Hydrogen exchange of disordered proteins in Escherichia coli

A truly disordered protein lacks a stable fold and its backbone amide protons exchange with solvent at rates predicted from studies of unstructured peptides. We have measured the exchange rates of two model disordered proteins, FlgM and α-synuclein, in buffer and in Escherichia coli using the NMR experiment, SOLEXSY. The rates are similar in buffer and cells and are close to the rates predicted from data on small, unstructured peptides. This result indicates that true disorder can persist inside the crowded cellular interior and that weak interactions between proteins and macromolecules in cells do not necessarily affect intrinsic rates of exchange.     

Hydrogen exchange of monomeric alpha-synuclein shows unfolded structure persists at physiological temperature and is independent of molecular crowding in Escherichia coli

Amide proton NMR signals from the N-terminal domain of monomeric α-synuclein (αS) are lost when the sample temperature is raised from 10°C to 35°C at pH 7.4. Although the temperature-induced effects have been attributed to conformational exchange caused by an increase in α-helix structure, we show that the loss of signals is due to fast amide proton exchange. At low ionic strength, hydrogen exchange rates are faster for the N-terminal segment of αS than for the acidic C-terminal domain. When the salt concentration is raised to 300 mM, exchange rates increase throughout the protein and become similar for the N- and C-terminal domains. This indicates that the enhanced protection of amide protons from the C-terminal domain at low salt is electrostatic in nature. Cα chemical shift data point to <10% residual α-helix structure at 10°C and 35°C. Conformational exchange contributions to R2 are negligible at both temperatures. In contrast to the situation in vitro, the majority of amide protons are observed at 37°C in ¹H-¹⁵N HSQC spectra of αS encapsulated within living Escherichia coli cells. Our finding that temperature effects on αS NMR spectra can be explained by hydrogen exchange obviates the need to invoke special cellular factors. The retention of signals is likely due to slowed hydrogen exchange caused by the lowered intracellular pH of high-density E. coli cultures. Taken together, our results emphasize that αS remains predominantly unfolded at physiological temperature and pH—an important conclusion for mechanistic models of the association of αS with membranes and fibrils.     

Measurement of intrinsic exchange rates of amide protons in a 15N-labeled peptide

Summary We have used a modified version of a previously proposed technique, MEXICO [Gemmecker et al. (1993) J. Am. Chem. Soc., 115, 11620], and improved data analysis procedures in order to measure rapid hydrogen exchange (HX) rates of amide protons in peptides labeled only with ¹⁵N. The requirement of ¹³C-/¹⁵N-labeled material has been circumvented by adjusting conditions so that NOE effects associated with amide protons can be neglected (i.e., _0c~1). The technique was applied to an unstructured ¹⁵N-labeled 12-residue peptide to measure intrinsic HX rates, which are the essential reference for examining protein and peptide structure and dynamics through deceleration of HX rates. The method provided accurate HX rates from 0.5 to 50 s^-1 under the conditions used. The measured rates were in good agreement with those predicted using correction factors determined by Englander and co-workers [Bai et al. (1993) Proteins, 17, 75], with the largest deviations from the predicted rates found for residues close to the N-terminus. The exchange rates were found to exhibit significant sensitivity to the concentration of salt in the sample.     

Water–Protein Hydrogen Exchange in the Micro-Crystalline Protein Crh as Observed by Solid State NMR Spectroscopy

We report site-resolved observation of hydrogen exchange in the micro-crystalline protein Crh. Our approach is based on the use of proton T₂^′ -selective ¹H–¹³C–¹³C correlation spectra for site-specific assignments of carbons nearby labile protein protons. We compare the proton T₂^′ selective scheme to frequency selective water observation in deuterated proteins, and discuss the impacts of deuteration on ¹³C linewidths in Crh. We observe that in micro-crystalline proteins, solvent accessible hydroxyl and amino protons show comparable exchange rates with water protons as for proteins in solution, and that structural constraints, such as hydrogen bonding or solvent accessibility, more significantly reduce exchange rates.     

Phosphatidylethanolamine synthesis in adult Dirofilaria immitis females

Srivastava Arvind K., Jaffe Julian J. and Lambert Roger A. 1985. Phosphatidylethanolamine synthesis in adult Dirofilaria immitis females. International Journal for Parasitology15: 429–433. Adult Dirofiliaria immitis females were found able to synthesize phosphatidylethanolamine (PE) by way of the following three pathways: (1) phosphorylethanolamine, cytidine diphosphoethanolamine and 1,2-diacylglycerol; (2) decarboxylation of phosphatidylserine (PS); and (3) direct exchange of ethanolamine for choline or serine in preformed phosphatidylcholine or PS. The latter two pathways were confined to the paniculate fraction of worm homogenates. Under stated assay conditions, the respective rates of PE formation by way of these pathways in the order given were around 250, 8500 and 2–3 pmol min^?1 mg^?1 protein.     

The role of leaf and canopy-level gas exchange in the replacement of Quercus virginiana (Fagaceae) by Juniperus ashei (Cupressaceae) in semiarid savannas

Photosynthesis, transpiration, and leaf area distribution were sampled in mature Quercus virginiana and Juniperus ashei trees to determine the impact of leaf position on canopy-level gas exchange, and how gas exchange patterns may affect the successful invasion of Quercus communities by J. ashei. Sampling was conducted monthly over a 2-yr period in 12 canopy locations (three canopy layers and four cardinal directions). Photosynthetic and transpiration rates of both species were greatest in the upper canopy and decreased with canopy depth. Leaf photosynthetic and transpiration rates were significantly higher for Q. virginiana (4.1–6.7 μmol CO₂·m⁻²·s⁻¹ and 1.1–2.1 mmol H₂O·m⁻²·s⁻¹) than for J. ashei (2.1–2.8 μmol CO₂·m⁻²·s⁻¹ and 0.7–1.0 mmol H₂O·m⁻²·s⁻¹) in every canopy level and direction. Leaves on the south and east sides of both species had higher gas exchange rates than leaves on the north and west sides. Although Quercus had a greater mean canopy diameter than Juniperus (31.3 vs. 27.7 m²), J. ashei had significantly greater leaf area (142 vs. 58 m²/tree). A simple model combining leaf area and gas exchange rates for different leaf positions demonstrated a significantly greater total canopy carbon dioxide uptake for J. ashei compared to Q. virginiana (831 vs. 612 g CO₂·tree⁻¹·d⁻¹, respectively). Total daily water loss was also greater for Juniperus (125 vs. 73 Ltree⁻¹·d⁻¹). Differences in leaf gas exchange rates were poor predictors of the relationship between the invasive J. ashei and the codominant Q. virginiana. Leaf area and leaf area distribution coupled with leaf gas exchange rates were necessary to demonstrate the higher overall competitive potential of J. ashei.     

Effects of flow rate and temperature on cyclic gas exchange in tsetse flies (Diptera, Glossinidae)

Air flow rates may confound the investigation and classification of insect gas exchange patterns. Here we report the effects of flow rates (50, 100, 200, 400 ml min⁻¹) on gas exchange patterns in wild-caught Glossina morsitans morsitans from Zambia. At rest, G. m. morsitans generally showed continuous or cyclic gas exchange (CGE) but no evidence of discontinuous gas exchange (DGE). Flow rates had little influence on the ability to detect CGE in tsetse, at least in the present experimental setup and under these laboratory conditions. Importantly, faster flow rates resulted in similar gas exchange patterns to those identified at lower flower rates suggesting that G. m. morsitans did not show DGE which had been incorrectly identified as CGE at lower flow rates. While CGE cycle frequency was significantly different among the four flow rates (p < 0.05), the direction of effects was inconsistent. Indeed, inter-individual variation in CGE cycle frequency exceeded flow rate treatment variation. Using a laboratory colony of closely related, similar-sized G. morsitans centralis we subsequently investigated the effects of temperature, gender and feeding status on CGE pattern variation since these factors can influence insect metabolic rates. At 100 ml min⁻¹ CGE was typical of G. m. centralis at rest, although it was significantly more common in females than in males (57% vs. 43% of 14 individuals tested per gender). In either sex, temperature (20, 24, 28 and 32 °C) had little influence on the number of individuals showing CGE. However, increases in metabolic rate with temperature were modulated largely by increases in burst volume and cycle frequency. This is unusual among insects showing CGE or DGE patterns because increases in metabolic rate are usually modulated by increases in frequency, but either no change or a decline in burst volume.     

Off-resonance rotating-frame amide proton spin relaxation experiments measuring microsecond chemical exchange in proteins

NMR spin relaxation in the rotating frame (R_1ρ) is a unique method for atomic-resolution characterization of conformational (chemical) exchange processes occurring on the microsecond time scale. Here, we use amide ¹H off-resonance R_1ρ relaxation experiments to determine exchange parameters for processes that are significantly faster than those that can be probed using ¹⁵N or ¹³C relaxation. The new pulse sequence is validated using the E140Q mutant of the C-terminal domain of calmodulin, which exhibits significant conformational exchange contributions to the transverse relaxation rates. The ¹H off-resonance R_1ρ data sample the entire relaxation dispersion profiles for the large majority of residues in this protein, which exchanges between conformations with a time constant of approximately 20 μs. This is in contrast to the case for ¹⁵N, where additional laboratory-frame relaxation data are required to determine the exchange parameters reliably. Experiments were performed on uniformly ¹⁵N-enriched samples that were either highly enriched in ²H or fully protonated. In the latter case, dipolar cross-relaxation with aliphatic protons were effectively decoupled to first order using a selective inversion pulse. Deuterated and protonated samples gave the same results, within experimental errors. The use of deuterated samples increases the sensitivity towards exchange contributions to the ¹H transverse relaxation rates, since dipolar relaxation is greatly reduced. The exchange correlation times determined from the present ¹H off-resonance R_1ρ experiments are in excellent agreement with those determined previously using a combination of ¹⁵N laboratory-frame and off-resonance R_1ρ relaxation data, with average values of and 21 ± 3 μs, respectively.     

Gas exchange in San Francisco Bay

Gas exchange across the air-water interface is one of the most important processes controlling the concentrations of dissolved gases in estuarine systems. A brief review of principles and equations to predict gas exchange indicates that both current shear and wind shear are possible sources of turbulence for controlling gas exchange rates in estuaries. Rates of exchange determined by constructing a mass balance for radon-222 indicate that wind shear is dominant in San Francisco Bay. Because many estuaries have wind shear and current speeds comparable to this system, this conclusion may be true for other systems as well. A compilation of gas exchange rates measured in San Francisco Bay with those for other wind-dominated systems updates previous compilations and yields an equation for predicting gas exchange: K _l = 34.6 R _v (D_m20)^1/2 (U₁₀)^1.5 where R is the ratio of the kinematic viscosity of pure water at 20° C to the kinematic viscosity of water at the measured temperature and salinity, D_m20 is the molecular diffusivity of the gas of interest at 20°C in cm² s^–1, U₁₀ is the wind speed at 10 meters above the surface in m s^–1, and K_L is the liquid phase gas transfer coefficient in m d^–1. This relationship fits the available field data within 20% for wind speeds between 3 and 12 m s^–1. It is used to show that the residence time of dissolved oxygen in San Francisco Bay should range from 2 days during windy summer periods to as much as 15 days during calm winter periods. Because these times are short compared to time constants for other processes controlling oxygen distribution in this system, dissolved oxygen concentrations in San Francisco Bay are usually near atmospheric equilibrium. Other systems, such as Chesapeake Bay, may differ. There, despite ample air-water gas exchange rates, some bottom waters become anoxic during summer months due to slow vertical mixing.     

Comparison of amide proton exchange in reduced and oxidizedRhodobacter capsulatus cytochrome c2: A1H−15N NMR study

Summary The hydrogen-deuterium exchange rates of the reduced and oxidized forms ofRhodobacter' capsulatus cytochrome c₂ were studied by¹H–¹⁵N homonuclear multiple quantum correlation spectroscopy. Minimal differences were observed for the N- and C-terminal helices on changing redox state suggesting that although these helices are structurally important they do not affect the relative stability of the two redox states and hence may not be important in determining the redox potential differences observed amongst the class I c-type cytochromes. However, significant differences were observed for other regions of the protein. For example, all slow exchanging protons of the helix spanning Phe⁸² to Asp⁸⁷ are similarly affected on reduction indicating that the unfolding equilibrium of this helix is altered between the two redox states. Other regions are not as simple to interpret; however, the difference in NH exchange rates between the redox states for a number of residues including His¹⁷, Leu³⁷, Arg⁴³, Ala⁴⁵, Gly⁴⁶, Ile⁵⁷, Val⁵⁸, Leu⁶⁰, Gly⁶¹ and Leu¹⁰⁰ suggest that interactions affecting the causes of these differences may be important factors in determining redox potential.Abbreviations NMR nuclear magnetic resonance - HMQC homonuclear multiple quantum correlation - NOESY nuclear Overhauser effect spectroscopy     

Measurement of amide proton exchange rates and NOEs with water in 13C/15N-enriched calcineurin B

Summary A rapid and sensitive 2D approach is presented for measuring amide proton exchange rates and the NOE interaction between amide protons and water. The approach is applicable to uniformly ¹³C/¹⁵N-enriched proteins and can measure magnetization exchange rates in the 0.02 to >20s^–1 range. The experiments rely on selective excitation of the water resonance, coupled with purging of underlying H resonances, followed by NOESY-or ROESY-type transfer to amide protons, which are dispersed by the amide ¹⁵N frequencies in an HSQC-type experiment. Two separate but interleaved experiments, with and without selective inversion of the H₂O resonance, yield quantitative results. The method is demonstrated for a sample of the calcium-binding protein calcineurin B. Results indicate rapid amide exchange for the five calcineurin B residues that are analogous to the five rapidly exchanging residues in the central helix of the homologous protein calmodulin.     

Structure and dynamics of the neutrophil defensins NP-2, NP-5, and HNP-1: NMR studies of amide hydrogen exchange kinetics

The exchange kinetics for the slowly exchanging amide hydrogens in three defensins, rabbit NP-2, rabbit NP-5, and human HNP-1, have been measured over a range of pH at 25°C using 1D and 2D NMR methods. These NHs have exchange rates 10² to 10⁵ times slower than rates from unstructured model peptides. The observed distribution of exchange rates under these conditions can be rationalized by intramolecular hydrogen bonding of the individual NHs, solvent accessibility of the NHs, and local fluctuations in structure. The temperature dependencies of NH chemical shifts (NH temperature coefficients) were measured for the defensins and these values are consistent with the defensin structure. A comparison is made between NH exchange kinetics, NH solvent accessibility, and NH temperature coefficients of the defensins and other globular proteins. Titration of the histidine side chain in NP-2 was examined and the results are mapped to the three-dimensional structure. © 1994 Wiley-Liss, Inc.     

Removing Constraints on the Biomass Production of Freshwater Macroalgae by Manipulating Water Exchange to Manage Nutrient Flux

Freshwater macroalgae represent a largely overlooked group of phototrophic organisms that could play an important role within an industrial ecology context in both utilising waste nutrients and water and supplying biomass for animal feeds and renewable chemicals and fuels. This study used water from the intensive aquaculture of freshwater fish (Barramundi) to examine how the biomass production rate and protein content of the freshwater macroalga Oedogonium responds to increasing the flux of nutrients and carbon, by either increasing water exchange rates or through the addition of supplementary nitrogen and CO₂. Biomass production rates were highest at low flow rates (0.1–1 vol.day⁻¹) using raw pond water. The addition of CO₂ to cultures increased biomass production rates by between 2 and 25% with this effect strongest at low water exchange rates. Paradoxically, the addition of nitrogen to cultures decreased productivity, especially at low water exchange rates. The optimal culture of Oedogonium occurred at flow rates of between 0.5–1 vol.day⁻¹, where uptake rates peaked at 1.09 g.m⁻².day⁻¹ for nitrogen and 0.13 g.m⁻².day⁻¹ for phosphorous. At these flow rates Oedogonium biomass had uptake efficiencies of 75.2% for nitrogen and 22.1% for phosphorous. In this study a nitrogen flux of 1.45 g.m⁻².day⁻¹ and a phosphorous flux of 0.6 g.m⁻².day⁻¹ was the minimum required to maintain the growth of Oedogonium at 16–17 g DW.m⁻².day⁻¹ and a crude protein content of 25%. A simple model of minimum inputs shows that for every gram of dry weight biomass production (g DW.m⁻².day⁻¹), Oedogonium requires 0.09 g.m⁻².day⁻¹ of nitrogen and 0.04 g.m⁻².day⁻¹ of phosphorous to maintain growth without nutrient limitation whilst simultaneously maintaining a high-nutrient uptake rate and efficiency. As such the integrated culture of freshwater macroalgae with aquaculture for the purposes of nutrient recovery is a feasible solution for the bioremediation of wastewater and the supply of a protein resource.     

Measurement of the exchange rates of rapidly exchanging amide protons: Application to the study of calmodulin and its complex with a myosin light chain kinase fragment

Summary A technique is described for measuring the approximate exchange rates of the more labile amide protons in a protein. The technique relies on a comparison of the intensities in¹H–¹⁵N correlation spectra recorded with and without presaturation of the water resonance. To distinguish resonance attenuation caused by hydrogen exchange from attenuation caused by cross relation, the experiment is repeated at several different pH values and the difference in attenuation of any particular amide resonance upon presaturation is used for calculating its exchange rate. The technique is demonstrated for calmodulin and for calmodulin complexed with its binding domain of skeletal muscle myosin light chain kinase. Upon complexation, increased amide exchange rates are observed for residues Lys⁷⁵ through Thr⁷⁹ located in the central helix of calmodulin, and for the C-terminal residues Ser¹⁴⁷ and Lys¹⁴⁸. In contrast, a decrease in amide exchange rate is observed at the C-terminal end of the F helix, from residues Thr¹¹⁰ through Glu¹¹⁴.Istituto Guido Donegani, Novara, Italy     

Effects of net charge on the hydrogen-deuterium exchange parameters of lysozyme.

Possible effects of changes in net charge on protein hydrogen exchange rates were investigated by desalting hen egg-white lysozyme, which allowed its net charge to increase with decreasing pH in the acid region. Chloride ion-binding ratios, expressed as ratios of free to total Cl^?, were measured with a chloride-specific electrode at pH 5 on a 2.4% solution of a five-time-desalted product. This ratio was used to show a 97% reduction of the 11% Cl^? present in a commercial lysozyme preparation upon three passes of the enzyme through a column of ion-retardation resin. Net charges on the purified product were assigned from a combination of electrophoretic mobility and proton titration data gathered under minimal ionic strength conditions. The net charge on the desalted product increased by 1.64 units between pH 5.0 and 3.0. Hydrogendeuterium exchange studies on the purified lysozyme in D₂O were obtained using the near-infrared region of a Cary 14R spectrophotometer. The rate-pD profile for k₂, the rate constant for the intermediate class of exchanging hydrogens, showed a decrease in the apparent pD of minimum exchange rate of 0.3 units, when compared to that obtained earlier in 0.2 m added NaCl. However, the rate of exchange at pD minimum and the number of hydrogens in the class remained largely unaffected. A similar shift was observed for the rate-pD profile of the class 1 hydrogens. Thus, the effect of an increase in net positive charge is to shift the rate-pD profile to a lower pD. Moreover, the effect extended to the interior peptide hydrogens of this globular protein. Consequently, the exchange rates of all the observable hydrogens are altered by the net charge changes, and the effect appeared uniform. The shift can be accounted for quantitatively by applying electrostatic interaction terms to the acid and base catalytic constants characterizing the exchange process. The calculated electrostatic interaction factors in minimal salt and 0.2 m added NaCl were found to be 29 and 18% lower, respectively, than those obtained theoretically. Therefore, under conditions where changes in net charge may occur for a globular protein, the effect on hydrogen exchange rates can be estimated fairly well theoretically, especially at moderate ionic strengths.     

Conformational studies of the manganese transport regulator (MntR) from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> using deuterium exchange mass spectrometry

The manganese transport regulator (MntR) of Bacillus subtilis is a metalloregulatory protein responsible for regulation of genes involved in manganese uptake by this organism. MntR belongs to the iron-responsive DtxR family, but is allosterically regulated by manganese and cadmium ions. Having previously characterized the metal binding affinities of this protein as well as the DNA-binding activation profiles for the relevant metal ions, we have focused the current study on investigating the structural changes of MntR in solution upon binding divalent transition metal ions. Deuterium exchange mass spectrometry was utilized to investigate the deuterium exchange dynamics between apo-MntR, Co²⁺-MntR, Cd²⁺-MntR, and Mn²⁺-MntR. Comparing the rates of deuteration of each metal-bound form of MntR reveals that the N-terminal DNA-binding motif is more mobile in solution than the C-terminal dimerization domain. Furthermore, significant protection from deuterium exchange is observed in the helices that contribute metal-chelating amino acids to form the metal binding site of MntR. In contrast, the bulk of the DNA-binding winged helix–turn–helix motif shows no difference in deuterium exchange upon metal binding. Mapping of the deuteration patterns onto the crystal structures of MntR yields insight into how metal binding affects the protein structure and complements earlier studies on the mechanism of MntR. Metal binding acts to rigidify MntR, thereby limiting the mobility of the protein and reducing the entropic cost of DNA binding. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Studies on the reactivity of malondialdehyde. I. Effects of acid concentration and solvent composition on deuterium exchange reactions

The rates of deuterium exchange reactions of malondialdehyde (MDA) and deuterated malondialdehyde (MDAd) have been studied as a function of acidity and the content of dimethyl sulfoxide (DMSO) in binary mixtures with D₂O . MDA incorporates deuterium from D₂O solutions in a first-order reaction with a rate constant (k_obs) that depends on the acid concentration. From this dependence, a catalytic constant, k_cat, can be derived (k_cat^MDA = 2.25 × 10⁵M^?s^?1). Similar kinetic behavior was found for MDAd in H₂O solutions, and in this case, k_cat^MDA = 1.56 × 10⁵M^?1s^?1. Results from reactions of MDA and MDAd in identical H₂OD₂O mixtures show that primary and secondary isotope effects are small (k_H/k_D = 1.13) and that solvent isotope effects cause most of the differences found between reactions in D₂O and H₂O. Reactions in binary DMSOd₆D₂O mixtures show a six-fold rate increase as the proportion of DMSOd₆ increases from 50% to 90%. These results also illustrate the relatively high reactivity of MDA at pH values well above its pK_a and the importance of medium composition on its reaction rate.     

The transbilayer movement of phosphatidylcholine in vesicles reconstituted with intrinsic proteins from the human erythrocyte membrane

Vesicles have been prepared from 18 : 1_c/18 : 1_c-phosphatidylcholine with or without purified glycophorin or partially purified band 3 (obtained by organomercurial gel chromatography). The vesicles have been characterized by freeze-fracture electron microscopy, binding studies to DEAE-cellulose, ³¹P-NMR and K⁺ trap measurements. Pools of phosphatidylcholine available for exchange have been investigated using phosphatidylcholine exchange protein from bovine liver. The protein-containing vesicles both exhibit exchangeable pools larger than the fraction of phosphatidylcholine in the outer monolayer, whereas in the protein-free vesicles the exchangeable pool is consistent with the outer monolayer. The results indicate that both glycophorin and the partially purified band 3 preparation enhance the transbilayer movement of phosphatidylcholine.     

Distribution of ATPase and ATP-to-ADP phosphate exchange activities in magnesium chelatase subunits of Chlorobium vibrioforme and Synechocystis PCC6803

Insertion of magnesium into protoporphyrin IX is a complex ATP-dependent reaction catalysed by the enzyme Mg-chelatase. Three separate proteins (Mg-chelatase subunits), designated as D, H and I, are involved in the chelation reaction. The genes encoding the Mg-chelatase subunits of the green sulfur bacterium Chlorobium vibrioforme and of the cyanobacterium Synechocystis strain PCC6803 were expressed in Escherichia coli. The recombinant proteins were purified, tested for ATPase and phosphate exchange activities, and compared with the activities of the corresponding subunits of Rhodobacter sphaeroides. The Synechocystis strain PCC6803 I subunit and the C. vibrioforme H and I subunits hydrolysed ATP at the rates of 2.0, 1.8 and 0.16 nmol (mg protein)^–1 min^–1, respectively. The ATPase activity of the C. vibrioforme H subunit was similar to that reported for the R. sphaeroides H subunit. The Synechocystis strain PCC6803 H subunit failed to hydrolyse ATP. The I subunit of Synechocystis strain PCC6803 and C. vibrioforme catalysed a transfer of PO₄ from ATP to ADP (exchange activity) at the rate of 1.75 ± 0.15 nmol (mg protein)^–1 min^–1. This exchange rate was 300-fold lower than that reported for the R. sphaeroides I subunit. The PO₄ exchange activities were correlated with the presence of the sequence GXRGTGKSTXVRALA in the primary structure of the three I subunits. Mg-chelatase activity was reconstituted by combining the three subunits of the same bacterium [rates of 41–89 pmol Mg-deuteroporphyrin (mg protein)^–1 min^–1]. Heterologous subunit combinations resulted in low or no Mg-chelatase activity. Received: 25 May 1998 / Revision received: 24 November 1998 / Accepted: 27 November 1998     

On-line screening of soil VOCs exchange responses to moisture, temperature and root presence

The exchanges of volatile organic compounds (VOCs) between soils and the atmosphere are poorly known. We investigated VOC exchange rates and how they were influenced by soil moisture, temperature and the presence of plant roots in a Mediterranean forest soil. We measured VOC exchange rates along a soil moisture gradient (5%–12.5%–20%–27.5% v/v) and a temperature gradient (10°C–15°C–25°C–35°C) using PTR-MS. Monoterpenes were identified with GC-MS. Soils were a sink rather than a source of VOCs in both soil moisture and temperature treatments (−2.16 ± 0.35 nmol m⁻² s⁻¹ and −4.90 ± 1.24 nmol m⁻² s⁻¹ respectively). Most compounds observed were oxygenated VOCs like alcohols, aldehydes and ketones and aromatic hydrocarbons. Other volatiles such as acetic acid and ethyl acetate were also observed. All those compounds had very low exchange rates (maximum uptake rates from −0.8 nmol m⁻² s⁻¹ to −0.6 nmol m⁻² s⁻¹ for methanol and acetic acid). Monoterpene exchange ranged only from −0.004 nmol m⁻² s⁻¹ to 0.004 nmol m⁻² s⁻¹ and limonene and α-pinene were the most abundant compounds. Increasing soil moisture resulted in higher soil sink activity possibly due to increases in microbial VOCs uptake activity. No general pattern of response was found in the temperature gradient for total VOCs. Roots decreased the emission of many compounds under increasing soil moisture and under increasing soil temperature. While our results showed that emission of some soil VOCs might be enhanced by the increases in soil temperature and that the uptake of most soil VOCs uptake might be reduced by the decreases of soil water availability, the low exchange rates measured indicated that soil-atmosphere VOC exchange in this system are unlikely to play an important role in atmospheric chemistry. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.