Neal J. Dawson

Real-time protein unfolding: a method for determining the kinetics of native protein denaturation using a quantitative real-time thermocycler

Protein stability can be monitored by many different techniques. However, these protocols are often lengthy, consume large amounts of protein, and require expensive and specialized instruments. Here we present a new protocol to analyze protein unfolding kinetics using a quantified real-time thermocycler. This technique enables the analysis of a wide range of denaturants (and their interactions with temperature change) on protein stability in a multi-well platform, where samples can be run in parallel under virtually identical conditions and with highly sensitive detection. Using this set-up, researchers can evaluate the half-maximal rate of protein denaturation (K(nd)), maximum rate of denaturation (D(max)), and the cooperativity of individual denaturants in protein unfolding (µ-coefficient). Both lysozyme and hexokinase are used as model proteins and urea as a model denaturant to illustrate this new method and the kinetics of protein unfolding that it provides. Overall, this method allows the researcher to explore a large number of denaturants, at either constant or variable temperatures, within the same assay, providing estimates of denaturation kinetics that have been previously inaccessible.

Free-radical first responders: The characterization of CuZnSOD and MnSOD regulation during freezing of the freeze-tolerant North American wood frog, Rana sylvatica

BACKGROUND The North American wood frog, Rana sylvatica, is able to overcome subzero conditions through overwintering in a frozen state. Freezing imposes ischemic and oxidative stress on cells as a result of cessation of blood flow. Superoxide dismutases (SODs) catalyze the redox reaction involving the dismutation of superoxide (O(2)(-)) to molecular oxygen and hydrogen peroxide. METHODS The present study investigated the regulation of CuZnSOD and MnSOD kinetics as well as the transcript, protein and phosphorylation levels of purified enzyme from the muscle of control and frozen R. sylvatica. RESULTS CuZnSOD from frozen muscle showed a significantly higher V(max) (1.52 fold) in comparison to CuZnSOD from the muscle of control frogs. MnSOD from frozen muscle showed a significantly lower Km for O(2)(-) (0.66 fold) in comparison to CuZnSOD from control frogs. MnSOD from frozen frogs showed higher phosphorylation of serine (2.36 fold) and tyrosine (1.27 fold) residues in comparison to MnSOD from control animals. Susceptibility to digestion via thermolysin after incubation with increasing amount of urea (C(m)) was tested, resulting in no significant changes for CuZnSOD, whereas a significant change in MnSOD stability was observed between control (2.53 M urea) and frozen (2.92 M urea) frogs. Expressions of CuZnSOD and MnSOD were quantified at both mRNA and protein levels in frog muscle, but were not significantly different. CONCLUSION The physiological consequence of freeze-induced SOD modification appears to adjust SOD function in freezing frogs. GENERAL SIGNIFICANCE Augmented SOD activity may increase the ability of R. sylvatica to overcome oxidative stress associated with ischemia.

Purification and Properties of White Muscle Lactate Dehydrogenase from the Anoxia-Tolerant Turtle, the Red-Eared Slider, Trachemys scripta elegans

Lactate dehydrogenase (LDH; E.C. 1.1.1.27) is a crucial enzyme involved in energy metabolism in muscle, facilitating the production of ATP via glycolysis during oxygen deprivation by recycling NAD+. The present study investigated purified LDH from the muscle of 20 h anoxic and normoxic T. s. elegans, and LDH from anoxic muscle showed a significantly lower (47%) K m for L-lactate and a higher V max value than the normoxic form. Several lines of evidence indicated that LDH was converted to a low phosphate form under anoxia: (a) stimulation of endogenously present protein phosphatases decreased the K m of L-lactate of control LDH to anoxic levels, whereas (b) stimulation of kinases increased the K m of L-lactate of anoxic LDH to normoxic levels, and (c) dot blot analysis shows significantly less serine (78%) and threonine (58%) phosphorylation in anoxic muscle LDH as compared to normoxic LDH. The physiological consequence of anoxia-induced LDH dephosphorylation appears to be an increase in LDH activity to promote the reduction of pyruvate in muscle tissue, converting the glycolytic end product to lactate to maintain a prolonged glycolytic flux under energy-stressed anoxic conditions.

A hydrogen peroxide safety valve: The reversible phosphorylation of catalase from the freeze-tolerant North American wood frog, Rana sylvatica

BACKGROUND The North American wood frog, Rana sylvatica, endures whole body freezing while wintering on land and has developed multiple biochemical adaptations to elude cell/tissue damage and optimize its freeze tolerance. Blood flow is halted in the frozen state, imparting both ischemic and oxidative stress on cells. A potential build-up of H2O2 may occur due to increased superoxide dismutase activity previously discovered. The effect of freezing on catalase (CAT), which catalyzes the breakdown of H2O2 into molecular oxygen and water, was investigated as a result. METHODS The present study investigated the purification and kinetic profile of CAT in relation to the phosphorylation state of CAT from the skeletal muscle of control and frozen R. sylvatica. RESULTS Catalase from skeletal muscle of frozen wood frogs showed a significantly higher Vmax (1.48 fold) and significantly lower Km for H2O2 (0.64 fold) in comparison to CAT from control frogs (5°C acclimated). CAT from frozen frogs also showed higher overall phosphorylation (1.73 fold) and significantly higher levels of phosphoserine (1.60 fold) and phosphotyrosine (1.27 fold) compared to control animals. Phosphorylation via protein kinase A or the AMP-activated protein kinase significantly decreased the Km for H2O2 of CAT, whereas protein phosphatase 2B or 2C action significantly increased the Km. CONCLUSION The physiological consequence of freeze-induced CAT phosphorylation appears to improve CAT function to alleviate H2O2 build-up in freezing frogs. GENERAL SIGNIFICANCE Augmented CAT activity via reversible phosphorylation may increase the ability of R. sylvatica to overcome oxidative stress associated with ischemia.

Purification and characterization of a urea sensitive lactate dehydrogenase from the liver of the African clawed frog, Xenopus laevis

The African clawed frog, Xenopus laevis, is able to withstand extremely arid conditions by estivating, in conjunction with dehydration tolerance and urea accumulation. Estivating X. laevis reduce their metabolic rate and recruit anaerobic glycolysis, driven by lactate dehydrogenase (LDH; E.C. 1.1.1.27) enzymes that reversibly convert pyruvate and NADH to lactate and NAD+, to meet newly established ATP demands. The present study investigated purified LDH from the liver of dehydrated and control X. laevis. LDH from dehydrated liver showed a significantly higher Km for l-lactate (1.74 fold), NAD+ (2.41 fold), and pyruvate (1.78 fold) in comparison to LDH from the liver of control frogs. In the presence of physiological levels of urea found in dehydrated animals, the Km values obtained for dehydrated LDH all returned to control LDH Km values. Dot blot analysis showed post-translational modifications may be responsible for the kinetic modification as the dehydrated form of LDH showed more phosphorylated serine residues (1.54 fold), less methylated lysine residues (0.43 fold), and a higher level of ubiquitination (1.90 fold) in comparison to control LDH. The physiological consequence of dehydration-induced LDH modification appears to adjust LDH function in conjunction with urea levels in dehydrated frogs. When urea levels are high during dehydration, LDH retains its normal function. Yet, as urea levels drop during rehydration, LDH function is reduced, possibly shunting pyruvate to the TCA cycle.

Characterization of Fructose-1,6-Bisphosphate Aldolase during Anoxia in the Tolerant Turtle, Trachemys scripta elegans: An Assessment of Enzyme Activity, Expression and Structure

One of the most adaptive facultative anaerobes among vertebrates is the freshwater turtle, Trachemys scripta elegans. Upon a decrease in oxygen supply and oxidative phosphorylation, these turtles are able to reduce their metabolic rate and recruit anaerobic glycolysis to meet newly established ATP demands. Within the glycolytic pathway, aldolase enzymes cleave fructose-1,6-bisphosphate to triose phosphates facilitating an increase in anaerobic production of ATP. Importantly, this enzyme exists primarily as tissue-specific homotetramers of aldolase A, B or C located in skeletal muscle, liver and brain tissue, respectively. The present study characterizes aldolase activity and structure in the liver tissue of a turtle whose survival greatly depends on increased glycolytic output during anoxia. Immunoblot and mass spectrometry analysis verified the presence of both aldolase A and B in turtle liver tissue, and results from co-immunoprecipitation experiments suggested that in the turtle aldolase proteins may exist as an uncommon heterotetramer. Expression levels of aldolase A protein increased significantly in liver tissue to 1.59±0.11-fold after 20 h anoxia, when compared to normoxic control values (P<0.05). A similar increase was seen for aldolase B expression. The overall kinetic properties of aldolase, when using fructose-1,6-bisphosphate as substrate, were similar to that of a previously studied aldolase A and aldolase B heterotetramer, with a Km of 240 and 180 nM (for normoxic and anoxic turtle liver, respectively). Ligand docking of fructose-1,6-bisphosphate to the active site of aldolase A and B demonstrated minor differences in both protein:ligand interactions compared to rabbit models. It is likely that the turtle is unique in its ability to regulate a heterotetramer of aldolase A and B, with a higher overall enzymatic activity, to achieve greater rates of glycolytic output and support anoxia survival.

Stable Suppression of Lactate Dehydrogenase Activity during Anoxia in the Foot Muscle of Littorina littorea and the Potential Role of Acetylation as a Novel Posttranslational Regulatory Mechanism

The intertidal marine snail, Littorina littorea, has evolved to withstand extended bouts of oxygen deprivation brought about by changing tides or other potentially harmful environmental conditions. Survival is dependent on a strong suppression of its metabolic rate and a drastic reorganization of its cellular biochemistry in order to maintain energy balance under fixed fuel reserves. Lactate dehydrogenase (LDH) is a crucial enzyme of anaerobic metabolism as it is typically responsible for the regeneration of NAD+, which allows for the continued functioning of glycolysis in the absence of oxygen. This study compared the kinetic and structural characteristics of the D-lactate specific LDH (E.C. 1.1.1.28) from foot muscle of aerobic control versus 24 h anoxia-exposed L. littorea. Anoxic LDH displayed a near 50% decrease in V max (pyruvate-reducing direction) as compared to control LDH. These kinetic differences suggest that there may be a stable modification and regulation of LDH during anoxia, and indeed, subsequent dot-blot analyses identified anoxic LDH as being significantly less acetylated than the corresponding control enzyme. Therefore, acetylation may be the regulatory mechanism that is responsible for the suppression of LDH activity during anoxia, which could allow for the production of alternative glycolytic end products that in turn would increase the ATP yield under fixed fuel reserves.

Native denaturation differential scanning fluorimetry: Determining the effect of urea using a quantitative real-time thermocycler

The effect of protein stability on kinetic function is monitored with many techniques that often require large amounts of expensive substrates and specialized equipment not universally available. We present differential scanning fluorimetry (DSF), a simple high-throughput assay performed in real-time thermocyclers, as a technique for analysis of protein unfolding. Furthermore, we demonstrate a correlation between the half-maximal rate of protein unfolding (Knd), and protein unfolding by urea (I50). This demonstrates that DSF methods can determine the structural stability of an enzymes active site and can compare the relative structural stability of homologous enzymes with a high degree of sequence similarity.

Regulation of tail muscle hexokinase in the anoxia-tolerant freshwater crayfish, Orconectes virilis

Abstract Hexokinase (HK)(E.C.2.7.1.1) is the enzyme responsible for catalyzing the first step of glucose metabolism, the phosphorylation of glucose to glucose-6-phosphate (G6P). The present study investigated HK from

Insights into the In Vivo Regulation of Glutamate Dehydrogenase from the Foot Muscle of an Estivating Land Snail

Land snails, Otala lactea, survive in seasonally hot and dry environments by entering a state of aerobic torpor called estivation. During estivation, snails must prevent excessive dehydration and reorganize metabolic fuel use so as to endure prolonged periods without food. Glutamate dehydrogenase (GDH) was hypothesized to play a key role during estivation as it shuttles amino acid carbon skeletons into the Krebs cycle for energy production and is very important to urea biosynthesis (a key molecule used for water retention). Analysis of purified foot muscle GDH from control and estivating conditions revealed that estivated GDH was approximately 3-fold more active in catalyzing glutamate deamination as compared to control. This kinetic difference appears to be regulated by reversible protein phosphorylation, as indicated by ProQ Diamond phosphoprotein staining and incubations that stimulate endogenous protein kinases and phosphatases. The increased activity of the high-phosphate form of GDH seen in the estivating land snail foot muscle correlates well with the increased use of amino acids for energy and increased synthesis of urea for water retention during prolonged estivation.