The K edges of the heme iron in the x-ray absorption spectra of native and carboxymethylated cytochrome c

The K-absorption edges of the heme iron have been determined from the x-ray absorption spectra of native (Nat) and carboxymethylated (Cm) cytochrome c. The data collected at pH 6.5 and 10 and in both the oxidation states of the hemes indicate that the latter parameter is the one responsible for the observed chemical shifts of the absorption edges in both the cytochromes. The implications regarding a proposed structural model for Cm cytochrome c are discussed     

Bis(monoacylglycero)phosphate Forms Stable Small Lamellar Vesicle Structures: Insights into Vesicular Body Formation in Endosomes

Bis(monoacylglycero)phosphate (BMP) is an unusually shaped lipid found in relatively high percentage in the late endosome. Here, we report the characterization of the morphology and molecular organization of dioleoyl-BMP (DOBMP) with dynamic light scattering, transmission electron microscopy, nuclear magnetic resonance (NMR) spectroscopy, and electron paramagnetic resonance spectroscopy. The morphology of hydrated DOBMP dispersions varies with pH and ionic strength, and DOBMP vesicles are significantly smaller in diameter than phosphatidylcholine dispersions. At neutral pH, DOBMP forms highly structured, clustered dispersions 500 nm in size. On the other hand, at acidic pH, spherically shaped vesicles are formed. NMR and spin-labeled electron paramagnetic resonance demonstrate that DOBMP forms a lamellar mesophase with acyl-chain packing similar to that of other unsaturated phospholipids. ³¹P NMR reveals an orientation of the phosphate group in DOBMP that differs significantly from that of other phospholipids. These macroscopic and microscopic structural characterizations suggest that the biosynthesis of BMP on the inner luminal membrane of maturing endosomes may possibly produce budded vesicles high in BMP content, which form small vesicular structures stabilized by the physical properties of the BMP lipid.     

Structural investigation of the capsular polysaccharide produced by a novel Klebsiella serotype (SK1). Location of O-acetyl substituents using NMR and MS techniques

The capsular polysaccharide of Klebsiella SK1 was investigated by methylation analysis, Smith degradation, and ¹H NMR spectroscopy. The oligosaccharides (P1 and P2) obtained by bacteriophage ΦSK1 degradation of the polymer were studied by methylation analysis, and 1D- and 2D-NMR spectroscopy. The resulting data showed that the patent repeating unit is a branched pentasaccharide having a structure identical to the revised structure recently proposed for Klebsiella serotype K8 capsular polysaccharide.

The 2D-NMR data showed that one third of the glucuronic acid residues in the SK1 polymer are acetylated at O-2, O-3, or O-4. FABMS studies confirmed the presence of monoacetylated glucuronic acid residues. Thus, the relationship between the Klebsiella K8 and SK1 polymers is akin to that found for Klebsiella polysaccharides K30 and K33, which have been typed as serologically distinct yet their structures differ only in the degree of acetylation.     

Nickel(II)—bleomycin: spectral investigation of the metal binding site

Bleomycin (blm) solutions containing the nickel(II) ion have been investigated through ¹H nmr, ligand field and circular dichroism spectroscopies. It has been found the blm binds the metal ion in a pH dependent fashion. The spectral data are consistent with the presence of at least two species. It is suggested that in the low pH region blm binds to nickel(II) through the β-aminoalanino residue, whereas in the high pH region, the 4-amino-pyrimidine, imidazole, and amido group of β-hydroxy-histidine are also involved in coordination.     

Thermodynamic characterization of an equilibrium folding intermediate of staphylococcal nuclease.

High-sensitivity differential scanning calorimetry and CD spectroscopy have been used to probe the structural stability and measure the folding/unfolding thermodynamics of a Pro117-->Gly variant of staphylococcal nuclease. It is shown that at neutral pH the thermal denaturation of this protein is well accounted for by a 2-state mechanism and that the thermally denatured state is a fully hydrated unfolded polypeptide. At pH 3.5, thermal denaturation results in a compact denatured state in which most, if not all, of the helical structure is missing and the beta subdomain apparently remains largely intact. At pH 3.0, no thermal transition is observed and the molecule exists in the compact denatured state within the 0-100 degrees C temperature interval. At high salt concentration and pH 3.5, the thermal unfolding transition exhibits 2 cooperative peaks in the heat capacity function, the first one corresponding to the transition from the native to the intermediate state and the second one to the transition from the intermediate to the unfolded state. As is the case with other proteins, the enthalpy of the intermediate is higher than that of the unfolded state at low temperatures, indicating that, under those conditions, its stabilization must be of an entropic origin. The folding intermediate has been modeled by structural thermodynamic calculations. Structure-based thermodynamic calculations also predict that the most probable intermediate is one in which the beta subdomain is essentially intact and the rest of the molecule unfolded, in agreement with the experimental data. The structural features of the equilibrium intermediate are similar to those of a kinetic intermediate previously characterized by hydrogen exchange and NMR spectroscopy.     

Highly aligned lipid membrane systems in the physiologically relevant "excess water" condition

The "excess water" condition in biologically relevant systems is met when a membrane mesophase coexists with excess bulk water. Further addition of water to such a system results in no change to any of the system's physical properties (e.g., transition temperature, repeat spacing, and structural mesophases). Moreover, because biological membranes are anisotropic systems, many of their properties are best studied using aligned samples. Although model membrane systems are routinely aligned, they have traditionally been hydrated with water vapor. It is well known that membranes exposed to water vapor at 100% humidity do not imbibe the same quantity of water as a sample in contact with liquid water. As such, membranes that have been hydrated with water vapor have physical properties different from those of membranes dispersed in water. Because of this shortcoming, aligned membranes have not been utilized to their full potential. Here we present a novel and simple method of aligning model membrane systems under conditions of excess water, which will make possible, for the first time, a variety of techniques (e.g., neutron and x-ray diffraction, nuclear magnetic resonance, electron spin resonance, attenuated total reflection infrared spectroscopy, etc.) for studying such systems under physiologically relevant conditions. In addition, when dealing with samples of limited availability, the system allows for the conditions (buffer pH and ionic strength) to be altered without any effect on the sample's alignment.     

Bicelle samples for solid-state NMR of membrane proteins

Magnetically aligned bicelles are an excellent medium for structure determination of isotopically labeled membrane proteins by solid-state NMR spectroscopy. Bicelles are a mixture of long- and short-chain phospholipids that form bilayers in an aqueous medium and align spontaneously in a high magnetic field, for example that of an NMR spectrometer with a 1H resonance frequency between 400 and 900 MHz. Importantly, membrane proteins have been shown to be fully functional in these fully hydrated, planar bilayers under physiological conditions of pH and temperature. We describe a protocol for preparing stable protein-containing bicelles samples that yield high-resolution solid-state NMR spectra. Depending on the details of the protein and its behavior in the lipids, the time for sample preparation can vary from a few hours to several days.     

Initial structural and dynamic characterization of the M2 protein transmembrane and amphipathic helices in lipid bilayers

Amphipathic helices in membrane proteins that interact with the hydrophobic/hydrophilic interface of the lipid bilayer have been difficult to structurally characterize. Here, the backbone structure and orientation of an amphipathic helix in the full-length M2 protein from influenza A virus has been characterized. The protein has been studied in hydrated DMPC/DMPG lipid bilayers above the gel to liquid-crystalline phase transition temperature by solid-state NMR spectroscopy. Characteristic PISA (Polar Index Slant Angle) wheels reflecting helical wheels have been observed in uniformly aligned bilayer preparations of both uniformly 15N labeled and amino acid specific labeled M2 samples. Hydrogen/deuterium exchange studies have shown the very slow exchange of some residues in the amphipathic helix and more rapid exchange for the transmembrane helix. These latter results clearly suggest the presence of an aqueous pore. A variation in exchange rate about the transmembrane helical axis provides additional support for this claim and suggests that motions occur about the helical axes in this tetramer to expose the entire backbone to the pore.     

Control of the yeast cell cycle by protein synthesis

The increased synthesis of ribosomal RNA (rRNA) is correlated with enhanced cell proliferation, and it has been suggested that rRNA metabolism may have a regulatory role in the progression of the cell cycle. Alternatively, it might be the ensuing more active protein synthesis that drives the cell cycle progression. We have found that treatment with low doses of cycloheximide dissociates rRNA and protein synthesis. In fact, after the addition of cycloheximide the protein synthesis rate is strongly inhibited, whereas the rate of rRNA synthesis is unaffected for some time. The progression of the cell cycle, monitored as analysis of DNA distribution by flow cytometry and as bud emergence, is quickly and largely inhibited, thus indicating that a sustained rRNA metabolism is not sufficient to allow continuous cycle progression. The effects of cycloheximide on the daughter and mother duplication times, on the mean cell volume, and on the volume at budding were also analyzed. The results suggest that protein synthesis, rather than rRNA synthesis, may have a key role in the control of cell cycle progression in Saccharomyces cerevisiae.     

The epr spectra of the inhibitor derivatives of cobalt carbonic anhydrase

The epr spectra at 4.2 K of inhibitor derivatives of cobalt carbonic anhydrase have been recorded. The spectra can be grouped into two classes according to whether the low-field signal is broad or sharp and with g ranges of 6.1–6.8, 2.3–2.9, 1.6–1.8, and 5.8–6.2, 2.2–2.8, 1.5–1.8, respectively. The two kinds of spectra have been empirically related to the features of the room-temperature solution electronic spectra. A third kind of epr spectrum with a single broad signal is obtained when the inhibitor is in large excess.The possibility of using the epr spectra for deducing the geometry of cobalt enzymes is discussed.     

pH-Dependent Interactions Guide the Folding and Gate the Transmembrane Pore of the β-Barrel Membrane Protein OmpG

The physical interactions that switch the functional state of membrane proteins are poorly understood. Previously, the pH-gating conformations of the β-barrel forming outer membrane protein G (OmpG) from Escherichia coli have been solved. When the pH changes from neutral to acidic the flexible extracellular loop L6 folds into and closes the OmpG pore. Here, we used single-molecule force spectroscopy to structurally localize and quantify the interactions that are associated with the pH-dependent closure. At acidic pH, we detected a pH-dependent interaction at loop L6. This interaction changed the (un)folding of loop L6 and of β-strands 11 and 12, which connect loop L6. All other interactions detected within OmpG were unaffected by changes in pH. These results provide a quantitative and mechanistic explanation of how pH-dependent interactions change the folding of a peptide loop to gate the transmembrane pore. They further demonstrate how the stability of OmpG is optimized so that pH changes modify only those interactions necessary to gate the transmembrane pore.     

Chemical intermediates in dopamine oxidation by tyrosinase,and kinetic studies of the process

A minor pathway for dopamine oxidation to dopaminochrome, by tyrosinase, is proposed. Characterization of intermediates in this oxidative reaction and stoichiometric determination have both been undertaken. After oxidizing dopamine with mushroom tyrosinase or sodium periodate in a pH range from 6.0 to 7.0, it was spectrophotometrically possible to detect o-dopaminoquinone-H⁺ as the first intermediate in this pathway. The steps for dopamine transformation to dopaminochrome are as follows: dopamine → o-dopaminequinone-H⁺ → o-dopaminequinone → leuko-dopaminochrome → dopaminochrome. No participation of oxygen was detected in the conversion of leukodopaminochrome to dopaminochrome. Scanning spectroscopy and graphical analysis of the obtained spectra also verified that dopaminequinone-H⁺ was transformed into aminochrome in a constant ratio. The stoichiometry equation for this conversion is 2 o-dopaminequinone-H⁺ → dopamine + dopaminochrome. The pathway for dopamine oxidation to dopaminochrome by tyrosinase has been studied as a system of various chemical reactions coupled to an enzymatic reaction. A theoretical and experimental kinetic approach is proposed for such a system; this type of mechanism has been named “Enzymatic-chemical-chemical” (E_ZCC). Rate constants for the implied chemical steps at different pH and temperature values have been evaluated from the measurement of the lag period arising from the accumulation of dopaminochrome that took place when dopamine was oxidized at acid pH. The thermodynamic activation parameters of the chemical steps, the deprotonation of dopaminequinone-H⁺ to dopaminequinone, and the internal cyclization of dopaminequinone to leukodopaminochrome have been calculated.     

Role of head group structure in the phase behavior of amino phospholipids. 1. Hydrated and dehydrated lamellar phases of saturated phosphatidylethanolamine analogues

Analogues of dimyristoylphosphatidylethanolamine (DMPE) have been prepared with head groups modified by N-alkylation, alkylation of carbon 2 of the ethanolamine group, or interposition of extra methylene segments between the phosphoryl and amino groups. The phases formed by these lipids in aqueous dispersions have been examined by high-sensitivity differential scanning calorimetry and Raman spectroscopy. All of the DMPE analogues examined, excepting N-methyl-DMPE but including N-ethyl-DMPE, form hydrated gel phases that are metastable with respect to a dehydrated "high-melting" solid phase that has been observed previously for DMPE itself. The properties and the conditions of formation of this high-melting phase are qualitatively distinct from those of the "subgel" phase, which is observed for dipalmitoylphosphatidylcholine and for some of the DMPE analogues examined in this study. The high-melting phases of different DMPE analogues all exhibit similarly tight packing of the acyl chains, which however do not pack according to a single type of subcell that can be universally and specifically associated with this phase. Increasing the size of the PE head group invariably decreases the melting temperature of the hydrated gel phase, even when the normal hydrogen-bonding capability of the head group is preserved. By contrast, addition of larger alkyl substituents to either the amino group or carbon 2 of the ethanolamine moiety substantially increases the transition temperature of the high-melting solid phase, indicating that the contributions of the head group to the energies of the hydrated gel and the high-melting phases are fundamentally different. Our results suggest that the head group structural requirements for a neutral phospholipid to form stable hydrated bilayers are rather stringent, a fact that may explain the overwhelming predominance of only a few such head group structures in most natural membranes.     

Thermal and structural behavior of natural cerebroside 3-sulfate in bilayer membranes

Differential scanning calorimetry (DSC), polarizing microscopy and X-ray diffraction studies have been performed on dry and hydrated natural bovine brain sulfatides. Dry sulfatide fractions exhibit a high temperature transition (delta H = 6.6 kcal/mol sulfatide) at 87.3 degrees C. X-ray diffraction shows this transition to be associated with a hydrocarbon chain order-disorder transformation between two lamellar phases. Hydrated sulfatide dispersions undergo a complex chain order-disorder transition (delta H = 7.5 kcal/mol sulfatide) at 32 degrees C with two peak temperatures at 35 degrees C and 47 degrees C. Structural studies performed on hydrated liquid-crystal sulfatide dispersions at 75 degrees C verify the existence of a bilayer structure over the 16 wt.% to 50 wt.% phosphate buffer (pH = 7.4) range. The interbilayer separation between galactosyl-3-sulfate groups averages 48 A as the multilamellar bilayers swell with the addition of phosphate buffer. The formation of micellar phases is not observed at high water contents. The comparison of the structural characteristics of dry and hydrated sulfatides with structural data for dry and hydrated bovine brain non-sulfated glycolipid (cerebroside) is discussed in molecular terms.     

Glycosidase inhibitors of gem-diamine 1-N-iminosugars. structures in media of enzyme assays

The relationships between structures and inhibitory activities of glycosidase inhibitors of gem-diamine 1-N-iminosugars in media of enzyme assays have been investigated. It has been proved that gem-diamine 1-N-iminosugar smoothly undergoes a structural change to a hydrated ketone or its derivative via a hemiaminal in the media (pH 5.0-6.3), and that the products generated in the media as well as the parent gem-diamine 1-N-iminosugars potently inhibit glycosidases.     

Bis(monoacylglycero)phosphate and ganglioside GM1 spontaneously form small homogeneous vesicles at specific concentrations

The morphology and size of hydrated lipid dispersions of bis(monoacylglycero)phosphate (BMP) mixed with varying mole percentages of the ganglioside GM1 were investigated by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Electron paramagnetic resonance (EPR) spectroscopy of these same mixtures, doped at 0.5 mol% with doxyl labeled lipids, was used to investigate acyl-chain packing. Results show that for 20-30% GM1, hydrated BMP:GM1 mixtures spontaneously form small spherical vesicles with diameters ∼100 nm and a narrow size distribution profile. For other concentrations of GM1, hydrated dispersions with BMP have non-spherical shapes and heterogeneous size profiles, with average vesicle diameters >400 nm. All samples were prepared at pH 5.5 to mimic the lumen acidity of the late endosome where BMP is an essential component of intraendosomal vesicle budding, lipid sorting and trafficking. These findings indicate that GM1 and BMP under a limited concentration range spontaneously form small vesicles of homogeneous size in an energy independent manner without the need of protein templating. Because BMP is essential for intraendosomal vesicle formation, these results imply that lipid-lipid interactions may play a critical role in the endosomal process of lipid sorting and trafficking.     

Downhill versus Barrier-Limited Folding of BBL 2: Mechanistic Insights from Kinetics of Folding Monitored by Independent Tryptophan Probes

Barrier-free downhill folding has been proposed for the peripheral subunit-binding domain BBL. To date, ultrafast kinetic experiments on BBL, which are crucial for a mechanistic understanding of folding, have been hampered by the lack of good intrinsic spectroscopic probes. Here, we present a detailed kinetic characterization of three single-point tryptophan mutants of BBL that have suitable fluorescence properties for following microsecond and nanosecond folding kinetics using temperature jump fluorescence spectroscopy. Experiments were performed at pH 7, which is optimal for stability and minimizes complications that arise from the presence of an alternative native-state conformation of BBL at lower pH. We examined the dependence of rate and equilibrium constants on concentration of denaturant and found that they follow well-established laws allowing kinetic transients to be related to events in folding and compared with equilibrium data. Logarithms of rate constants versus denaturant concentration yielded plots (chevrons) that are characteristic of barrier-limited folding for all mutants investigated, including a truncated sequence that was previously used in the proposal of downhill folding. The thermodynamic quantities calculated from the rate constants were in excellent agreement with those directly determined from equilibrium denaturation based on empirical two-state equations. We found that sequence truncation of BBL as used in studies proposing downhill folding leads to a large loss in helical content and protein stability, which were exacerbated at the low pH used in those studies. The kinetics and equilibria of folding of BBL fit to conventional barrier-limited kinetics.     

Direct visualization of lipid domains in human skin stratum corneum's lipid membranes: effect of pH and temperature

The main function of skin is to serve as a physical barrier between the body and the environment. This barrier capacity is in turn a function of the physical state and structural organization of the stratum corneum extracellular lipid matrix. This lipid matrix is essentially composed of very long chain saturated ceramides, cholesterol, and free fatty acids. Three unsolved key questions are i), whether the stratum corneum extracellular lipid matrix is constituted by a single gel phase or by coexisting crystalline (solid) domains; ii), whether a separate liquid crystalline phase is present; and iii), whether pH has a direct effect on the lipid matrix phase behavior. In this work the lateral structure of membranes composed of lipids extracted from human skin stratum corneum was studied in a broad temperature range (10 degrees C-90 degrees C) using different techniques such as differential scanning calorimetry, fluorescence spectroscopy, and two-photon excitation and laser scanning confocal fluorescence microscopy. Here we show that hydrated bilayers of human skin stratum corneum lipids express a giant sponge-like morphology with dimensions corresponding to the global three-dimensional morphology of the stratum corneum extracellular space. These structures can be directly visualized using the aforementioned fluorescence microscopy techniques. At skin physiological temperatures (28 degrees C-32 degrees C), the phase state of these hydrated bilayers correspond microscopically (radial resolution limit 300 nm) to a single gel phase at pH 7, coexistence of different gel phases between pH 5 and 6, and no fluid phase at any pH. This observation suggests that the local pH in the stratum corneum may control the physical properties of the extracellular lipid matrix by regulating membrane lateral structure and stability.     

Study of hydration of cross-linked high amylose starch by solid state 13C NMR spectroscopy

Starch is subjected to chemical treatments such as cross-linking or hydroxypropylation to meet the material requirements for food uses or controlled release in the pharmaceutical industries. In this work, two types of cross-linking formulations have been employed for the preparation of high amylose starch for use as an excipient for sustained drug release. The structural differences and chain dynamics of the modified starches in the dry and hydrated states have been compared by the use of variable contact time cross polarization-magic angle spinning solid state (13)C NMR spectroscopy.     

Electric behaviour of natural and demineralized bones. Dielectric properties up to 1 GHz

In this paper we have measured the dielectric spectrum of water-saturated bones in native and demineralized states up to 1 GHz in the time domain. A novel method of analysis of the time domain spectroscopy data has been used. The results show a dielectric dispersion centered around 400 MHz for native samples and around 200 MHz for demineralized ones. The proposed mechanism for this dispersion is the movement of polar side chains, which is in agreement with what happens in hydrated collagen fibres.