Effect of phase transitions in hydrated 1,2-dipalmitoylphosphatidylethanolamine bilayers on the spin probe order parameter

The ‘main’ phase transition L_β→L_α of hydrated 1,2-dipalmitoylphosphatidylethanolamine (DPPE) bilayers in excess water affects the ESR order parameter S₃₃ of N-cetyl-N,N-dimethyl-N-tempoylammonium bromide (CAT-16), 5-doxylstearic acid (5-DSA) and 16-doxylstearic acid (16-DSA) spin probes. The ‘pretransition’ and ‘subtransition’ suggested to occur in hydrated DPPE by Chowdhry et al. [(1984) Biophys. J. 45, 901–904] and Silvius et al. [(1986) Biochemistry 25, 4249–4258], respectively, affect exclusively the S₃₃ of CAT-16, but not that of 5-DSA and 16-DSA spin probes. The subtransition occurs about 15 ± 1°C below the main transition.     

Effects of including an N-terminal insertion region and arginine-mimetic side chains in helical peptoid analogues of lung surfactant protein B

Surfactant protein B (SP-B) is one of two helical, amphipathic proteins critical for the biophysical functioning of lung surfactant (LS) and hence is an important therapeutic protein. This small, complex 79mer has three internal disulfide bonds and homodimerizes via another disulfide bridge. A helical, amphipathic 25mer from the amino terminus (SP-B(1-25)) exhibits surface-active properties similar to those of full-length, synthetic SP-B. In previous work, we created helical, non-natural mimics of SP-B(1-25) based on sequence-specific peptoid 17mers and demonstrated their biomimetic surface activity. Like SP-B(1-25), the peptoids were designed to adopt helical structures with cationic and nonpolar faces. Here, we compare the surface activities of six different helical peptoid analogues of SP-B(1-25) to investigate the importance of mimicking its N-terminal insertion domain as well as its two arginine residues, both thought to be important for the peptide's proper function. Although the peptoid analogues of SP-B(1-25) studied here share many similar features and all functionally mimic SP-B(1-25) to some degree, it is notable that small differences in their sequences and side chain chemistries lead to substantial differences in their observed interactions with a lipid film. A peptoid comprising a hydrophobic, helical insertion region with aromatic side chains shows more biomimetic surface activity than simpler peptoids, and even better activity, by comparison to natural LS, than SP-B(1-25). However, the substitution of lysine-like side chains for arginine-like side chains in the peptoid has little effect on biomimetic surface activity, indicating that interactions of the guanidino groups with lipids may not be critical for the function of these SP-B mimics.     

Acute antihypertensive action of nitroxides in the spontaneously hypertensive rat

Tempol is an amphipathic radical nitroxide (N) that acutely reduces blood pressure (BP) and heart rate (HR) in the spontaneously hypertensive rat (SHR). We investigated the hypothesis that the response to nitroxides is determined by SOD mimetic activity or lipophilicity. Groups (n = 6-10) of anesthetized SHRs received graded intravenous doses of Ns: tempol (T), 4-amino-tempo (AT), 4-oxo-tempo (OT), 4-trimethylammonium-2,2,6,6-tetramethylpiperidine-1-oxyl iodide (CAT-1), 3-carbamoyl-proxyl (3-CP), or 3-carboxy-proxyl (3-CTPY). Others received native or liposomal (L) Cu/Zn SOD. T and OT are uncharged, AT is positively charged and cell-permeable, and CAT-1 is positively charged and cell-impermeable. 3-CP and 3-CTPY have five-member pyrrolidine rings, whereas T, AT, OT, and CAT-1 have six-member piperidine rings. T and AT reduced mean arterial pressure (MAP) similarly (-48 +/- 2 mmHg and -55 +/- 8 mmHg) but more (P < 0.05) than OT and CAT-1. 3-CP and 3-CTPY were ineffective. The group mean change in MAP with piperidine Ns correlated with SOD activity (r = -0.94), whereas their ED(50) correlated with lipophilicity (r = 0.89). SOD and L-SOD did not lower BP acutely but reduced it after 90 min (-32 +/- 5 and -31 +/- 6 mmHg; P < 0.05 vs. vehicle). Pyrrolidine nitroxides are ineffective antihypertensive agents. The antihypertensive response to piperidine Ns is predicted by SOD mimetic action, and the sensitivity of response is by hydrophilicity. SOD exerts a delayed hypotensive action that is not enhanced by liposome encapsulation, suggesting it must diffuse to an extravascular site.     

Structure of mini-B, a functional fragment of surfactant protein B, in detergent micelles

Surfactant protein B (SP-B) is essential for normal lung surfactant function, which is in itself essential to life. However, the molecular basis for SP-B's activity is not understood and a high-resolution structure for SP-B has not been determined. Mini-B is a 34-residue peptide with internal disulfide linkages that is composed of the N- and C-terminal helical regions of SP-B. It has been shown to retain similar activity to full-length SP-B in certain in vitro and in vivo studies. We have used solution NMR to determine the structure of Mini-B in the presence of micelles composed of the anionic detergent sodium dodecyl sulfate (SDS). Under these conditions, Mini-B forms two alpha-helices connected by an unstructured loop. Mini-B possesses a strikingly amphipathic surface with a large positively charged patch on one face of the peptide and a large hydrophobic patch on the opposite face. A tryptophan side chain extends outward from the peptide in a position to interact with lipids at the polar/apolar interface. Interhelix interactions are stabilized by both disulfide bonds and by interleaving of hydrophobic side chains from the two helices.     

Interaction of dipyridamole with micelles of lysophosphatidylcholine and with bovine serum albumin: fluorescence studies.

The interaction of the coronary vasodilator dipyridamole with biological systems, protein and membranes has been studied through optical absorption and fluorescence spectroscopies. Using the analysis of the spectra and fluorescence intensity of dipyridamole (DIP) in solution, the interaction of this compound with the transport protein albumin (BSA) and with a model of cell membranes, namely micelles of lysophosphatidylcholine (L-PC), was investigated. Measurements were performed at pH 5.0 and pH 7.0 where the molecule of DIP is fully protonated and partially protonated, respectively. The quenching of fluorescence with nitroxide-stable radicals 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) as well as with acrylamide and iodide allowed the localization of the drug in the polar interface of micelles. Quenching by acrylamide and iodide in L-PC micelles demonstrated the effect of micelle protonation which increased the accessibility of iodide to the chromophore. An effective association constant was obtained both at pH 7.0 (7.5 x 10(3) M-1) and pH 5.0 (2.5 x 10(3) M-1) and a very good agreement with the proposed binding model was observed. The quantum yields of fluorescence data agree very well with the fluorescence lifetimes. The measurement of lifetimes was important to understand the kinetic data obtained from Stern-Volmer plots both of radical, acrylamide and iodide quenching of fluorescence. It was observed that, in the presence of micelles, the kq value increased for TEMPO while decreased for TEMPOL. This result, together with the vanishing solubility of DIP in saturated hydrocarbons and the preferential partition of TEMPO in micelles, suggested the localization of DIP in the polar micellar interface. This is also supported by the enhanced iodide quenching at pH 5.0, constancy of acrylamide quenching in the range of pH 7.0-5.0 and the partition of TEMPO and TEMPOL in SDS micelles. The association constant of DIP to BSA was also estimated both at pH 7.0 (2 x 10(4) M-1) and pH 5.0 (4 x 10(3) M-1). Quenching studies with nitroxide radicals, acrylamide and iodide also suggested the binding of the drug to a hydrophobic region of the protein. At pH 5.0, the protein undergo a conformational change which leads to a loosening of the overall structure so that the accessibility of the nitroxide radicals for DIP is increased at this pH. The differences in kq values at pH 7.0 and pH 5.0 suggested that at pH 7.0 the chromophore is protected in the protein site.(ABSTRACT TRUNCATED AT 400 WORDS)     

An EPR spin probe study of liposomes from sunflower and soybean phospholipids

Comparative properties of lecithin-based liposomes prepared from the mixed phospholipids of sunflower seeds, soybean and egg yolk were investigated by electron paramagnetic resonance (EPR) spectroscopy. For these investigations, stable nitroxide radicals, 1-oxyl-2,2,6,6-tetramethylpiperidin-4-yl 5,7-dimethyladamantane-1-carboxylate (DMAC-TEMPO), 5-doxylstearic acid (5-DSA) and 16-doxylstearic acid (16-DSA) were used as spin probes. Binding of the spin probes to the liposome membranes resulted in a substantial increase of the apparent rotational diffusion correlation times. The EPR spectra of the incorporated nitroxides underwent temperature-dependent changes. For every spin probe, values of apparent enthalpy and entropy of activation were calculated from the temperature dependence of rotational diffusion correlation times via Arrhenius equation. In case of DMAC-TEMPO, the data point to differences between the phospholipid bilayer of liposomes derived from sunflower and soy lecithin, and some similarity between the sunflower and egg yolk liposomes. Anisotropic hyperfine interaction constants of DMAC-TEMPO and 16-DSA included in the liposomes have been analyzed and attributed to different micropolarity of the surroundings of the spin probes. The kinetics of EPR signal decay of DMAC-TEMPO in the presence of 2,2′-azobis(2-amidinopropane) suggest the better stability of the sunflower liposomes to lipid peroxidation as compared to the liposomes prepared from soy lecithin.     

Structure of the antimicrobial peptide tritrpticin bound to micelles: a distinct membrane-bound peptide fold

Tritrpticin is a member of the cathelicidin family, a group of diverse antimicrobial peptides found in neutrophil granules. The three Trp and four Arg residues in the sequence VRRFPWWWPFLRR make this a Trp-rich cationic peptide. The structure of tritrpticin bound to membrane-mimetic sodium dodecyl sulfate micelles has been determined using conventional two-dimensional NMR methods. It forms two adjacent turns around the two Pro residues, a distinct fold for peptide-membrane interaction. The first turn involves residues 4-7, followed immediately by a second well-defined 3(10)-helical turn involving residues 8-11. The hydrophobic residues are clustered together and are clearly separated from the basic Arg residues, resulting in an amphipathic structure. Favorable interactions between the unusual amphipathic fold and the micelle surface are probably key to determining the peptide structure. NMR studies of the peptide in the micelle in the presence of the spin-label 5-doxylstearic acid determined that tritrpticin lies near the surface of the micelle, where its many aromatic side chains appear to be equally partitioned into the hydrophilic-hydrophobic interface. Additional fluorescence studies confirmed that the tryptophan residues are inserted into the micelle and are partially protected from the effects of the soluble fluorescence quencher acrylamide.     

Detection, characterization, and quenching of the intrinsic fluorescence of bovine heart cytochrome c oxidase

The intrinsic fluorescence of lauryl maltoside solubilized bovine heart cytochrome c oxidase has been determined to arise from tryptophan residues of the oxidase complex. The magnitude of the fluorescence is approximately 34% of that from n-acetyltryptophanamide (NATA). This level of fluorescence is consistent with an average heme to tryptophan distance of 30 A. The majority of the fluorescent tryptophan residues are in a hydrophobic environment as indicated by the fluorescence emission maximum at 328 nm and the differing effectiveness of the quenching agents: Cs+, I-, and acrylamide. Cesium was ineffective up to a concentration of 0.7 M, whereas quenching by the other surface quenching agent, iodide, was complex. Below 0.2 M, KI was ineffective whereas between 0.2 and 0.7 M 15% of the tryptophan fluorescence was found to be accessible to iodide. This pattern indicates that protein structural changes were induced by iodide and may be related to the chaotropic character of KI. Acrylamide was moderately effective as a quenching agent of the oxidase fluorescence with a Stern-Volmer constant of 2 M-1 compared with acrylamide quenching of NATA and the water-soluble enzyme aldolase having Stern-Volmer constants of 12 M-1 and 0.3 M-1, respectively. There was no effect of cytochrome c on the tryptophan emission intensity from cytochrome c oxidase under conditions where the two proteins form a tight, 1:1 complex, implying that the tryptophan residues near the cytochrome c binding site are already quenched by energy transfer to the homes of the oxidase. The lauryl maltoside concentration used to solubilize the enzyme did not affect the fluorescence of NATA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Luminescence studies of saccharide binding to wheat germ agglutinin (lectin).

The fluorescence and phosphorescence emission of wheat germ agglutinin are reported. Fluorescent tryptophan residues of wheat germ agglutinin are found highly exposed to solvent: fluorescence quenching induced by temperature fits with a single Arrhenius critical energy close to that of tryptophan in solution; the whole fluorescence emission is susceptible to iodide ion quenching and data reveal the homogeneity of fluorescence arising from only one type of tryptophan exposition. Energy transfers are analyzed at singlet and triplet state level. Tyrosine fluorescence at 25 degrees C is very weak. Results obtained from the relative excitation fluorescence quantum yield and from intrinsic fluorescence polarization show that a large amount of energy absorbed by tyrosine at 280 nm is transferred to tryptophan residues. However, tyrosine fluorescence is highly increased at 70 degrees C although disulfide bridges are not reduced. The phosphorescence spectrum at 77 K in 50% ethylene glycol is finely structured with several resolved vibrational bands at 405, 432 and 455 nm. Phosphorescence decay can be fitted with a single exponential. Lifetime is independent of excitation wave-length. Its value is very close to that of free tryptophan. Influence of tri-N-acetyl-chitotriose binding on luminescence properties are investigated. Results are analyzed in terms of steric tryptophan-ligand relationships. It is shown that all the fluorescent chromophores are concerned by the ligand binding but all fluorescence emission is still susceptible to iodide ion quenching. There is no change induced in energy transfer at the singlet state level and no modification in triplet state population.     

Analysis of a conserved hydrophobic pocket important for the thermostability of Bacillus pumilus chloramphenicol acetyltransferase (CAT-86)

Site-directed mutagenesis was carried out on Bacillus pumilus chloramphenicol acetyltransferase (CAT-86) to determine the effects of substitution at a conserved hydrophobic pocket identified earlier as important for thermostability. Mutations were introduced that would substitute residues at consensus positions 33, 191 and 203 in the enzyme, both individually and in combination. Two mutants, SDM1 (CAT-86 Y33F, A203V) and SDM5 (CAT-86 A203I), were more thermostable than wild-type and two mutants, SDM4 (CAT-86 I191V) and SDM7 (CAT-86 A203G), were less stable. Reconstruction of the residues of this hydrophobic pocket to that of a more thermostable CAT-R387 enzyme pocket (as a Y33F, I191V, A203V triple mutant) increased the thermostability of the enzyme above the wild-type, but its stability was less than that of SDM1 and SDM5. The K(m) values of the mutant enzymes for chloramphenicol and acetyl-CoA were essentially unaltered (in the ranges 15-30 and 26-35 microM respectively) and the specific activity of purified enzyme was in the range 270-710 units/mg protein. The possible effects of the amino acid substitutions on the CAT-86 structure were determined by homology modelling. A reduction in conformational strain and optimized hydrophobic interactions are predicted to be responsible for the increased thermostability of the SDM1 and SDM5 mutants.     

The role of charged amphipathic helices in the structure and function of surfactant protein B.

Surfactant protein B (SP-B) is essential for normal lung surfactant function. Theoretical models predict that the disulfide cross-linked, N- and C-terminal domains of SP-B fold as charged amphipathic helices, and suggest that these adjacent helices participate in critical surfactant activities. This hypothesis is tested using a disulfide-linked construct (Mini-B) based on the primary sequences of the N- and C-terminal domains. Consistent with theoretical predictions of the full-length protein, both isotope-enhanced Fourier transform infrared (FTIR) spectroscopy and molecular modeling confirm the presence of charged amphipathic alpha-helices in Mini-B. Similar to that observed with native SP-B, Mini-B in model surfactant lipid mixtures exhibits marked in vitro activity, with spread films showing near-zero minimum surface tensions during cycling using captive bubble surfactometry. In vivo, Mini-B shows oxygenation and dynamic compliance that compare favorably with that of full-length SP-B. Mini-B variants (i.e. reduced disulfides or cationic residues replaced by uncharged residues) or Mini-B fragments (i.e. unlinked N- and C-terminal domains) produced greatly attenuated in vivo and in vitro surfactant properties. Hence, the combination of structure and charge for the amphipathic alpha-helical N- and C-terminal domains are key to SP-B function.     

Lipid bilayer surface association of lung surfactant protein SP-B, amphipathic segment detected by flow immunofluorescence.

Lung surfactant protein, SP-B, and synthetic amphipathic peptides derived from SP-B were studied in model lung surfactant lipid bilayers by immunofluorescent labeling. Liposomes were formed by hydrating a lipid film on the glass viewing port of a temperature controlled flow chamber. Membrane associated peptides were detected by epifluorescence optical microscopy of the binding of anti-peptide polyclonal monospecific antibodies and FITC-conjugated secondary antibodies added to buffer contained in the flow chamber. Liposomes were bound by antibody to residues 1-25 of SP-B if formed from lipid films containing the 1-25 peptide, (SP-B(1-25)), or if SP-B(1-25) was added to already formed liposomes in buffer solution. The distribution of antigen-antibody complex was temperature dependent with aggregation occurring at greater than or equal to 30 degrees C. Surface association was not detected in liposomes formed from lipid films containing the 49-66 peptides (SP-B(49-66)), using an antibody to the 49-66 peptide, or to a synthetic version of the SP-B protein, (SP-B(1-78)), using both antibodies to the 49-66 peptide and the 1-25 peptide. The detection of SP-B(1-78) with antibody to the 49-66 sequence was only possible after reducing SP-B(1-78) with dithiothreitol, suggesting that the COOH-terminus of the full monomer protein is accessible to the bulk aqueous environment unlike the COOH-terminal peptide. The size, number of layers, and fluidity of the liposomes were not altered by protein or peptides, although they were affected by lipid composition and temperature.     

Effect of thiol-reactive reagents on the permeability of fish erythrocyte membrane for spin-labelled non-electrolytes

Carp erythrocytes were treated with p-chloromercuribenzoate or N-ethylmaleimide. It was observed that these thiol-group inhibitors decreased the transport of spin-labelled hydrophilic compound, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, and increased the transport rate of more hydrophobic 2,2,6,6-tetramethylpiperidine-1-oxyl.     

The spectroscopic analysis for binding of amphipathic and antimicrobial model peptides containing pyrenylalanine and tryptophan to lipid bilayer

The binding of basic amphipathic fluorescent peptides to lipid bilayers was studied in relation to their antimicrobial activity. Four fluorescent peptides containing pyrenylalanine or tryptophan in an amphipathic basic peptide (4(4] consisting of four repeated units of tetrapeptide, -L-Leu-L-Ala-L-Arg-L-Leu-, were found to have antimicrobial activities against Gram-positive bacteria and to take conformations with fairly high alpha-helical content both in aqueous solutions and liposomes. The fluorescence spectroscopic data suggested that the pyrenylalanine-peptide existed as a monomer in methanol or liposomes but as an oligomer in aqueous solutions to form an excimer between pyrenylalanyl residues. Upon binding with liposomes, the fluorescence spectra of the tryptophan-containing peptide shifted to a shorter wavelength, indicating the change in the state of tryptophan from hydrophilic environment to hydrophobic one. The analytical data for the quenching of tryptophan fluorescence by I- anion suggest that the tryptophan residue in the peptide is not deeply buried in the hydrophobic core of the bilayers. Based on these findings, it is suggested that the peptides may interact with liposomes in such a manner that they lie parallel to the surface of the lipid bilayers with their hydrophobic regions shallowly in the amphipathic moiety of the bilayers.     

Amphipathic alpha-helix bundle organization of lipid-free chicken apolipoprotein A-I

Apolipoprotein A-I (apoA-I), the major protein component of plasma high-density lipoprotein (HDL), exists in alternate lipid-free and lipid-bound states. Among various species, chicken apoA-I possesses unique structural properties: it is a monomer in the lipid-free state and it is virtually the sole protein component of HDL. Near-UV circular dichroism (CD) spectroscopic studies provide evidence that chicken apoA-I undergoes a major conformational change upon binding to lipid, while far-UV CD data indicate its overall alpha-helix content is maintained during this transition. The fluorescence emission wavelength maximum (excitation 295 nm) of the tryptophans in apoA-I (W74 and W107) displayed a marked blue shift in both the lipid-free (331 nm) and HDL-bound (329 nm) states, compared to free tryptophan in solution. The effect of aqueous quenchers on tryptophan fluorescence was determined in lipid-free, dimyristoylphosphatidylcholine (DMPC)- and HDL-bound states. The most effective quencher in the lipid-free and HDL-bound states was acrylamide, giving rise to Ksv values of 1.6 +/- 0.1 and 1.2 +/- 0.1 M-1, respectively. Together, these data suggest that a hydrophobic environment around the two tryptophan residues (W74 and W107) is maintained in alternate conformations of the protein. To further probe the molecular organization of lipid-free apoA-I, its effect on the fluorescence properties of 8-anilino-1-naphthalenesulfonic acid (ANS) was determined. Human and chicken apoA-I induced a similar increase in ANS fluorescence quantum yield, in keeping with the hypothesis that these proteins adopt a similar global fold in the absence of lipid. When considered with near- and far-UV CD experiments, the data support a model in which lipid-free chicken apoA-I is organized as an amphipathic alpha-helix bundle. In other studies, lipid-soluble quenchers, 5-, 7-, 10-, and 12-DOXYL stearic acid (DSA), were employed to investigate the depth of penetration of apoA-I into the surface monolayer of spherical HDL particles. 5-DSA was the most effective quencher, suggesting that apoA-I tryptophan residues localize near the surface monolayer, providing a structural rationale for the reversibility of apoA-I-lipoprotein particle interactions.     

Examination of sodium/glucose cotransport by using a visible glucose analogue

The glucose derivative, 2,2,6,6-tetramethylpiperidine-1-oxylglucose (TEMPO-glucose) was synthesized and examined for its ability to substitute for glucose as a substrate for the intestinal brush border membrane Na+/glucose cotransporter. TEMPO-glucose inhibited Na(+)-dependent phlorizin binding with an apparent KI of 18 microM and Na(+)-dependent glucose uptake with an apparent KI of 70 microM. The transport competence of TEMPO-glucose was examined by using two measures of transport. The first involved comparing the reversal of trans Na+ inhibition by D-glucose and TEMPO-glucose. The second directly examined Na(+)-dependent TEMPO-glucose uptake by using TEMPO-glucose quenching of intravesicular fluorescein sulfonate fluorescence. Tryptophan fluorescence was sensitive to TEMPO-glucose in a Na(+)-dependent, glucose-inhibitable manner. The bulk of these tryptophans appeared to be located in hydrophobic environments based on Cs(+)-insensitivity. With the reconstituted cotransporter, TEMPO-glucose, and tryptophan quench reagents, the cotransporter was compared in three transport modes: zero trans uptake, zero trans uptake in the presence of a shunt of membrane potential, and substrate exchange. The results suggest that the cotransporter conformation varies depending on its mode of operation and that TEMPO-glucose may be a useful probe for localizing amino acid residues involved in glucose transport.     

Cucurbitacin delta 23-reductase from the fruit of Cucurbita maxima var. Green Hubbard. Physicochemical and fluorescence properties and enzyme-ligand interactions.

Cucurbitacin delta 23-reductase from Cucurbita maxima var. Green Hubbard fruit displays an apparent Mr of 32,000, a Stokes radius of 263 nm and a diffusion coefficient of 8.93 X 10(-7) cm2 X s-1. The enzyme appears to possess a homogeneous dimeric quaternary structure with a subunit Mr of 15,000. Two tryptophan and fourteen tyrosine residues per dimer were found. Emission spectral properties of the enzyme and fluorescence quenching by iodide indicate the tryptophan residues to be buried within the protein molecule. In the pH range 5-7, where no conformational changes were detected, protonation of a sterically related ionizable group with a pK of approx. 6.0 markedly influenced the fluorescence of the tryptophan residues. Protein fluorescence quenching was employed to determine the dissociation constants for binding of NADPH (Kd 17 microM), NADP+ (Kd 30 microM) and elaterinide (Kd 227 microM). Fluorescence energy transfer between the tryptophan residues and enzyme-bound NADPH was observed.     

Human surfactant polypeptide SP-B. Disulfide bridges, C-terminal end, and peptide analysis of the airway form.

Human hydrophobic surfactant polypeptide, SP-B, purified from lung tissue by exclusion chromatography in organic solvents, has been characterized. The polypeptide is 79 residues long, has a C-terminal methionine, and contains seven Cys residues. Native human SP-B lacks free thiol groups. Three intrachain disulfide bridges were defined, linking Cys8 to Cys77, Cys11 to Cys71 and Cys35 to Cys46. The remaining Cys48 is concluded to link the protein chains into homodimers via an interchain disulfide to its counterpart in a second SP-B polypeptide. These SS bridges are identical to those in the porcine form and confirm a consestant and unique disulfide pattern for SP-B polypeptides in general.     

Myb-DNA recognition: role of tryptophan residues and structural changes of the minimal DNA binding domain of c-Myb

The Myb oncoprotein specifically binds DNA by a domain composed of three imperfect repeats, R1, R2, and R3, each containing 3 tryptophans. The tryptophan fluorescence of the minimal binding domain, R2R3, of c-Myb was used to monitor structural flexibility changes occurring upon DNA binding to R2R3. The quenching of the Trp fluorescence by DNA titration shows that four out of the six tryptophans are involved in the formation of the specific R2R3-DNA complex and the environment of the tryptophan residues becomes more hydrophobic in the complex. The fluorescence intensity quenching of the tryptophans by binding of R2R3 to DNA is consistent with the decrease of the decay time: 1.46 ns for free R2R3 to 0.71 ns for the complexed protein. In the free R2R3, the six tryptophans are equally accessible to the iodide and acrylamide quenchers with a high collisional rate constant (4 x 10(9) and 3 x 10(9) M-1 s-1, respectively), indicating that R2R3 in solution is very flexible. In the R2R3-DNA complex, no Trp fluorescence quenching is observed with iodide whereas all tryptophan residues remain accessible to acrylamide with a collisional rate constant slightly slower than that in the free state. These results indicate that (i) a protein structural change occurs and (ii) the R2R3 molecule keeps a high mobility in the complex.The complex formation presents a two-step kinetics: a fast step corresponding to the R2R3-DNA association (7 x 10(5) M-1 s-1) and a slower one (0.004 s-1), which should correspond to a structural reorganization of the protein including a reordering of the water molecules at the protein-DNA interface.     

Production in Escherichia coli of a recombinant C-terminal truncated precursor of surfactant protein B (rproSP-B Delta C). Structure and interaction with lipid interfaces

SP-B, a protein absolutely required to maintain the lungs open after birth, is synthesized in the pneumocytes as a precursor containing C-terminal and N-terminal domains flanking the mature sequence. These flanking-domains are cleaved to produce mature SP-B, coupled with its assembly into pulmonary surfactant lipid-protein complexes. In the present work we have optimized over-expression in Escherichia coli and purification of rproSP-B(DeltaC), a recombinant form of human proSP-B lacking the C-terminal flanking peptide, which is still competent to restore SP-B function in vivo. rProSP-B(DeltaC) has been solubilized, purified and refolded from bacterial inclusion bodies in amounts of about 4 mg per L of culture. Electrophoretic mobility, immunoreactivity, N-terminal sequencing and peptide fingerprinting all confirmed that the purified protein had the expected mass and sequence. Once refolded, the protein was soluble in aqueous buffers. Circular dichroism and fluorescence emission spectra of bacterial rproSP-B(DeltaC) indicated that the protein is properly folded, showing around 32% alpha-helix and a mainly hydrophobic environment of its tryptophan residues. Presence of zwitterionic or anionic phospholipids vesicles caused changes in the fluorescence emission properties of rproSP-B(DeltaC) that were indicative of lipid-protein interaction. The association of this SP-B precursor with membranes suggests an intrinsic amphipathic character of the protein, which spontaneously adsorbs at air-liquid interfaces either in the absence or in the presence of phospholipids. The analysis of the structure and properties of recombinant proSP-B(DeltaC) in surfactant-relevant environments will open new perspectives on the investigation of the mechanisms of lipid and protein assembly in surfactant complexes.