Effect of temperature on intramolecular dynamics and conformational state of bacterial alkaline phosphatase

The slow internal dynamics and the conformational state of Escherichia coli alkaline phosphatase by the action of temperature in the range 0-100 degrees C have been investigated by tryptophan room temperature phosphorescence and fluorescence. It has been shown that heating an alkaline phosphatase solution in the interval 0-70 degrees C leads to a substantial increase in the slow internal dynamics. A further increase in temperature to 95 degrees C causes a reversible enhancement of internal dynamics and a partial unfolding of the globule. Heating the protein solution in a narrow temperature range 97-100 degrees C induces an irreversible conformational transition, which is characterized by total unfolding of the globule, a drastic increase in internal dynamics, and the loss of enzymatic activity.     

Catalytically active alkaline molten globular enzyme: Effect of pH and temperature on the structural integrity of 5-aminolevulinate synthase

5-Aminolevulinate synthase (ALAS), a pyridoxal-5′phosphate (PLP)-dependent enzyme, catalyzes the first step of heme biosynthesis in mammals. Circular dichroism (CD) and fluorescence spectroscopies were used to examine the effects of pH (1.0–3.0 and 7.5–10.5) and temperature (20 and 37 °C) on the structural integrity of ALAS. The secondary structure, as deduced from far-UV CD, is mostly resilient to pH and temperature changes. Partial unfolding was observed at pH 2.0, but further decreasing pH resulted in acid-induced refolding of the secondary structure to nearly native levels. The tertiary structure rigidity, monitored by near-UV CD, is lost under acidic and specific alkaline conditions (pH 10.5 and pH 9.5/37 °C), where ALAS populates a molten globule state. As the enzyme becomes less structured with increased alkalinity, the chiral environment of the internal aldimine is also modified, with a shift from a 420 nm to 330 nm dichroic band. Under acidic conditions, the PLP cofactor dissociates from ALAS. Reaction with 8-anilino-1-naphthalenesulfonic acid corroborates increased exposure of hydrophobic clusters in the alkaline and acidic molten globules, although the reaction is more pronounced with the latter. Furthermore, quenching the intrinsic fluorescence of ALAS with acrylamide at pH 1.0 and 9.5 yielded subtly different dynamic quenching constants. The alkaline molten globule state of ALAS is catalytically active (pH 9.5/37 °C), although the k_cat value is significantly decreased. Finally, the binding of 5-aminolevulinate restricts conformational fluctuations in the alkaline molten globule. Overall, our findings prove how the structural plasticity of ALAS contributes to reaching a functional enzyme.     

Probing the Impact of Temperature and Substrates on the Conformational Dynamics of the Neurotransmitter:Sodium symporter LeuT

The crucial function of neurotransmitter:sodium symporters (NSS) in facilitating the reuptake of neurotransmitters into neuronal cells makes them attractive drug targets for treating multiple mental diseases. Due to the challenges in working with eukaryotic NSS proteins, LeuT, a prokaryotic amino acid transporter, has served as a model protein for studying structure–function relationships of NSS family proteins. With hydrogen–deuterium exchange mass spectrometry (HDX-MS), slow unfolding/refolding kinetics were identified in multiple regions of LeuT, suggesting that substrate translocation involves cooperative fluctuations of helical stretches. Earlier work has solely been performed at non-native temperatures (25 °C) for LeuT, which is evolutionarily adapted to function at high temperatures (85 – 95 °C). To address the effect of temperature on LeuT dynamics, we have performed HDX-MS experiments at elevated temperatures (45 °C and 60 °C). At these elevated temperatures, multiple regions in LeuT exhibited increased dynamics compared to 25 °C. Interestingly, coordinated slow unfolding/refolding of key regions could still be observed, though considerably faster. We have further investigated the conformational impact of binding the efficiently transported substrate alanine (Ala) relative to the much slower transported substrate leucine (Leu). Comparing the HDX of the Ala-bound versus Leu-bound state of LeuT, we observe distinct differences that could explain the faster transport rate (k_cat) of Ala relative to Leu. Importantly, slow unfolding/refolding dynamics could still be observed in regions of Ala-bound LeuT . Overall, our work brings new insights into the conformational dynamics of LeuT and provides a better understanding of the transport mechanism of LeuT and possibly other transporters bearing the LeuT fold.     

Luminescence analysis of the structure of human alpha-1-microglobulin

Absorption, fluorescence, and fluorescence excitation spectra in UV and visible regions are studied for alpha-1-microglobulin preparations isolated from human urine by gel chromatography and immunoaffinity chromatography with charcoal adsorption. The possible nature of low-molecular-weight compounds that impart yellow-brown color to alpha-1-microglobulin preparations and their role in the stabilization of the structure of protein globule is discussed. The effect of urea (1–10 M) and guanidine hydrochloride (0.25–6 M) on the conformational state and fast internal dynamics of alpha-1-microglobulin is studied by tryptophan fluorescence. The unfolding of the protein under the action of denaturants is attended with pronounced activation of its nanosecond internal dynamics. Alpha-1-microglobulin can regain the initial conformation and internal dynamics typical of native protein after denaturation unfolding of the globule with 10 M urea or 6 M guanidine hydrochloride. Alpha-1-microglobulin isolated by gel chromatography can exist in a partially folded thermodynamically stable state in 4–6 M urea.     

Population dynamics of <Emphasis Type="Italic">Daphnia longispina</Emphasis> (O.F. Müller, 1785) and <Emphasis Type="Italic">Diaphanosoma brachyurum</Emphasis> (Lievin, 1848) (Crustacea,Cladocera) under stable and graded temperature regimes

Laboratory experiments have been performed to study the effects of different temperature regimes—constant (16.3±0.8, 20.3±0.8, and 24.6±0.7°C) and graded, with temperatures changing at intervals of 4.4 or 8.8°C within the range of 15.1–25.4°C—on the population dynamics of two dominant species of littoral zooplankton, Daphnia longispina and Diaphanosoma brachyurum. The results show that aperiodic stepwise changes in temperature within the ranges of 16.3–20.4, 20.4–25.1, and 16.3–25.1°C, which may take place in water bodies of the temperate zone in the summer period, can exert either stimulating (direct or delayed) or inhibitory effect on the natural development of D. longispina populations. The development of D. brachyurum, a more stenothermic thermophile, is stimulated only by a stable elevated temperature (24.5±0.9°C). All temperature changes within the above range, except for rapid heating from 16.3 to 25.1°C, either inhibit the development of D. brachyurum populations or have no significant effect on them.     

Selective Extraction of Alkaline Phosphatase and 51 -Nucleotidase from Milk Fat Globule Membranes by a Single Phase n-Butanol Procedure

ABSTRACT

A single phase extraction procedure employing 8% (v/v) n-butanol at room temperature extracted over 90% of alkaline phosphatase activity and over 60% of 5'-nucleotidase activity from bovine milk fat globule membranes (MFGM). For 5'-nucleotidase, higher n-butanol concentrations lead to loss of activity, while lower concentrations were ineffective in extracting the enzyme. When extractions were performed at 0°C, similar yields were obtained for alkaline phosphatase extraction with 8% (v/v) n-butanol, but 5¹- nucleotidase extraction required 10% (v/v) n-butanol for similar yields. However, 5'-nucleotidase was less susceptible to denaturation during extraction at 0°C. The Km values and substrate specificities for both alkaline phosphatase and 5'-nucleotidase were unchanged by extraction with 8% (v/v) n-butanol. The 8% (v/v) n-butanol extraction procedure provides a 3-fold purification step, and an enzyme preparation suitable for further purification.     

Raman spectroscopy of the milk globule membrane and triglycerides

The acyl chain mobilities of the lipids of bovine milk fat globules and the component triglycerides have been determined by Raman spectroscopy as a function of temperature from -100°C to 80°C. A broad transition is observed centered about 2–6°C. The C-H and C-C stretching bands in the spectra of liquid and crystalline triglycerides are used comparatively to show that the lipids of the milk globule membrane are 30–40% more ordered than the lipids of the intact milk fat globules at 20°C. Synthetic triglyceride melts, quenched rapidly, are used to illustrate the effect of the thermal history of a sample on lipid order as determined spectroscopically.Strong infrared amide I and amide II bands at 1646 and 1543 cm^?1, respectively, indicate that the protein conformation of the globule membrane is not characterized by extensive regions of beta-sheet structure. Raman spectra of the globule triglycerides indicate cis unsaturation of $\text{39 ± 5\%}$ by comparison to triolein and trielaidin.     

Pressure effects on thermal preference behaviour in gammarid amphipods from 600–1000m in Lake Baikal

Temperature preference behaviour of gammarid crustaceans from depths between 600–2000 m in Lake Baikal was studied in a system which provided a stable temperature gradient at pressures ranging from 50–150 atm. At the pressure of their habitat, these animals show well-defined modal distributions of sojourn temperatures around mean values from 3.0–5.5°C, av. 3.9 ± 0.3°C; mean modal T_p is estimated at 3.5°C. Year-round habitat temperatures are 3.0–3.6°C. The effect of changing pressure upon sojourn temperatures was explored over the range 50–150 atm. The slope of the mean sojourn temperature/pressure curves was 2.1°C/100 atm, significantly greater than 0. Mean nodal temperature estimates indicate that the corresponding slope in the range of 50–100 atm is 3°C/100 atm, and in the range of 100–150 atm, is likely to exceed 5°C/100 atm.     

Existence of acid and alkaline phosphohydrolase activity in the phytoflagellate Ochromonas danica

Cell homogenates of light-grown Ochromonas danica contained distinct non-specific non-phosphate-repressible acid and alkaline phosphohydrolase activities. Acid phosphohydrolase activity had a broad pH range of 2.0–5.0 and the optimum for alkaline phosphohydrolase activity was pH 8.6 Acid phosphohydrolase (pH 3.6) activity had an optimum temperature of 55°C; the alkaline enzyme activity had an optimum temperature of 37–40°C.     

Purification and characterization of an extracellular chitosanase produced by Amycolatopsis sp. CsO-2

Extracellular chitosanase produced by Amycolatopsis sp. CsO-2 was purified to homogeneity by precipitation with ammonium sulfate followed by cation exchange chromatography. The molecular weight of the chitosanase was estimated to be about 27,000 using SDS-polyacrylamide gel electrophoresis and gel filtration. The maximum velocity of chitosan degradation by the enzyme was attained at 55°C when the pH was maintained at 5.3. The enzyme was stable over a temperature range of 0–50°C and a pH range of 4.5–6.0. About 50% of the initial activity remained after heating at 100°C for 10 min, indicating a thermostable nature of the enzyme. The isoelectric point of the enzyme was about 8.8. The enzyme degraded chitosan with a range of deacetylation degree from 70% to 100%, but not chitin or CM-cellulose. The most susceptible substrate was 100% deacetylated chitosan. The enzyme degraded glucosamine tetramer to dimer, and pentamer to dimer and trimer, but did not hydrolyze glucosamine dimer and trimer.     

Pressure perturbation calorimetry of apolipoproteins in solution and in model lipoproteins

High‐density lipoproteins (HDLs) are complexes of lipids and proteins (termed apolipoproteins) that remove cell cholesterol and protect from atherosclerosis. Apolipoproteins contain amphipathic α‐helices that have high content (≥1/3) and distinct distribution of charged and apolar residues, adopt molten globule‐like conformations in solution, and bind to lipid surfaces. We report the first pressure perturbation calorimetry (PPC) study of apolipoproteins. In solution, the main HDL protein, apoA‐I, shows relatively large volume contraction, ΔV_unf = ?0.33%, and an apparent reduction in thermal expansivity upon unfolding, Δα_unf ≤ 0, which has not been observed in other proteins. We propose that these values are dominated by increased charged residue hydration upon α‐helical unfolding, which may result from disruption of multiple salt bridges. At 5°C, apoA‐I shows large thermal expansion coefficient, α(5°) = 15·10^?4 K^?1, that rapidly declines upon heating from 5 to 40°C, α(40°) ? α(5°) = ?4·10^?4 K^?1; apolipoprotein C‐I shows similar values of α(5°) and α(40°). These values are larger than in globular proteins. They indicate dominant effect of charged residue hydration, which may modulate functional apolipoprotein interactions with a broad range of their protein and lipid ligands. The first PPC analysis of a protein–lipid complex is reported, which focuses on the chain melting transition in model HDL containing apoA‐I or apoC‐I, dimyristoyl phosphatidylcholine, and 0–20% cholesterol. The results may provide new insights into volumetric properties of HDL that modulate metabolic lipoprotein remodeling during cholesterol transport. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

On the possibility for a protein to exist in a manifold of partially folded states

With the help of the methods of tryptophan fluorescence and room-temperature phosphorescence and using Escherichia coli alkaline phosphatase as an example, the ability of a protein to exist in a manifold of partially folded thermodynamically stable states differing in conformation, the internal dynamics, and functional activity was shown. Such intermediate (between native and unfolded) structures may form during unfolding or folding of the protein. It was shown that the degree of destruction of the native structural organization of the globule depends on both the nature and the mode of action of the destroying agent and the structure of the protein. Conformational transitions of the globule can change the kind of the internal dynamics (fast, slow), and shifts of dynamics can initiate conformation changes of the protein and precede them. A scheme of the structural and functional transformations of the protein during denaturation is presented, which takes into account the possibility of globule transitions into a manifold of functional active and inactive partially folded states. The role of partially folded forms of cell proteins in the development of pathology is discussed.     

Adaptive responses of embryos and larvae of the heart-shaped sea urchin <Emphasis Type="Italic">Echinocardium cordatum</Emphasis> to temperature and salinity changes

The combined effects of temperature (8, 12, 14, 17, 20, 22 and 25°C) and a salinity decrease from 36 to 12‰ on the development of the sea urchin Echinocardium cordatum (Pennant) were studied. Embryonic development proved to be the process most vulnerable to a salinity decrease. It was completed successfully at 8–20°C within a narrow salinity range of 36–28‰ Larvae at the most resistant stage, the blastula, survived at 12–22°C and a salinity of 36–18‰. Larvae at the most sensitive stage, pluteus I with the first pair of arms, died even in a favorable environment, a temperature of 17–20°C and a salinity of 34–28‰. That may be related to qualitative alterations during skeleton formation and to transition to phytoplankton feeding. The resistance of larvae to variations in environmental factors gradually increased in the pluteus II and III stages; however, it significantly decreased before the settling of the larvae. Larvae that were 37 days old survived at a temperature of 14–20°C within a salinity range of 36–22‰ and at 22 and 25°C, they survived at a salinity of 36–24‰; however, all the larvae became abnormal at 25°C. The larvae settled earlier on sand inhabited by adult individuals of E. cordatum than on sand from other locations, and they settled faster at 20–25°C, than at 14 and 17°C. The juveniles, if lacking an opportunity to burrow in the sand, died within 14 days after settling.     

Kinetics of conformational changes in tRNAPhe (yeast) as studied by the fluorescence of the y-base and of formycin substituted for the 3'-terminal adenine

The kinetics of the melting transitions of tRNA^phe (yeast) were followed by the fluorescence of the Y-base and of formycin substituted for the 3'-terminal adenine. As judged from differential UV absorbance melting cutves the formycin label had virtually no influence on the conformation of the tRNA. A temperature jump apparatus was modified to allow the simultaneous observation of transmission and fluorescence intensities by two independent optical channels. The design of a temperature jump cell with an all quartz center piece is given. The cell is resistant to temperatures up to 90°C; it provides high optical sensitivity, low stray light intensity and the possibility of measuring fluorescence polarization. The T-jump experiments allowed to discriminate between fast unspecific fluorescence quenching (τ <5 μsec) and slow co-operative conformational changes. In the central part of the temperature range of UV-melung (midpoint temperature 30°C in 0.01 M Na⁺ and 39°C in 0.03 M Na⁺, pH 6.8) two resolvable relaxation processes were observed. The coirssponding relaxation times were 20 msec and 800 msec at 30°C in 0.01 M Na⁺, and 4 msec and 120 msec at 39°C in 0.03 M Na⁺. The Y-base fluorescence shows both of the relaxation effects, which almost cancel in equilibrium fluorescence melting, because their amplitudes have opposite signs. From this finding the existence of some residual tertiary structure is inferred which persists after the unfolding of the main part of tertiary structure durirg early melting (midpoint temperature 24°C in 0.03 M Na⁺). In the fluorescence sigXXX of the formycin also the two relaxation effects appear. Both of them are connected with a decrease of the fluorescence intensity. From the results a coupled opening of the anticodon and acceptor branches is concluded.Enzymes: phenylalanyl-tRNA synthetase, PRS (EC 6.1.1.-20); ATP (CTP) tRNA nucleotidyl transferase, NT (EC 2.7.7.-20); alkaline phosphatase (EC 3-1-3.1).     

Enzyme activities in the serum of rainbow trout,Salmo gairdneri Richardson; the effects of water temperature*

Activities of lactate dehydrogenase, hydroxy butyric dehydrogenase, glutamic oxalacetic transaminase, glutamic pyruvic transaminase, glutamate dehydrogenase, creatinine kinase, alkaline phosphatase, and leucine amino peptidase were determined in the sera of rainbow trout. The animals had previously been adapted to temperatures of 3.5, 6, 8, 10, 12.5, 15, 17, 19, 21.5 and 23° C. Most of the enzyme activity increased with the rise in temperature. The activity of alkaline phosphatase decreased in the range 6–19° C, while the changes in the glutamate dehydrogenase activity took a complex course. The results are compared with the findings of other authors.     

Responses of the sand dollar <Emphasis Type="Italic">Scaphechinus mirabilis</Emphasis> to extreme environmental changes

The survival ability of the adult sand dollar Scaphechinus mirabilis (Agassiz) in varying environments was studied. In an experiment on a hard substrate, 88% of the animals survived for 40 days (August–September) during variation of sea water temperature from 21.0 to 16.5°C and variation of salinity from 33.3 to 31.5‰. At 17°C, the salinity tolerance range was 34–24‰. At the same temperature. 100% of the animals remained alive for 14 days within a salinity range from 34 to 18‰; at 16 and 14‰ the ratio of surviving sea urchins was 30 and 20% respectively. Thus, S. mirabilis has considerable adaptive capabilities and is able to survive for a long time under extreme environmental changes.     

Further Studies on Factors Affecting the Efflux of Betacyanin from Beet Root: A Note on Thermal Effects

As judged by betacyanin efflux, beet root tissue differs in stability toward O₂ at low and high temperatures (45–60°C and 60–100°C respectively). The effect of temperature itself can he divided into a high activation energy (93 Kcal mol^?1) process in the lower temperature range and a low activation energy process (19 Kcal mor^?1) in the higher range (> 60°C). From these data it is suggested that initially, elevating temperatures bring about reversible conformational changes in the membrane. With continuing increase in temperature in the presence of O₂, membrane chemical groups susceptible to oxidation are exposed and, upon oxidation, render conformational changes irreversible.     

Understanding the relevance of local conformational stability and dynamics to the aggregation propensity of an IgG1 and IgG2 monoclonal antibodies

Aggregation of monoclonal antibodies is often a multi‐step process involving structural alterations in monomeric proteins and subsequent formation of soluble or insoluble oligomers. The role of local conformational stability and dynamics of native and/or partially altered structures in determining the aggregation propensity of monoclonal antibodies, however, is not well understood. Here, we investigate the role of conformational stability and dynamics of regions with distinct solvent exposure in determining the aggregation propensity of an IgG1 and IgG2 monoclonal antibody. The temperatures employed span the pre‐unfolding range (10–40°C) and the onset temperatures (T_onset) for exposure of apolar residues (～50°C), alterations in secondary structures (～60°C) and initiation of visible aggregate formation (～60°C). Solvent‐exposed regions were found to precede solvent‐shielded regions in an initiation of aggregation for both proteins. Such a process was observed upon alterations in overall tertiary structure while retaining the secondary structures in both the proteins. In addition, a greater dynamic nature of solvent‐shielded regions in potential intermediates of IgG1 and the improved conformational stability increased its resistance to aggregation when compared to IgG2. These results suggest that local conformational stability and fluctuations of partially altered structures can influence the aggregation propensity of immunoglobulins.     

Luminescent analysis of the structure of human alpha-1-microglobulin

The spectra of absorption, fluorescence, and excitation of fluorescence of preparations of alpha-1-microglobulin isolated from human urea by two methods, gel chromatography and immunoaffinity chromatography with additional purification by activated charcoal, have been investigated in ultraviolet and visible regions. A possible nature of low-molecular compounds coloring alpha-1-microglobulin yellow-brown and their role in stabilizing the structure of protein globule are discussed. The action of urea (1.0-10 M) and guanidine hydrochloride (0.25-6 M) on the conformational state and the fast (nanosecond) internal dynamics of alpha-1-microglobulin has been investigated by the method of tryptophan fluorescence. It has been shown that the unfolding of alpha-1-microglobulin under the action of these denaturants is associated with a significant increase in the nanosecond internal dynamics of protein. The ability of alpha-1-microglobulin to restore the initial conformation characteristic for the native protein and the internal dynamics after the unfolding of the globule by 10 M urea and 6 M guanidine hydrochloride has been ascertained. It has been found that alpha-1-microglobulin isolated by the method of gel chromatography can exist in solution of 4-6 M urea in a thermodynamically stabile partialy folded state.     

A measure of conformational entropy change during thermal protein unfolding using neutron spectroscopy

Thermal unfolding of proteins at high temperatures is caused by a strong increase of the entropy change which lowers Gibbs free energy change of the unfolding transition (DeltaG(unf) = DeltaH - TDeltaS). The main contributions to entropy are the conformational entropy of the polypeptide chain itself and ordering of water molecules around hydrophobic side chains of the protein. To elucidate the role of conformational entropy upon thermal unfolding in more detail, conformational dynamics in the time regime of picoseconds was investigated with neutron spectroscopy. Confined internal structural fluctuations were analyzed for alpha-amylase in the folded and the unfolded state as a function of temperature. A strong difference in structural fluctuations between the folded and the unfolded state was observed at 30 degrees C, which increased even more with rising temperatures. A simple analytical model was used to quantify the differences of the conformational space explored by the observed protein dynamics for the folded and unfolded state. Conformational entropy changes, calculated on the basis of the applied model, show a significant increase upon heating. In contrast to indirect estimates, which proposed a temperature independent conformational entropy change, the measurements presented here, demonstrated that the conformational entropy change increases with rising temperature and therefore contributes to thermal unfolding.