Secondary structure transitions and aggregation induced in dynorphin neuropeptides by the detergent sodium dodecyl sulfate

Dynorphins, endogeneous opioid neuropeptides, function as ligands to the opioid kappa receptors and also induce non-opioid effects in neurons, probably related to direct membrane interactions. We have characterized the structure transitions of dynorphins (big dynorphin, dynorphin A and dynorphin B) induced by the detergent sodium dodecyl sulfate (SDS). In SDS titrations monitored by circular dichroism, we observed secondary structure conversions of the peptides from random coil to α-helix with a highly aggregated intermediate. As determined by Fourier transform infrared spectroscopy, this intermediate exhibited β-sheet structure for dynorphin B and big dynorphin. In contrast, aggregated dynorphin A was α-helical without considerable β-sheet content. Hydrophobicity analysis indicates that the YGGFLRR motif present in all dynorphins is prone to be inserted in the membrane. Comparing big dynorphin with dynorphin A and dynorphin B, we suggest that the potent neurotoxicity of big dynorphin could be related to the combination of amino acid sequences and secondary structure propensities of dynorphin A and dynorphin B, which may generate a synergistic effect for big dynorphin membrane perturbing properties. The induced aggregated α-helix of dynorphin A is also correlated with membrane perturbations, whereas the β-sheet of dynorphin B does not correlate with membrane perturbations.     

Inactivation of prions by acidic sodium dodecyl sulfate

Prompted by the discovery that prions become protease-sensitive after exposure to branched polyamine dendrimers in acetic acid (AcOH) (S. Supattapone, H. Wille, L. Uyechi, J. Safar, P. Tremblay, F. C. Szoka, F. E. Cohen, S. B. Prusiner, and M. R. Scott, J. Virol. 75:3453-3461, 2001), we investigated the inactivation of prions by sodium dodecyl sulfate (SDS) in weak acid. As judged by sensitivity to proteolytic digestion, the disease-causing prion protein (PrPSc) was denatured at room temperature by SDS at pH values of < or =4.5 or > or =10. Exposure of Sc237 prions in Syrian hamster brain homogenates to 1% SDS and 0.5% AcOH at room temperature resulted in a reduction of prion titer by a factor of ca. 10(7); however, all of the bioassay hamsters eventually developed prion disease. When various concentrations of SDS and AcOH were tested, the duration and temperature of exposure acted synergistically to inactivate both hamster Sc237 prions and human sporadic Creutzfeldt-Jakob disease (sCJD) prions. The inactivation of prions in brain homogenates and those bound to stainless steel wires was evaluated by using bioassays in transgenic mice. sCJD prions were more than 100,000 times more resistant to inactivation than Sc237 prions, demonstrating that inactivation procedures validated on rodent prions cannot be extrapolated to inactivation of human prions. Some procedures that significantly reduced prion titers in brain homogenates had a limited effect on prions bound to the surface of stainless steel wires. Using acidic SDS combined with autoclaving for 15 min, human sCJD prions bound to stainless steel wires were eliminated. Our findings form the basis for a noncorrosive system that is suitable for inactivating prions on surgical instruments, as well as on other medical and dental equipment.     

Denaturation of MM-creatine kinase by sodium dodecyl sulfate

The denaturation of dimeric cytoplasmic MM-creatine kinase by sodium dodecyl sulfate (SDS) has been investigated using activity measurements, far-ultraviolet circular dichroism, SEC-HPLC, electric birefringence, intrinsic probes (cysteine and tryptophan residues), and an extrinsic fluorescent probe (ANS). Our results show that inactivation is the first detectable event; the inactivation curve midpoint is located around 0.9 mM SDS. The second event is dissociation and it occurs in parallel to tertiary and secondary perturbations, as demonstrated by the coincidence (near 1.3 mM) of the midpoints of the transition curves monitoring dissociation and structural changes. At high total SDS concentration (concentration higher than 2.5 mM), the monomer had bound 170 mol of SDS per mol of protein. In these conditions, electric birefringence experiments suggest that the SDS-CK complex may be described as a prolate ellipsoid with an axial ratio of 1.27 (14 nm×11 nm). These results are compatible with recent models of SDS-protein complexes: the protein decorated micelle structure or the necklace structure.     

Stability of aprA-subtilisin in sodium dodecyl sulfate

The effect of sodium dodecyl sulfate (SDS) on the structure and activity of aprA-subtilisin, a secreted bacterial serine protease which is 85% homologous to subtilisin BPN', was examined. The addition of SDS resulted in the slow conversion of the subtilisin from the intact protein to the completely unfolded form of the enzyme. No intermediates between these two populations were detected. This conversion was accompanied by decreased activity, disruption of tertiary structure, a change in the mobility of the protein when subjected to SDS-polyacrylamide gel electrophoresis, and an increase in the apparent Stokes radius of the protein. After 2 h in 1% SDS at 20 degrees C, 25% of the subtilisin was still intact and active. The amount of protein existing in the unfolded form was increased by increasing the length of time in SDS, by increasing the concentration of SDS, and by increasing the temperature of the subtilisin-SDS solution. Analysis of the dependence of the rate of unfolding on SDS concentration indicated that one SDS micelle can destroy two protein molecules. The activation energy for the SDS-induced denaturation of aprA-subtilisin was 20 kcal mol-1, indicating that unfolding of the protein could be the rate-limiting step.     

Ontogeny-related changes in the peptide profiles of the germinal epithelium: analysis by sodium dodecyl sulfate gradient gel electrophoresis

The premise that one manifestation of the nexus between Sertoli cells and germ cells may be an orderly and sequential change in their protein profiles has been examined in relation to the ontogeny of spermatogenesis in the colony-bred albino rat. Viable "Sertoli cell-germ cell associations" isolated from the testes of animals of defined postnatal age and incubated in an appropriate medium were separated into a Sertoli cell and a germ cell fraction and processed for analysis by sodium dodecyl sulfate gradient gel electrophoresis. The resulting stained bands were "mapped" and assigned relative mobility values by comparison with standard marker proteins. This enabled identification by serial number of individual bands from an overall total of 163. For purposes of detailed analysis, they were classified into high, medium-high, medium, and low molecular weight bands. Two major categories were delineated: 1) those associated uniquely with a specified day of ontogeny and 2) those appearing intermittently. Significantly enough, not one of the bands was encountered on all days examined. The relevance of the patterns observed to the possible exchange of "information" between Sertoli cells and germ cells during spermatogenesis is mooted.     

Detection of histones by DNA binding ability after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate

Histones can be detected on the basis of their binding to DNA after electrophoresis in sodium dodecyl sulfate-polyacrylamide gels containing DNA. The method depends on the ability of individual histone components to 1) renature, 2) bind to DNA, and 3) prevent ethidium bromide binding and fluorescence. This technique can provide qualitative and, possibly, quantitative information on histones in crude extracts.     

Overlapping between fluorescence modifications and activation of prostate transglutaminase induced by sodium dodecyl sulfate.

The transglutaminase from rat coagulating gland secretion has been proposed as a new member of the transglutaminase family. Its basal activity is about 11-fold lower than those of other transglutaminases (e.g., the cytosolic tissue transglutaminase), but reaches levels comparable to those of other transglutaminases on addition of specific surfactant agents. There is no study devoted to understanding the molecular basis of this apparently anomalous activation, which is maximal at approximately 1.5 mM sodium dodecyl sulfate. We provide evidence that in the presence of this detergent modifications of the intrinsic fluorescence as well as energy transfer of the protein fluorescence to a micellar probe parallel the activation of the enzyme. As the sodium dodecyl sulfate concentration inducing maximal activation equals the critical micellar concentration, the biological activity of this transglutaminase appears to be modulated by the binding of micellar aggregates. In fact, the enzyme is modified by posttranslational modifications consisting of some lipid tails. At least two of these tails could act as aggregation nuclei of the enzyme with detergents. This behavior is different from that typical of molecular forms purified from other sources.     

Structural changes of a sodium dodecyl sulfate (SDS) micelle induced by alcohol molecules

Coarse-grained dynamical simulations have been performed to investigate the behavior of a surfactant micelle in the presence of six different alcohols: hexanol, octanol, decanol, dodecanol, tetradecanol, and hexadecanol. The self-assembly of sodium dodecyl sulfate (SDS) is modified by the alcohol molecules into cylindrical and bilayer micelles as a function of the alcohol/SDS mass ratio. Therefore, in order to understand, from a molecular point of view, how SDS and alcohol molecules self-organize to form the new micelles, different studies were carried out. Analysis of micelle structures, density profiles, and parameters of order were conducted to characterize the shape and size of those micelles. The density profiles revealed that the alcohol molecules were located at the water–micelle interface next to the SDS molecules at low alcohol/SDS mass ratio. At high alcohol/SDS mass ratios, alcohol molecules moved to the middle of the micelle by increasing their size and by producing a structural change. Moreover, micelle structures and sizes were influenced not only by the alcohol/SDS mass ratio but also by the order of the SDS and alcohol tails. Finally, the size of the micelles and enthalpy calculations were used as order parameters to determine a structural phase diagram of alcohol/SDS mixtures in water.

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Graphical Abstract Structural transition of SDS/alcohol mixtures

     

The importance of coulombic interactions for the induction of beta structure in lysine oligomers by sodium dodecyl sulfate.

Circular dichroism spectra have been obtained for tri(L -lysine), tetra(L -lysine), and penta(L -lysine) in aqueous sodium dodecyl sulfate at 25°C. None of the oligomers are affected significantly by sodium dodecyl sulfate at detergent concentrations exceeding 0.01 M. Literature results show that the high-molecular-weight polymer forms a β strucure under these conditions. At detergent concentrations near 3.5 × 10^?4 M the penta(L -lysine), but not the smaller oligomers, undergoes a conformational change. Its circular dichroism under these conditions is essentially identical to that observed with poly(L -lysine) when it forms a β structure in sodium dodecyl sulfate. Solutions of the penta(L -lysine), which exhibit this modified circular dichroism, are also turbid, leading to the conclusion that the oligomer has formed an intermolecular β structure. When these experiments are conducted in the presence of 0.1 M sodium hydroxide, the sodium dodecyl sulfate produces neither turbidity nor a modified circular dichroism spectrum. These observations provide compelling evidence that Coulombic interaction between the anionic detergent head and the cationic lysyl amino groups is essential for the conformational change induced in penta(L -lysine) by sodium dodecyl sulfate.     

Gas chromatographic determination of sodium dodecyl sulfate

A gas chromatographic method has been developed for the determination of sodium dodecyl sulfate. The method is sensitive, reasonably accurate, and uninfluenced by the presence of protein. The method depends upon the formation of 1-dodecanol and inorganic sulfate by acidic hydrolysis of sodium dodecyl sulfate (4 n HCl, 2 hr, 100°C). The ether extracted 1-dodecanol is analyzed by standard gas chromatographic techniques.     

Structure of the bovine antimicrobial peptide indolicidin bound to dodecylphosphocholine and sodium dodecyl sulfate micelles

Indolicidin is a cationic, 13-residue antimicrobial peptide (ILPWKWPWWPWRR-NH(2)) which is unusually rich in tryptophan and proline. Its antimicrobial action involves the bacterial cytoplasmic membrane. Fluorescence and circular dichroism spectra demonstrated the structural similarity of indolicidin in complexes with large unilamellar phospolipid vesicles and with detergent micelles. The structure of indolicidin bound to zwitterionic dodecylphosphocholine (DPC) and anionic sodium dodecyl sulfate (SDS) micelles was determined using NMR methods and shown to represent a unique membrane-associated peptide structure. The backbone structure in DPC, well defined between residues 3 and 11, was extended, with two half-turns at residues Lys-5 and Trp-8. The backbone structure in SDS, well defined between residues 5 and 11, was also extended, but lacked the bend in the C-terminal half. Indolicidin in complexes with DPC had a central hydrophobic core composed of proline and tryptophan, which was bracketed by positively charged regions near the peptide termini. The tryptophan side chains, with one exception, folded flat against the peptide backbone, thus giving the molecule a wedge shape. Indolicidin in complexes with SDS had an arrangement of hydrophobic and cationic regions similar to that found in the presence of DPC. The tryptophan side chains were less well defined than for indolicidin in DPC and extended away from the peptide backbone. The preferred location of indolicidin in DPC micelles and lipid bilayers, analyzed using spin-label probes, was at the membrane interface.     

Prestaining of glycoproteins in sodium dodecyl sulfate polyacrylamide gels by dansylhydrazine

A new fluorescent prestaining method for gel‐separated glycoproteins in 1D and 2D SDS‐PAGE was developed by using dansylhydrazine in this study. The prestained gels could be easily imaged after electrophoresis without any time‐consuming steps needed for poststains. As low as 4–8 ng glycoproteins (transferrin, α1‐acid glycoprotein) could be selectively detected, which is comparable to that of Pro‐Q Emerald 488, one of the most commonly used glycoprotein stain. In addition, a subsequent study of deglycosylation, glycoprotein affinity isolation, and LC‐MS/MS analysis was performed to confirm the specificity of the newly developed method.     

Activation of tobacco leaf polyphenol oxidase by sodium dodecyl sulfate

The effect of sodium dodecyl sulfate (SDS) on purified tobacco leaf PPO (PPO II) was investigated at various pHs and temperatures. SDS increased the activity of PPO II due to the formation of SDS-PPO II complex, leading to conformational changes, thus making access to active center easier. The relationship between the activity and the molar ratio of SDS-PPO II to PPO II showed that the critical point reached a plateau of activity at the molar ratio of about 1.2. The pH had a significant effect on interaction between SDS and PPO II, as compared to PPO II. The optimum catalytic temperature of the complex rose by 10 degrees C, suggesting that stabilization of the structure had been improved by the formation of complex.     

Electrophoretic behavior of protein dodecyl sulfate complexes in the presence of various amounts of sodium dodecyl sulfate.

This report describes the relationship between the amount of sodium dodecyl sulfate present in a sample solution and the electrophoretic mobility of the protein-dodecyl sulfate complexes. In order to determine the extent of any conformational changes in the proteins and to establish a correlation between any of these structural changes and the electrophoretic behavior, visible absorption spectra and circular dichroism spectra were obtained for heme proteins in the presence of the same amounts of surfactants as used in electrophoresis.From the results obtained, it is apparent that the amount of sodium dodecyl sulfate present in the sample solution must be taken into consideration when performing a separation. Optimum experimental conditions are chosen for attaining enhanced separation and a maximized linear range of molecular weights of proteins that can be accurately determined.