Conformational transitions of calmodulin as studied by vacuum-uv CD

CD measurements were made for calmodulin and its calcium (Ca²⁺) complexes at different ionic strengths and Ca²⁺ concentrations. Calmodulin at an ionic strength of 0.00M and in the absence of Ca²⁺ exists as an α-helical protein with a negligible amount of β-sheet. An increase in ionic strength, whether or not Ca²⁺ is present, increases α-helix at the expense of “other” (coil) structure. The changes in β-sheet and β-turns are insignificant. Binding of Ca²⁺ at low ionic strength occurs in stages with at least one folding intermediate before attaining the final stable state. Binding of Ca²⁺ at an ionic strength of 0.165M causes only a slight increase in α-helix, so that the secondary structure of the protein depends on ionic strength and is insensitive to the nature of the cation (i.e., Ca²⁺). Thus, the activation of calmodulin by Ca²⁺ must be due to a structural reorientation rather than to a major secondary structural alteration. The CD estimation of secondary structure with 4 mol Ca²⁺/calmodulin (61% α-helix, 2% antiparallel β-sheet, 2% parallel β-sheet, 21% β-turns, and 14% other) is in excellent agreement with the x-ray results.     

Circular dichroism of the parallel β helical proteins pectate lyase C and E

The pectate lyases, PelC and PelE, have an unusual folding motif, known as a parallel β-helix, in which the polypeptide chain is coiled into a larger helix composed of three parallel β-sheets connected by loops having variable lengths and conformations. Since the regular secondary structure consists almost entirely of parallel β-sheets these proteins provide a unique opportunity to study the effect of parallel β-helical structure on circular dichroism (CD). We report here the CD spectra of PelC and PelE in the presence and absence of Ca²⁺, derive the parallel β-helical components of the spectra, and compare these results with previous CD studies of parallel β-sheet structure. The shape and intensity of the parallel β-sheet spectrum is distinctive and may be useful in identifying other proteins that contain the parallel β-helical folding motif. © 1995 Wiley-Liss, Inc.     

Metal-controlled interdomain cooperativity in parvalbumins

Conformational behavior of five homologous proteins, parvalbumins (PAs) from northern pike (α and β isoforms), Baltic cod, and rat (α and β isoforms), was studied by scanning calorimetry, circular dichroism, and bis-ANS fluorescence. The mechanism of the temperature-induced denaturation of these proteins depends dramatically on both the peculiarities of their amino acid sequences and on their interaction with metal ions. For example, the pike α-PA melting can be described by two successive two-state transitions with mid-temperatures of 90 and 120 °C, suggesting the presence of two thermodynamic domains. The intermediate state populated at the end of the first transition was shown to bind Ca²⁺ ions, and was characterized by the largely preserved secondary structure and increased solvent exposure of hydrophobic groups. Mg²⁺- and Na⁺-loaded forms of pike α-PA demonstrated a single two-state transition. Therefore, the mechanism of the PA thermal denaturation is controlled by metal binding. It ranged from the absence of detectable first-order transition (apo-form of pike PA), to the two-state transition (e.g., Mg²⁺- and Na⁺-loaded forms of pike α-PA), to the more complex mechanisms (Ca²⁺-loaded PAs) involving at least one partially folded intermediate. Analysis of isolated cavities in the protein structures revealed that the interface between the CD and EF subdomains of Ca²⁺-loaded pike α-PA is much more loosely packed compared with PAs manifesting single heat-sorption peak. The impairment of interactions between CD and EF subdomains may cause a loss of structural cooperativity and appearance of two separate thermodynamic domains. One more peculiar feature of pike α-PA is that depending on its interactions with metal ions, it can be an intrinsically disordered protein (apo-form), an ordered protein of mesophilic (Na⁺-bound state), thermophilic (Mg²⁺-form), or even of the hyperthermophilic origin (Ca²⁺-form).     

Existence of γ-helix in natural proteins

Serum albumin is known to act as a carrier for a variety of molecules and metal ions. This property of the protein could be due to the presence of different types of secondary structures in its molecules. The most commonly known are the α-helix, β-sheets, β-turns and random coil forms. A rigorous analysis of human serum albumin has been carried out by using four different approaches. Comparative studies have revealed that the segment Asp 107 to Val 122 of this protein assumes a γ-helical structure. Under favourable circumstances, two prolines at the i and (i + 5)th positions can initiate a γ-helix. Further requirements for the formation and stability of γ-helix are discussed.     

The influence of nearest-neighboring amino acid residues on aspects of secondary structure of proteins. Attempts to locate -helices and -sheets

The influence of nearest-neighbor pairs of amino acids (n ? 1) and (n + 1) on the conformation of amino acid (n) in proteins has been studied. From experimental data on eleven proteins of known three dimensional structures, our definition of an α-helical domain in the Φ,Ψ plot has been reexamined and found to be satisfactory. On the same principle, a regular β-sheet domain has been delineated. We then revised our 20 × 20 table of frequencies of occurrences of various conformations tabulating three values: α-helical, β-sheet, and neither. These frequencies were then used to locate the helixbreaking positions in cytochrome b₅, papain, thermolysin, and calcium-binding protein. In conjuction with the helical wheel method, they were useful for predicting the locations of most α-helical segments. Similarly the β-sheet breaking positions in papain were located and most of the β-sheets found by X-ray diffraction were close to or between these positions. Data on β-sheets are extremely sparse so that extensive tests were not possible. The application of this method to abnormal hemoglobins suggested possible distortions of helices and in several instances correlated with abnormal properties of the hemoglobins and association with disease. The variable region of human immunoglobin heavy chains was found to have a very low α-helical content though β-sheet structures might exist.     

Physicochemical and immunological characterization of the type E botulinum neurotoxin binding protein purified fromClostridium botulinum

Type E botulinum neurotoxin is produced byClostridium botulinum along with a neurotoxin binding protein which helps protect the neurotoxin from adversepH, temperature, and proteolytic conditions. The neurotoxin binding protein has been purified as a 118-kDa protein. Secondary structure content of the neurotoxin binding protein as revealed by far-UV circular dichroism spectroscopy was 19% α-helix, 50%β-sheets, 28% random coils, and 3%β-turns. This compared to 22% α-helix, 44%β-sheets, 34% random coils, and noβ-turns of the type E botulinum neurotoxin. The complex of the two proteins revealed 25%α-helix, 45%β-sheets, 27% random coils, and 3%β-turns, suggesting a significant alteration at least in theα-helical folding of the two proteins upon their interaction. Tyrosine topography is altered considerably (28%) when the neurotoxin and its binding protein are separated, indicating strong interaction between the two proteins. Gel filtration results suggested that type E neurotoxin binding protein clearly complexes with type E neurotoxin. The interaction is favored at lowpH as indicated by an initial binding rate of 8.4 min^?1 atpH 5.7 compared to 4.0 min^?1 atpH 7.5 as determined using a fiber optic-based biosensor. The neurotoxin and its binding protein apparently are of equivalent antigenicity, as both reacted equally on enzyme-linked immunosorbent assay to polyclonal antibodies raised against the toxoid of their complex.     

Characteristics of Relationships Between Structure of Gluten Proteins and Dough Rheology – Influence of Dietary Fibres Studied by FT-Raman Spectroscopy

The aim of this research was to study which kind of conformational changes in gluten proteins were induced by addition of four dietary fibre (apple-cranberry, cacao, carob and oat) by using FT-Raman spectroscopy and to find relationships between conformational changes and rheological behaviour of bread dough in mixing and extensional tests. Structural studies showed that all fibres induced formation of β-like structures between two protein molecules (pseudo-β-sheets) with the band at 1616 cm^?1 in the Raman spectrum. According to Principal Component Analysis, the strongest dependence was between changes in gluten structure and two extensographic parameters (resistance to extension and extensibility). Resistance to extension was positively correlated with content of α-helix and pseudo-β-sheets, while a negative correlation was observed between the parameter and content of β-sheets and β-turns. Gauche-gauche-gauche conformation of disulphide bridges and ability of tyrosine residues to hydrogen bonds creation improved mixing properties as stability of dough.     

Parvalbumin as a metal-dependent antioxidant

Parvalbumin (PA) is a Ca²⁺-binding protein of vertebrates massively expressed in tissues with high oxygen uptake and respectively elevated level of reactive oxygen species (ROS). To characterize antioxidant properties of PA, antioxidant capacity (AOC) of intact rat α-PA has been explored. ORAC, TEAC and hydrogen peroxide AOC assays evidence conformation-dependent oxidation of the PA. AOC value for the apo-PA 4-11-fold exceeds that for the Ca²⁺-loaded protein. Despite folded conformation of apo-PA, it has AOC equivalent to that of the proteolized protein. The most populated under resting conditions PA form, Mg²⁺-bound PA, has AOC similar to that of apo-PA. ROS-induced changes in absorption spectrum of PA evidence an oxidation of PA's phenylalanines in the ORAC assay. Sensitivity of PA oxidation to its conformation enabled characterization of its metal affinity and pH-dependent behavior: a transition with pK_a of 7.6 has been revealed for the Ca²⁺-loaded PA. Since total AOC of PA under in vivo conditions may reach the level of reduced glutathione, we propose that PA might modulate intracellular redox equilibria and/or signaling in a calcium-dependent manner. We speculate that the oxidation-mediated damage of some of PA-GABAergic interneurons observed in schizophrenia is due to a decline in total AOC of the reduced glutathione–PA pair.     

Beta-turns in proteins

The X-ray atomic co-ordinates from 29 proteins of known sequence and structure were utilized to elucidate 459 β-turns in regions of chain reversals. Tetrapeptides whose αC_iαC_{(i + 3)} distances were below 7 Å and not in a helical region were characterized as β-turns. In addition, β-turns were considered to have hydrogen bonding if their computed O_(i)N_{(i + 3)} distances were ≤3.5 Å. The torsion angles of 26 proteins containing 421 β-turns were examined and classified into 11 bend types based on the (φ, ψ) dihedral angles of the i + 1 and i + 2 bend residues. The average frequency of β-turns is 32% as compared to the 38% helices and 20% β-sheets in the 29 proteins. The most frequently occurring bend residues are Asn, Cys, Asp in the first position, Pro, Ser, Lys in the second position, Asn, Asp, Gly in the third position, and Trp, Gly, Tyr in the fourth position. Residues with the highest β-turn potential in all four positions are Pro, Gly, Asn, Asp, and Ser with the most hydrophobic residues (i.e. Val, IIe, and Leu) showing the lowest bend potential. However, in the region just beyond the β-turns, hydrophobic residues occur with greater frequency than do hydrophilic residues. An environmental analysis of β-turn neighboring residues shows that reverse chain folding is stabilized by anti-parallel β-sheets as well as helix-helix and α-β interactions. The β-turn potential at the 12 positions adjacent to and including the bend were plotted for the 20 amino acids and showed dramatic positional preferences, which may be classified according to the nature of the side-chains. An examination of the 27 β-turns in elastase showed that 21 were found in identical positions as those in α-chymotrypsin. However, only 37 of the 84 bend residues were conserved, indicating that structural similarity may persist despite differences in sequence homology. A survey of residues occupying bend types I′, II′ and III′ showed that Gly appeared most frequently in the third position in bend types I′ and III′ as well as in the second position in bend types II′ and III′. Fourteen hydrogenbonded type II bends were found without a Gly at the third position, contrary to the energy calculations. Eight type VI bends with a cis Pro at the third position were also elucidated.     

Optical properties of an outer membrane lipoprotein from Escherichia coli

The infrared spectrum of a structural lipoprotein from the Escherichia coli outer membrane indicated the lipoprotein had and α-helical conformation but no sign for the existence of β-structures. From circular dichroism spectra of the lipoprotein, the α-helical content of the protein was found to be as high as 88% in 0.01–0.03% sodium dodecyl sulfate in the presence of 10^?5 M Mg²⁺ at pH 7.1 and 23° C. When sodium dodecyl sulfate concentration increased higher than 0.1%, the α-helical content of the lipoprotein decreased to about 57%. Divalent cations, such as Mg²⁺ and Mn²⁺, were found to increase the helical content of the lipoprotein. The high α-helical content of the lipoprotein was observed in a wide range of temperatures (23 to 55° C). The significance of the high α-helical content of the lipoprotein is discussed in light of the three-dimensional molecular models of the lipoprotein proposed previously.     

Stabilizing effects of 2-methylalanine residues on β-turns and α-helices

¹³C-, ¹H-nmr, CD, and x-ray crystallography revealed β-turns of type III for Boc-Gly-L-Ala-Aib-OMe, Boc-L-Ala-Aib-L-Ala-OMe; the 3₁₀-helix for Boc-Aib-L-Ala-Aib-L-Ala-Aib-OMe; and antiparallel arranged α-helices for Boc-L-Ala-Aib-Ala-Aib-Ala-Glu(OBzl)-Ala-Aib-Ala-Aib-Ala-OMe. An N-terminal rigid α-helical segment is found in the polypeptide antibiotics alamethicin, suzukacillin, and trichotoxin. The α-helix dipole is essential for their voltage-dependent pore formation in lipid bilayer membranes, which is explained by a flip-flop gating mechanism based on dipole–dipole interactions of parallel and antiparallel arranged α-helices within oligomeric structures.     

Analysis and prediction of the packing of α-helices against a β-sheet in the tertiary structure of globular proteins

The packing of α-helices and β-sheets in six $\text{α}\text{β}$ proteins (e.g. flavodoxin) has been analysed. The results provide the basis for a computer algorithm to predict the tertiary structure of an $\text{α}\text{β}$ protein from its amino acid sequence and actual assignment of secondary structure.The packing of an individual α-helix against a β-sheet generally involves two adjacent ± 4 rows of non-polar residues on the α-helix at the positions i, i + 4, i + 8, i + 1, i + 5, i + 9. The pattern of interacting β-sheet residues results from the twisted nature of the sheet surface and the attendant rotation of the side-chains. At a more detailed level, four of the α-helical residues (i + 1, i + 4, i + 5 and i + 8) form a diamond that surrounds one particular β-sheet residue, generally isoleucine, leucine or valine. In general, the α-helix sits 10 Å above the sheet and lies parallel to the strand direction.The prediction follows a combinational approach. First, a list of possible β-sheet structures (10⁶ to 10¹⁴) is constructed by the generation of all β-sheet topologies and β-strand alignments. This list is reduced by constraints on topology and the location of non-polar residues to mediate the sheet/helix packing, and then rank-ordered on the extent of hydrogen bonding. This algorithm was uniformly applied to 16 $\text{α}\text{β}$ domains in 13 proteins. For every structure, one member of the reduced list was close to the crystal structure; the root-mean-square deviation between equivalenced C^α atoms averaged 5.6 Å for 100 residues. For the $\text{α}\text{β}$ proteins with pure parallel β-sheets, the total number of structures comparable to or better than the native in terms of hydrogen bonds was between 1 and 148. For proteins with mixed β-sheets, the worst case is glyceraldehyde-3-phosphate dehydrogenase, where as many as 3800 structures would have to be sampled. The evolutionary significance of these results as well as the potential use of a combinatorial approach to the protein folding problem are discussed.     

Secondary structure of proteins associated in thin films

The solid state secondary structure of myoglobin, RNase A, concanavalin A (Con A), poly(L -lysine), and two linear heterooligomeric peptides were examined by both far-uv CD spectroscopy¹ and by ir spectroscopy. The proteins associated from water solution on glass and mica surfaces into noncrystalline, amorphous films, as judged by transmission electron microscopy of carbon-platinum replicas of surface and cross-fractured layer. The association into the solid state induced insignificant changes in the amide CD spectra of all α-helical myoglobin, decreased the molar ellipticity of the α/β RNase A, and increased the molar ellipticity of all-β Con A with no change in the positions of the bands' maxima. High-temperature exposure of the films induced permanent changes in the conformation of all proteins, resulting in less α-helix and more β-sheet structure. The results suggest that the protein α-helices are less stable in films and that the secondary structure may rearrange into β-sheets at high temperature. Two heterooligomeric peptides and poly (L -lysine), all in solution at neutral pH with “random coil” conformation, formed films with variable degrees of their secondary structure in β-sheets or β-turns. The result corresponded to the protein-derived Chou-Fasman amino acid propensities, and depended on both temperature and solvent used. The ir and CD spectra correlations of the peptides in the solid state indicate that the CD spectrum of a “random” structure in films differs from random coil in solution. Formic acid treatment transformed the secondary structure of the protein and peptide films into a stable α-helix or β-sheet conformations. The results indicate that the proteins aggregate into a noncrystalline, glass-like state with preserved secondary structure. The solid state secondary structure may undergo further irreversible transformations induced by heat or solvent. © 1993 John Wiley & Sons, Inc.     

Involvement of Ca2+-stimulated adenosine 5′-triphosphatase in the Ca2+ releasing mechanism of rat liver nuclei

The regulatory role of Ca²⁺-stimulated adenosine 5-triphosphatase (Ca²⁺-ATPase) in Ca²⁺ transport system of rat liver nuclei was investigated. Ca²⁺ uptake and release were determined with a Ca²⁺ electrode. Ca²⁺-ATPase activity was calculated by subtracting Mg²⁺-ATPase activity from (Ca²⁺–Mg²⁺)-ATPase activity. The release of Ca²⁺ from the Ca²⁺-loaded nuclei was evoked progressively after Ca²⁺ uptake with 1.0 mM ATP addition, while it was only slightly in the case of 2.0 mM ATP addition, indicating that the consumption of ATP causes a leak of Ca²⁺ from the Ca²⁺-loaded nuclei. The presence of N-ethylmaleimide (NEM; 0.1 mM) caused an inhibition of nuclear Ca²⁺ uptake and induced a promotion of Ca²⁺ release from the Ca²⁺-loaded nuclei. NEM (0.1 and 0.2 mM) markedly inhibited nuclear Ca²⁺-ATPase activity. This inhibition was completely blocked by the presence of dithiothreitol (DTT; 0.1 and 0.5 mM). Also, DTT inhibited the effect of NEM (0.1 mM) on nuclear Ca²⁺ uptake and release. Meanwhile, verapamil and diltiazem (10 M), a blocker of Ca²⁺ channels, did not prevent the NAD⁺ (1.0 and 2.0 mM), zinc sulfate (1.0 and 2.5 M) and arachidonic acid (10 M)-induced increase in nuclear Ca²⁺ release, suggesting that Ca²⁺ channels do not involve on Ca²⁺ release from the nuclei. These results indicates that an inhibition of nuclear Ca²⁺-ATPase activity causes the decrease in nuclear Ca²⁺ uptake and the release of Ca²⁺ from the Ca²⁺-loaded nuclei. The present finding suggests that Ca²⁺-ATPase plays a critical role in the regulatory mechanism of Ca²⁺ uptake and release in rat liver nuclei.     

Different mechanisms underlying the stimulation of myocardial alpha- and beta-adrenoceptors.

On isolated electrically driven rat left atria (1 Hz) experiments were undertaken in order to characterize further the mechanism of the positive inotropic effect underlying the stimulation of cardiac α-adrenoceptors.— The increase in extracellular Ca²⁺ from 1.25 to 2.5 mM increased the pD₂-value for the α-mimetic effect of phenylephrine by 0.6 log units. Under the same conditions the pD₂-value for its β-mimetic positive inotropic effect increased by only 0.19 log units.—When the amplitude of contraction was diminished by carbachol which is known to inhibit the influx of Ca²⁺ the pD₂-value for the α-mimetic effect of phenylephrine was significantly increased while that for the β-mimetic effect was decreased.—After increasing concentrations of dinitrophenol the pD₂-value for the α-effect of phenylephrine was significantly increased by maximal 0.6 log units whereas that for the β-effect was lowered by 0.25 log units.—In conclusion, all measures favouring the conditions for an enhanced influx of Ca²⁺-elevation of the gradient for Ca²⁺, shortening of the plateau phase of action potential by carbachol, increased efflux of Ca²⁺ by dinitrophenol- are able to increase the pD₂-value, i.e. the affinity of phenylephrine to cardiac α-adrenoceptors, thus demonstrating the importance of an increased Ca²⁺-influx induced by stimulation of α-adrenoceptors.     

α-Adrenergic stimulation of trans-sarcolemma electron efflux in perfused rat heart. Possible regulation of Ca2+-channels by a sarcolemma redox system

The role of trans-sarcolemma membrane electron efflux in the α-adrenergic control of Ca²⁺ influx in perfused rat heart was examined. Electron efflux was measured by monitoring the rate of reduction of extracellular ferricyanide and compared with changes in contractility, as an indirect assessment of changes in cytoplasmic Ca²⁺ concentration. Methoxamine and phenylephrine each increased the rate of ferricyanide reduction from 80 to approx. 114 nmol/min per g wet wt. of heart, with half-maximal activation occurring at 10 μM for each agonist. Activation of the rate of ferricyanide reduction by both 10 μM methoxamine and 10 μM phenylephrine was blocked by the α-adrenergic antagonist, phenoxybenzamine, but not by the β-antagonist, propranolol. Stimulation of the rate of ferricyanide reduction by the α-agonist coincided with the increase in contractility, each reaching maximum values at approx. 80 s. Removal of the α-agonists led to parallel decreases in contractility and the rate of reduction, each returning to pre-stimulation values in approx. 400 s. In addition, the relationship between Ca²⁺ and ferricyanide reduction was examined. Perfusion of the heart with medium containing 6 mM CaCl₂ significantly increased contractility and the rate of ferricyanide reduction. Perfusion of the heart with low Ca²⁺ diminished contractility, did not affect the rate of ferricyanide reduction, but amplified the stimulatory effect of methoxamine on this rate. The increase in ferricyanide reduction by α-adrenergic agonists resulted from a change in the apparent V_max, indicative of an increase in electron efflux sites in the plasma membrane. It is concluded that α-adrenergic control of electron efflux closely parallels changes in contractility and therefore changes in the cytoplasmic concentration of Ca²⁺. The data suggest that α-agonist-mediated changes in electron efflux may lead to Ca²⁺ influx.     

Circular dichroism analysis of the secondary structures of bovine blood coagulation factor IX, factor X, and prothrombin

Analysis of the far-ultraviolet circular dichroism spectrum of bovine blood coagulation factor IX reveals the presence of approximately 14% helical structures 26% -sheets, 20% -turns, and 40% coils. These values are essentially the same for the activation products of this zymogen, factor IXa and factor IXa. Similar analysis for bovine factor X permits calculation of these secondary structural as approximately 11% helices, 31% -structures, 22% -turns, and 36% random structures. Bovine prothrombin contains approximately 12% helical structures, 35% -structures, 24% -turns, and 29% coils. None of these values is substantially altered as a result of increase of thepH from 7.4 to 10.5, or upon addition of Ca²⁺ to a concentration of at least 20 mM. Analysis of the near-ultraviolet spectra of factor IX and prothrombin suggests that several aromatic amino acid residues and the disulfide bond present in their -carboxyglutamic acid-containing regions are exposed to solvent and are perturbed by the abovepH adjustment and Ca²⁺ addition. Similar effects are observed in the case of factor X; in addition, the Trp residue at the amino terminus of the heavy chain appears to be influenced by the abovepH alteration. The results reported in this paper show that these vitamin K-dependent blood coagulation proteins are similar in their ordered secondary structures, which are dominated by -sheets and -turns. Their overall secondary structures are not influenced by Ca²⁺ binding and are stable to alkalinepH changes. However, these same environmental alterations appear to be effective probes of aromatic residues in the -carboxyglutamic acid regions.     

Mouse S100G protein exhibits properties characteristic of a calcium sensor

Bovine S100 G (calbindin D_9k, small Ca²⁺-binding protein of the EF-hand superfamily) is considered as a calcium buffer protein; i.e., the binding of Ca²⁺ practically does not change its general conformation. A set of experimental approaches has been used to study structural properties of apo- and Ca²⁺-loaded forms of mouse S100 G (81.4% identity in amino acid sequence with bovine S100 G). This analysis revealed that, in contrast to bovine S100 G, the removal of calcium ions increases α-helices content of mouse S100 G protein and enhances its accessibility to digestion by α-chymotrypsin. Furthermore, mouse apo-S100 G is characterized by a decreased surface hydrophobicity and reduced tendency for oligomerization. Such behavior is typical of calcium sensor proteins. Apo-state of mouse S100 G still has rather compact structure, which can be cooperatively unfolded by temperature and GdnHCl. Computational analysis of amino acid sequences of S100 G proteins shows that these proteins could be in a disordered state upon a removal of the bound calcium ions. The experimental data show that, although mouse apo-S100 G is flexible compared to the Ca²⁺-loaded state, the apo-form is not completely disordered and preserves some cooperatively meting structure. The origin of the unexpectedly high stability of mouse S100 G can be rationalized by an exceptionally strong association of its N- and C-terminal parts containing the EF-hands I and II, respectively.     

Structural transitions in the intrinsically disordered plant dehydration stress protein LEA7 upon drying are modulated by the presence of membranes

Dehydration stress-related late embryogenesis abundant (LEA) proteins have been found in plants, invertebrates and bacteria. Most LEA proteins are unstructured in solution, but some fold into amphipathic α-helices during drying. The Pfam LEA_4 (Group 3) protein LEA7 from the higher plant Arabidopsis thaliana was predicted to be 87% α-helical, while CD spectroscopy showed it to be largely unstructured in solution and only 35% α-helical in the dry state. However, the dry protein contained 15% β-sheets. FTIR spectroscopy revealed the β-sheets to be largely due to aggregation. β-Sheet content was reduced and α-helix content increased when LEA7 was dried in the presence of liposomes with secondary structure apparently influenced by lipid composition. Secondary structure was also affected by the presence of membranes in the fully hydrated state. A temperature-induced increase in the flexibility of the dry protein was also only observed in the presence of membranes. Functional interactions of LEA7 with membranes in the dry state were indicated by its influence on the thermotropic phase transitions of the lipids and interactions with the lipid headgroup phosphates.     

Mouse models to unravel the role of inhaled pollutants on allergic sensitization and airway inflammation

Background

Nicotinic acetylcholine receptors (nAChR) have been identified on a variety of cells of the immune system and are generally considered to trigger anti-inflammatory events. In the present study, we determine the nAChR inventory of rat alveolar macrophages (AM), and investigate the cellular events evoked by stimulation with nicotine.

Methods

Rat AM were isolated freshly by bronchoalveolar lavage. The expression of nAChR subunits was analyzed by RT-PCR, immunohistochemistry, and Western blotting. To evaluate function of nAChR subunits, electrophysiological recordings and measurements of intracellular calcium concentration ([Ca²⁺]_i) were conducted.

Results

Positive RT-PCR results were obtained for nAChR subunits α3, α5, α9, α10, β1, and β2, with most stable expression being noted for subunits α9, α10, β1, and β2. Notably, mRNA coding for subunit α7 which is proposed to convey the nicotinic anti-inflammatory response of macrophages from other sources than the lung was not detected. RT-PCR data were supported by immunohistochemistry on AM isolated by lavage, as well as in lung tissue sections and by Western blotting. Neither whole-cell patch clamp recordings nor measurements of [Ca²⁺]_i revealed changes in membrane current in response to ACh and in [Ca²⁺]_i in response to nicotine, respectively. However, nicotine (100 μM), given 2 min prior to ATP, significantly reduced the ATP-induced rise in [Ca²⁺]_i by 30%. This effect was blocked by α-bungarotoxin and did not depend on the presence of extracellular calcium.

Conclusions

Rat AM are equipped with modulatory nAChR with properties distinct from ionotropic nAChR mediating synaptic transmission in the nervous system. Their stimulation with nicotine dampens ATP-induced Ca²⁺-release from intracellular stores. Thus, the present study identifies the first acute receptor-mediated nicotinic effect on AM with anti-inflammatory potential.