REVIEW Adaptations of protein structure and function to temperature: there is more than one way to ‘skin a cat’ Peter A. Fields 1, *, Yunwei Dong 2 , Xianliang Meng 3 and George N. Somero 4 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, A 4 -lactate dehydrogenase (A 4 -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 A 4 -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. KEY WORDS: A 4 -lactate dehydrogenase, Cytosolic malate dehydrogenase, Protein stability, Temperature adaptation Introduction Environmental temperature is of overriding importance in determining whether ectotherms can survive, grow and reproduce in a particular habitat. As a result, temperature plays an important role in determining biogeographic range limits of many organisms, and therefore the species composition and diversity of communities (Pörtner, 2002; Somero, 1995; Angilletta, 2009). Underlying this sensitivity of biological systems to temperature is the impact that changes in the thermal energy of the environment have on biochemical and thus physiological processes (Hochachka and Somero, 2002). The effects of temperature on most chemical reactions, including those underlying metabolism, are encapsulated in a simple equation derived by Svante Arrhenius in the latter half of the nineteenth century: k ¼ Ae ÀE a =ðRT Þ ; ð1Þ in which the rate of a reaction, k, increases exponentially with temperature, T (where R is the universal gas constant, A is a reaction- specific pre-exponential constant and E a represents the Arrhenius activation energy of the reaction). Depending on the value of E a , rates of most metabolic processes will increase on the order of 2- to 3-fold with a 10°C increase in environmental temperature, giving rise to the familiar ‘Q 10 ’ relationship of thermal physiology. And indeed, when an enzyme is assayed in vitro across a range of temperatures encompassing the normal physiological temperatures of the organism, it usually will show the expected exponential increase in reaction rate, at least until a ‘break point’ is reached and activity begins to decline due to loss of the protein’s native structure. However, when metabolic rates of species adapted to different temperatures are compared, the Arrhenius relationship does not hold; that is, a cold-adapted polar fish living at 0°C does not have a metabolic rate 20-times lower than that of a desert lizard living at 40°C. Thus, there must be a compensatory mechanism available, acting on an evolutionary time scale, that allows natural selection to alter rates of metabolic reactions as organisms adapt to new environments. Because most metabolic reactions are universally shared, physiologists recognized that it was likely the catalysts themselves – that is, the enzymes – that would be altered to allow appropriate metabolic rates at different physiological temperatures. It was not until the 1960s, however, that experimental evidence began to accrue showing that enzyme orthologs [that is, enzymes encoded by a common (homologous) gene from different species] indeed did display modified function correlating with the physiological temperatures the species experienced. These early data consisted mainly of temperatures of maximal enzyme activity and thermal denaturation temperatures (T m ) (e.g. Licht, 1964; Vroman and Brown, 1963; Read, 1964). However, work later in the decade began to show that more functionally relevant kinetic properties, most notably ligand affinity as measured by apparent Michaelis–Menten constants (K m ), also were modified through selection to environmental temperatures (e.g. Hochachka and Somero, 1968). In the time since these first studies correlating enzyme function and adaptation temperature, research on temperature adaptation in enzymes has accelerated and has begun to incorporate a fuller understanding of the protein structural changes that underlie shifts in enzyme function. In this review, we describe studies spanning the past four decades confirming that small differences in habitat temperature can lead to selection for physiologically significant changes in enzyme function. We also describe studies that have pinpointed the location and amount of amino acid change necessary 1 Biology Department, Franklin & Marshall College, Lancaster, PA 17603, USA. 2 State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen 361005, China. 3 Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China. 4 Hopkins Marine Station, Department of Biology, Stanford University, Pacific Grove, CA 93940, USA. *Author for correspondence ( peter.fields@fandm.edu) 1801 © 2015. Published by The Company of Biologists Ltd | The Journal of Experimental Biology (2015) 218, 1801-1811 doi:10.1242/jeb.114298 The Journal of Experimental Biology