Ligand binding to heme proteins: relevance of low-temperature data

Binding of carbon monoxide to the beta chain of adult human hemoglobin has been studied by flash photolysis over the time range from about 100 ps to seconds and the temperature range from 40 to 300 K. Below about 180 K, binding occurs directly from the pocket (process I) and is nonexponential in time. Above about 180 K, some carbon monoxide molecules escape from the pocket into the protein matrix. Above about 240 K, escape into the solvent becomes measurable. Process I can be observed up to 300 K. The low-temperature data extrapolate smoothly to 300 K, proving that the results obtained below 180 K provide functionally relevant information. The experiments show again that the binding process even at physiological temperatures is regulated by the final binding step at the heme iron and that measurements at high temperatures are not sufficient to fully understand the association process.     

Insights into the binding of ferulic acid to the thermally treated xanthine oxidase

Ferulic acid (FA) is a biologically active compound used as an additive in the food industry, and possesses a wide range of therapeutic effects for treating different health problems. The interaction between FA and bovine xanthine oxidase (XOD) has been investigated by means of fluorescence spectroscopy methods. The numbers of binding sites and the binding constants were estimated at various temperatures and the results indicated the existence of one specific FA binding site of XOD. Detailed information on the interaction between molecules gathered after performing in silico molecular docking indicated the accommodation of the FA molecule in a XOD binding pocket, in close vicinity to the active site residues. The formation of the XOD–FA complex causes the quenching of protein fluorescence. The process followed a static mechanism at lower temperatures, and a dynamic mechanism at higher temperatures. The thermodynamic parameters calculated on the basis of different temperatures revealed that the association between FA and XOD is a spontaneous process driven by enthalpy and dominated by hydrogen bonding and van der Waals interaction. The results of synchronous fluorescence and 3D fluorescence spectra showed that the conformation of protein was altered in the presence of FA. Copyright © 2016 John Wiley & Sons, Ltd.     

Reactive line-shape narrowing in low-temperature inhomogeneous geminate recombination of CO to myoglobin

The temporal shift in the near-IR absorption peak of myoglobin (Mb) following flash photolysis of MbCO at cryogenic temperatures appears to be due largely to an inhomogeneous reactive process rather than to relaxation. This conclusion, which follows from a new analysis of the experimental data, is based on the following three points: First, at very low temperatures (60 K) a transient line-narrowing effect can be detected. Second, there is a universal, temperature-independent, correlation between spectral shift and survival probability in the rebinding kinetics, and third, the same quantitative model which accounts for rebinding accounts semiquantitatively for the temporal shift in the peak. A fit to the model indicates that the inhomogeneous broadening of the near-IR peak in myoglobin is 15-20% of the total width. The same rebinding process which governs the loss of intensity of this peak is therefore most likely responsible for the shift in its center wavelength.     

Molecular motions in chitosan studied by dielectric relaxation spectroscopy

Neutralized and nonneutralized chitosan films subject to different thermal treatments were studied by dielectric relaxation spectroscopy from -130 to +150 degrees C in the frequency range between 20 Hz and 1 MHz. Two main relaxation processes, both arrhenian type, were detected: process I at temperatures below 0 degrees C with a mean activation energy of 49 +/- 1 kJ mol(-1), which has the characteristics of a secondary relaxation process related with local chain dynamics, and process II observable at higher temperatures with an activation energy of 94 +/- 2 kJ mol(-1), correlated with dc conductivity, which is found in dried polysaccharides systems. Process I is always observed in neutralized chitosan, but it is strongly depleted in the wet nonneutralized form. Although the location of process I is independent of NH2/NH3+ side group, process II deviates to higher temperatures with dryness in both chitosan forms, being located at lower temperatures in nonneutralized chitosan.     

RNA Silencing Is Resistant to Low-Temperature in Grapevine

RNA silencing is a natural defence mechanism against viruses in plants, and transgenes expressing viral RNA-derived sequences were previously shown to confer silencing-based enhanced resistance against the cognate virus in several species. However, RNA silencing was shown to dysfunction at low temperatures in several species, questioning the relevance of this strategy in perennial plants such as grapevines, which are often exposed to low temperatures during the winter season. Here, we show that inverted-repeat (IR) constructs trigger a highly efficient silencing reaction in all somatic tissues in grapevines. Similarly to other plant species, IR-derived siRNAs trigger production of secondary transitive siRNAs. However, and in sharp contrast to other species tested to date where RNA silencing is hindered at low temperature, this process remained active in grapevine cultivated at 4°C. Consistently, siRNA levels remained steady in grapevines cultivated between 26°C and 4°C, whereas they are severely decreased in Arabidopsis grown at 15°C and almost undetectable at 4°C. Altogether, these results demonstrate that RNA silencing operates in grapevine in a conserved manner but is resistant to far lower temperatures than ever described in other species.     

Timing of diapause termination in relation to variation in winter climate

In temperate insects, winters are typically endured by entering diapause, which comprises a deep resting stage. Correct timing of diapause termination is vital for synchronization of emergence with conspecifics and for mobilizing resources when conditions for growth and reproduction become favourable. Although critical to survival, the intrinsic and extrinsic drivers of diapause termination timing are poorly understood. In the present study, we investigate diapause development under a range of durations (10–24 weeks) spent at different temperatures (?2 to 10 °C) in the pupal diapausing butterfly Pieris napi Linnaeus (Lepidoptera:Pieridae). We determine: (i) the maximum cold temperature for diapause development; (ii) if pupae in diapause count cold days or cold sums; and (iii) whether diapause termination is distinct or gradual. The results indicate large and idiosyncratic effects of high and low nonlethal temperatures on diapause development in P. napi. Although all temperatures tested lead to diapause termination, a thermal optimum between 2 and 4 °C is observed. Lower temperatures lead to decreased eclosion propensity, whereas higher temperatures slow down development and increase emergence desynchronization. These data suggest that, rather than a simple cold‐summing process with a distinct diapause termination point, there are trade‐offs between time and temperature at the low and high end of the thermal range, resulting in a nonlinear thermal landscape showing a ridge of increasing eclosion propensity at moderate temperatures. The present study suggests that the effects of temperature on diapause development should be included in projections on post‐winter phenology models of insects, including pest species.     

Heat and acid tolerance of Clostridium novyi Type A spores and their survival prior to preparation of heroin for injection

Clostridium novyi Type A was implicated as a cause of an outbreak of serious illness and deaths among drug users in the United Kingdom who injected heroin intramuscularly. A contaminated batch of heroin was believed to be the source of infection. To test the ability of the outbreak strain to survive certain processes associated with heroin use, it was tested for its ability to survive a range of temperature and pH and the process used in preparation of "street" heroin for injection. C. novyi spores survived temperatures of up to 100 degrees C in aqueous solution for 5 min and survived pH 2.0 at ambient temperatures for a similar time. However, a combination of low pH and raised temperatures reduced survival times. An experiment reconstructing the "street" preparation of heroin demonstrated that any C. novyi spores present would survive this process and thus be capable of initiating infection under the right conditions.     

Fast reactions in carbon monoxide binding to heme proteins.

Using fast flash photolysis, we have measured the binding of CO to carboxymethylated cytochrome c and to heme c octapeptide as a function of temperature (5 degrees-350 degreesK) over an extended time range (100 ns(-1) ks). Experiments used a microsecond dye laser (lambda = 540 nm), and a mode-locked frequency-doubled Nd-glass laser (lambda = 530 nm). At low temperatures (5 degrees-120 degreesK) the rebinding exhibits two components. The slower component (I) is nonexponential in time and has an optical spectrum corresponding to rebiding from an S = 2, CO-free deoxy state. The fast component (I*) is exponential in time with a lifetime shorter than 10 mus and an optical spectrum different from the slow component. In myoglobin and the separated alpha and beta chains of hemoglobin, only process I is visible. The optical absorption spectrum of I* and its time dependence suggest that it may correspond to recombination from an excited state in which the iron has not yet moved out of the heme plane. The temperature dependences of both processes have been measured. Both occur via quantum mechanical tunneling at the lowest temperatures and via over-the-barrier motion at higher temperatures.     

Calorimetric and structural studies of the nitric oxide carrier S-nitrosoglutathione bound to human glutathione transferase P1-1

The nitric oxide molecule (NO) is involved in many important physiological processes and seems to be stabilized by reduced thiol species, such as S-nitrosoglutathione (GSNO). GSNO binds strongly to glutathione transferases, a major superfamily of detoxifying enzymes. We have determined the crystal structure of GSNO bound to dimeric human glutathione transferase P1-1 (hGSTP1-1) at 1.4 A resolution. The GSNO ligand binds in the active site with the nitrosyl moiety involved in multiple interactions with the protein. Isothermal titration calorimetry and differential scanning calorimetry (DSC) have been used to characterize the interaction of GSNO with the enzyme. The binding of GSNO to wild-type hGSTP1-1 induces a negative cooperativity with a kinetic process concomitant to the binding process occurring at more physiological temperatures. GSNO inhibits wild-type enzyme competitively at lower temperatures but covalently at higher temperatures, presumably by S-nitrosylation of a sulfhydryl group. The C47S mutation removes the covalent modification potential of the enzyme by GSNO. These results are consistent with a model in which the flexible helix alpha2 of hGST P1-1 must move sufficiently to allow chemical modification of Cys47. In contrast to wild-type enzyme, the C47S mutation induces a positive cooperativity toward GSNO binding. The DSC results show that the thermal stability of the mutant is slightly higher than wild type, consistent with helix alpha2 forming new interactions with the other subunit. All these results suggest that Cys47 plays a key role in intersubunit cooperativity and that under certain pathological conditions S-nitrosylation of Cys47 by GSNO is a likely physiological scenario.     

A lipid-phase separation model of low-temperature damage to biological membranes

An hypothesis is proposed to explain the damage caused to biological membranes exposed to low temperatures. The thesis rests on the general observation that the lipid components of most membranes are heterogeneous and undergo phase transitions from gel-phase lamellae to liquid-crystalline lamellae and some to a non-lamellar, hexagonal-II phase over a wide range of temperatures. As a consequence of these phase transitions the lateral distribution of the lipids characteristic of the growth temperature is disturbed and redistribution takes place on the basis of the temperature at which phase transitions occur. When membranes are cooled, first the non-lamellar forming lipids pass through a transition to a fluid lamellar phase and are miscible with bilayer-forming lipids into which they diffuse. On further cooling the high-melting-point lipids begin to crystallize and separate into a lamellar gel phase, in the process excluding the low-melting point lipids and intrinsic proteins. The lipids in these remaining regions form a gel phase at the lowest temperature. It is suggested that, because the non-lamellar lipids tend to undergo a liquid-crystalline to gel-phase transition at higher temperatures than lamellar-forming lipids, these will tend to phase separate into a gel phase domain rich in these lipids. Damage results when the membrane is reheated, whereupon the hexagonal-II-forming lipids give rise to non-lamellar structures. These probably take the form of inverted micelles sandwiched within the lipid bilayer and they completely destroy the permeability barrier properties of the membrane. The model is consistent with the phase behavior of membrane lipids and the action of cryoprotective agents in modifying lipid phase properties.     

Autohydrolysis Pretreatment of Mixed Softwood to Produce Value Prior to Combustion

Autohydrolysis is a hot water pretreatment to extract soluble components from wood that can be used prior to converting the woody residuals into paper, wood products, fuel, or other goods. In this study, mixed softwood chips were autohydrolyzed in hot water at 150, 160, 170, and 180 °C for 1 and 2 h residence times. The objective was to understand the tradeoff between the extraction of fermentable sugar and the residual solid total energy of combustion quantitatively. This process strategy will be referred to as “value prior to combustion”. High-performance liquid chromatography was used to determine chemical compositions (sugars and byproducts such as acetic acid, furfural, and hydroxymethylfurfural) of the extracted liquid and residuals; a bomb calorimeter was used to measure the heating value of original wood and solid residue. As the autohydrolysis temperature increased, material balances of the system indicated higher volatile byproducts loss. More hemicelluloses were solubilized by the hot water extraction process at higher temperatures and longer residence times, and a greater degree of sugar degradation was also observed. The maximum sugar yield was determined to occur at conditions of 170 °C for 2 h, during which 13 g of sugar was recovered from the extract out of 100 g of oven-dried wood. The heating value of the solid residues after extraction was greater than the original wood. The total energy content of the solid residual after extraction ranged from 85 to 98 % of the original energy content of the feed with higher temperatures reducing the total energy content.     

Biochemical Adaptation to the Environment

SYNOPSIS. Biochemical adaptation to environmental parameterssuch as temperature appears to involve two distinct types ofchanges in the organism's chemistry. On the one hand, the quantitiesof certain molecular species present in the cells may change.Alternatively, the actual types of molecules present may vary.Rainbow trout (Salmo gairdneri) acclimated to warm and coldtemperatures exhibit a striking example of this latter typeof adaptation. For all enzymes we have examined in this species,distinct "warm" and "cold" isozymes are present. The isozymesfound in warmacclimated (18°C) trout function well onlyat temperatures above 10–12°C. The isozymes presentin cold-acclimated (4°C) trout function optimally at 2-5°C,temperatures this species normally encounters in winter. Thesedata, plus information on comparable changes in membrane lipids,lead us to propose that adult poikilotherms may undergo a considerabledegree of "biochemical restructuring" on a seasonal basis. Thefactors which control this "restructuring," and the rates atwhich the process occurs at high and low temperatures, are topicsfor future investigation.     

Process of Making Three-dimensional Microstructures using Vaporization of a Sacrificial Component

Vascular structures in natural systems are able to provide high mass transport through high surface areas and optimized structure. Few synthetic material fabrication techniques are able to mimic the complexity of these structures while maintaining scalability. The Vaporization of a Sacrificial Component (VaSC) process is able to do so. This process uses sacrificial fibers as a template to form hollow, cylindrical microchannels embedded within a matrix. Tin (II) oxalate (SnOx) is embedded within poly(lactic) acid (PLA) fibers which facilitates the use of this process. The SnOx catalyzes the depolymerization of the PLA fibers at lower temperatures. The lactic acid monomers are gaseous at these temperatures and can be removed from the embedded matrix at temperatures that do not damage the matrix. Here we show a method for aligning these fibers using micromachined plates and a tensioning device to create complex patterns of three-dimensionally arrayed microchannels. The process allows the exploration of virtually any arrangement of fiber topologies and structures.     

Dielectric characterization of neutralized and nonneutralized chitosan upon drying

Isothermal dielectric loss spectra of neutralized and nonneutralized chitosan were acquired in successive runs from -130 degrees C up to increasing final temperatures, in a frequency range between 20 Hz and 1 MHz. Essentially, three relaxation processes were detected in the temperature range covered: (i) a beta-wet process, detected when the sample has a higher water content that vanishes after heating to 150 degrees C; (ii) a beta process, which is located at temperatures below 0 degrees C, becoming better defined and maintaining its location after annealing at 150 degrees C independently of the protonation state of the amino side group; and (iii) a sigma process that deviates to higher temperatures with drying, being more mobile in the nonneutralized form. Moreover, in dried neutralized chitosan, a fourth process was detected in the low frequency side of the secondary beta process that diminishes after annealing. Whether this process is a distinct relaxation of the dried polymer or a deviated beta-wet process due to the loss of water residues achieved by annealing is not straightforward. Only beta and sigma processes persist after annealing at 150 degrees C. The changes in molecular mobility upon drying of these two relaxation processes were evaluated.     

Proline biosynthesis in winter plants due to exposure to low temperatures

The content of bound proline sharply increased in proteins of different organs of young plants of winter rape and winter wheat exposed for 72 h to temperatures from 0 to 2 °C while it decreased only in root tips of wheat plants. Free proline which at 20 °C occurs in all plant organs only in trace amounts, accumulated considerably after 72 h exposure to low temperatures in the above-ground organs and only slightly in the roots. Free proline did not accumulate during the first 24 h at 0 to 2 °C in detached leaves of winter wheat but it was incorporated into newly synthetized proteins in which proline content increased after 6 h incubation to its maximum ( + 11.75% in comparison to control); the content of free glutamate sharply decreased during the first 6 h of incubation and the accumulation of bound glutamate was belated in comparison to that of bound proline. Sucrose infiltrated into detached leaves of winter wheat strongly stimulated proline incorporation into proteins at low temperatures, but it did not influence glutamate incorporation. The results suggest that the main reason for thede novo proline biosynthesis during the first six hours of hardening of the plants is the synthesis of proteins rich in proline; free proline accumulates later predominantly in the above-ground organs as a surplus. The above-ground organs are dehydrated in the course of the hardening process approximately to the same extent both in the light and in the dark, but proline content increases much less in the dark than in tho light.     

Kinetics of folding of Escherichia coli OmpA from narrow to large pore conformation in a planar bilayer

The outer membrane protein of Escherichia coli, OmpA, is currently alleged to adopt two native conformations: a major two-domain conformer in which 171 N-terminal residues form a narrow eight beta-barrel pore and 154 C-terminal residues are in the periplasm and a minor one-domain conformer in which all 325 residues create a large pore. However, recent studies in planar bilayers indicate the conformation of OmpA is temperature-sensitive and that increasing temperature converts narrow pores to large pores. Here we examine the reversibility and kinetics of this transition for single OmpA molecules in planar bilayers of diphytanoylphosphatidylcholine (DPhPC). We find that the transition is irreversible. When temperatures are decreased, large pores close down, and when temperatures are stabilized they reopen in the large pore conformation, with gradually increasing open time. Large pores are converted to narrow pores only by denaturing agents. The transition from narrow to large pores requires temperatures >or= 26 degrees C and is a biphasic process with rates that rise steeply with temperature. The first phase, a flickering stepwise transition from a low-conductance to a high-conductance state requires approximately 7 h at 26 degrees C but only approximately 13 min at 42 degrees C, signifying an activation energy of 139 +/- 12 kJ/mol. This is followed by a gradual increase in conductance and open probability, interpreted as optimization of the large pore structure. The results indicate that the two-domain structure is a partially folded intermediate that is kinetically stable at lower temperatures and that mature fully folded OmpA is a large pore.     

Pathogenicity Process of Pseudomonas fuscovaginae, the Causal Agent of Sheath Brown Rot of Rice

Sheath brown rot of rice caused by Pseudomonas fuscovaginae has been described in areas where low temperatures occur during the rice booting and heading stages. To analyse the relationship between pathogenicity of P. fuscovaginae and low temperatures, pathogenicity process in rice at booting stage was studied in a growth chamber at midrange and low temperatures. Analysis performed at 13°C, 18°C and 23°C in two rice cultivars showed that pathogenicity of P. fuscovaginae was explained by the general model of the independent action. The inoculum dose necessary to obtain 50% of diseased sheaths decreased with increase of temperature Analysis of planta bacterial population dynamics and mean response time pointed out that low temperatures affected pathogen multiplication in host before and after symptoms development. In consideration of our results, it was concluded that low temperatures acted negatively on the pathogenicity process of P. fuscovaginae. Therefore, occurrence of P. fuscovaginae in areas where rice cultivation is restricted by low temperatures can not be explained by a direct effect of temperatures on pathogenicity.     

Apparent Inhibition of Chloroplast Protein Import by Cold Temperatures Is Due to Energetic Considerations Not Membrane Fluidity

The transport of proteins across virtually all types of biological membranes has been reported to be inhibited by low temperatures. Paradoxically, plants are able to acclimate to growth at temperatures below which protein import into chloroplasts is known to be blocked. In examining this incongruity, we made a number of unexpected observations. First, chloroplasts isolated from plants grown at 7/1[deg]C in light/dark and from plants grown at 25[deg]C were able to import proteins with the same efficiency over a temperature range from 5 to 21[deg]C, indicating that no functional adaptation had taken place in the protein import machinery of chloroplasts in these cold-grown plants. Second, chloroplasts from warm-grown plants were able to take up proteins at temperatures as low as 4[deg]C provided that they were illuminated. We determined that light mediates the import process at 5[deg]C by driving ATP synthesis in the stroma, the site of its utilization during protein transport. Direct measurement of the envelope phase transition temperature as well as the activity of the ATP/ADP translocator in the inner envelope membrane at 5 and 25[deg]C demonstrated that the cold block of protein import into chloroplasts observed in vitro is due primarily to energetic considerations and not to decreased membrane fluidity.     

Dynamics of ligand binding to myoglobin.

Myoglobin rebinding of carbon monoxide and dioxygen after photodissociation has been observed in the temperature range between 40 and 350 K. A system was constructed that records the change in optical absorption at 436 nm smoothly and without break between 2 musec and 1 ksec. Four different rebinding processes have been found. Between 40 and 160 K, a single process is observed. It is not exponential in time, but approximately given by N(t) = (1 + t/to)-n, where to and n are temperature-dependent, ligand-concentration independent, parameters. At about 170 K, a second and at 200 K, a third concentration-independent process emerge. At 210 K, a concentration-dependent process sets in. If myoglobin is embedded in a solid, only the first three can be seen, and they are all nonexponential. In a liquid glycerol-water solvent, rebinding is exponential. To interpret the data, a model is proposed in which the ligand molecule, on its way from the solvent to the binding site at the ferrous heme iron, encounters four barriers in succession. The barriers are tentatively identified with known features of myoglobin. By computer-solving the differential equation for the motion of a ligand molecule over four barriers, the rates for all important steps are obtained. The temperature dependences of the rates yield enthalpy, entropy, and free-energy changes at all barriers. The free-energy barriers at 310 K indicate how myoglobin achieves specificity and order. For carbon monoxide, the heights of these barriers increase toward the inside; carbon monoxide consequently is partially rejected at each of the four barriers. Dioxygen, in contrast, sees barriers of about equal height and moves smoothly toward the binding site. The entropy increases over the first two barriers, indicating a rupturing of bonds or displacement of residues, and then smoothly decreases, reaching a minimum at the binding site. The magnitude of the decrease over the innermost barrier implies participation of heme and/or protein. The nonexponential rebinding observed at low temperatures and in solid samples implies that the innermost barrier has a spectrum of activation energies. The shape of the spectrum has been determined; its existence can be explained by assuming the presence of many conformational states for myoglobin. In a liquid at temperatures above about 230 K, relaxation among conformational states occurs and rebinding becomes exponential.