizing to Fluctuating Temperatures in the Rocky Intertidal Environment Jacob R Extrinsic stabilizers of proteins: their roles in acclimatizing to fluctuating temperatures in the rocky intertidal environment Jacob R. Winnikoff May 5, 2016 1 EXTRINSIC STABILIZERS OF PROTEINS: THEIR ROLES IN ACCLIMATIZING TO FLUCTUATING TEMPERATURES IN THE ROCKY INTERTIDAL ENVIRONMENT An Honors Thesis Submitted to the Department of Biology in partial fulfillment of the Honors Program STANFORD UNIVERSITY by JACOB R. WINNIKOFF MAY 5, 2016 i Acknowledgements Many thanks to Dr. Paul H. Yancey for sharing his wealth of experience with organic osmolytes, and to members of the Yancey Lab at Whitman College for performing chromatography that was essential to this project. I am also tremendously grateful to Dr. Wes Dowd of Loyola Marymount University and Dr. Luke Miller of San José State University for the opportunity to harvest samples from their “wired” mussels. Dr. Jody Beers has been continually available with guidance throughout my time in the Somero Lab, and contributed much expertise and time to managing and heat-stressing mussels in the laboratory. Dr. George Somero provided invaluable advice on experimental design and extensive review of the manuscript. Dr. James Watanabe generously provided statistical consulting. Annette Verga-Lagier often helped troubleshoot and perform enzyme assays, and both she and Patrick Carilli maintained the supply of Mytilus californianus that made this study possible. This research was funded by a UAR Major Grant. iii Table of contents Abstract 1 1. Introduction 2 2. Materials and Methods 6 2.1 Experiment 1: Assay for Cytosolic Protection of Malate Dehydrogenase 6 2.2 Experiment 2: Laboratory heat stress followed by osmolyte analysis 7 2.3 Experiment 3: Field heat stress followed by osmolyte analysis 9 2.4 Statistical analysis 9 3. Results 11 3.1 Thermal denaturation of malate dehydrogenase 11 3.2 Effect of short-term heat stress in the laboratory on osmolyte composition 12 3.3 Effect of thermal stress in the field on osmolyte composition 12 4. Discussion 17 4.1 Induction of an osmolyte response 17 4.2 Function of an osmolyte response I: seeking direct evidence of protein stabilization 18 4.3. Function of an osmolyte response II: assessing the potential for thermoprotection 20 4.4 Conclusion 23 5. References 23 6. Appendix: linear model statistical summaries 28 6.1 Experiment 1: MDH denaturation linear regression statistics 28 6.2 Experiment 2: Laboratory heat stress ANOVA statistics 28 6.3 Experiment 3: Field heat stress linear regression statistics 29 6.3.1 Osmolyte concentration vs. MDM body temperature 29 6.3.2 Osmolyte concentration vs. standard deviation of body temperature 30 6.3.3 Osmolyte concentration vs. past day maximum body temperature 31 iv List of figures Figure 1. Organic osmolyte compositions of different marine species. Figure 2. Structures, names, and stress-related properties of compounds quantified by HPLC. Figure 3. Residual MDH activity over time at 42.5˚C in homogenate and intact tissue preparations. Figure 4. Estimated cellular concentrations of the five osmolytes measured after laboratory heat stress treatments in Experiment 2. Figure 5. Estimated cellular concentrations of the five osmolytes measured as functions of mean daily maximum (MDM) body temperature. Figure 6. Plot correlating standard deviation of body temperature to MDM body temperature. Figure 7. Estimated cellular concentrations of the five osmolytes measured as functions of the standard deviation of body temperature. Figure 8. Estimated cellular concentrations of the five osmolytes measured as functions of maximum body temperature on the day prior to dissection. v Abstract Organisms living in rocky intertidal habitats endure extremely variable conditions. Body temperatures of sessile invertebrates can vary by over 25°C in concert with the tidal cycle. Proteins are temperature-sensitive, and many denature at the highest temperatures intertidal species encounter. This study sought to elucidate whether and how the adjustment of organic cellular solutes known as “osmolytes” helps protect proteins in the California ribbed mussel Mytilus californianus against heat-denaturation. I approached these questions by directly measuring the extrinsic stability, i.e. stability afforded by co-solutes, of M. californianus malate dehydrogenase, and by measuring osmolyte concentrations after heat stress in the laboratory and in the field. Though malate dehydrogenase was much more stable in cytosol than in buffer as expected, prior heat stress did not strongly alter its stability in the cell (p=0.14). Changes in the osmolyte composition of gill tissue were observed after twenty-three days of heat stress in the intertidal zone, but not after one eight-hour heating-cooling cycle in the laboratory. Thus, induction of a putative osmolyte response requires multiple thermal stress episodes. Cellular concentrations of alanine, taurine, and glycine varied significantly (p<0.05) with mean daily maximum body temperature measured over twenty-three days in the field. Taurine, by far the most abundant osmolyte and a strong protein stabilizer, showed a significant increase with acclimatization to higher temperatures, consistent with an increase in extrinsic stabilization under heat stress. A weak trend (p=0.14) in the concentration of the stabilizer glycine betaine also supports a model in which osmolyte synthesis pathways are regulated in a way that protects proteins. I discuss the plausible role of osmolytes in mussels’ acclimatization to repetitive heat stress, and suggest further experiments to isolate and quantify this function. Keywords: Intertidal, heat stress, acclimatization, Mytilus californianus, proteins, osmolytes, extrinsic stability 1 1. Introduction The California ribbed mussel Mytilus californianus withstands large and rapid fluctuations in temperature during the tidal cycle. The highest temperatures it experiences might be expected to cause extensive protein denaturation (Liu and Bolen, 1995). This study aimed to augment our understanding of mussels’ ability to tolerate thermal extremes in the rocky intertidal habitat. Specifically, it investigated whether small organic solutes that are known to stabilize proteins in vitro contribute to maintaining protein structure in the face of environmental heat stress. This question’s ecological relevance lies in predicting animals’ tolerance of and response to climate change, as determined by their capacities for biochemical adaptation (Somero, 2011). The study has biomedical implications as well. Use of non-toxic stabilizers of peptides and proteins could diminish the need to refrigerate products such as insulin and vaccines, thus making them more accessible in remote areas. Determining how marine animals employ these solutes to cope with thermal stress thus has broad interest. To function optimally, proteins must possess the appropriate balance between stability and flexibility (Somero, 2010). This balance is commonly referred to as marginal stability, a concept that recognizes the importance of changes in protein three- dimensional shape (conformation) during function. If they are to function well, proteins must be flexible enough to undergo conformational changes. However, this flexibility can place proteins in jeopardy from physical and chemical stressors that perturb their folding. Because proteins are only marginally stable at physiological temperatures, fluctuations in temperature can have detrimental effects on protein structure and function. Two types of adaptations counter protein denaturation effects of thermal stress. During evolution, proteins undergo changes in amino acid sequence that modify their intrinsic stabilities. Throughout all three domains of life, a positive correlation has been found between evolutionary temperature and intrinsic thermal stability of a wide range of proteins (Hochachka and Somero, 2002). In addition to harboring adaptive changes in intrinsic stability, many organisms respond to rapid temperature variation by adjusting intracellular concentrations of molecular chaperones such as heat shock proteins and of small organic molecules (MW <500 Da) termed “thermoprotectants”. Thermoprotectants are extrinsic stabilizers that act to maintain appropriate marginal stability of proteins and 2 exert similar stabilizing effects across the proteome (Somero and Yancey, 1997; Yancey, 2005). Although several organic thermoprotectants have been discovered, many organic cellular solutes remain to be characterized, and additional types of thermoprotectants may remain to be discovered [Fig. 1]. In addition, the diversity of species in which thermoprotectants facilitate protein function over wide temperature ranges is not presently known. As is common in newly developing areas of biochemistry or molecular biology, our knowledge stems primarily from studying microbes, in this case thermophilic bacteria and archaea such as those found in hot springs, which accumulate high concentrations of thermoprotectants (Roberts, 2005). The role of extrinsic stabilizers in animals facing temperature fluctuations had not previously been investigated, providing part of the motivation for this study. On the rocky shores of Monterey Bay in California, the ribbed mussel M. californianus experiences temperatures between 0 and 38˚C (Denny et al., 2011). Heating rates can reach 7˚C/h (Denny et al., 2011) when still, clear weather coincides with an extreme low tide. In vitro at cytoplasm-like protein and buffer concentrations, temperature profiles like that above cause many proteins
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