Presence Of Neutral Detergents Research Articles

Contribution of hydrophobic interactions to the folding and fibrillation of histone H1 and its carboxy-terminal domain

Histone H1 is involved in chromatin structure and gene regulation. H1 also performs functions outside cell nuclei, which may depend on its properties as a lipid-binding protein. The H1 CTD behaves as an intrinsically disordered protein (IDP) with coupled binding and folding. Here, we used neutral detergents and anionic SDS to study the contribution of hydrophobic interactions to the folding of the CTD. In the presence of neutral detergents, the CTD folded with proportions of secondary structure motifs similar to those observed in the DNA complexes. These results identify a folding pathway for the CTD based on hydrophobic interactions, and independent of charge compensation. The CTD is phosphorylated to different extents by cyclin-dependent kinases. The general effect of phosphorylation in the presence of detergents was a decrease in the α-helix content and an increase in that of the β-structure. The greatest effect was observed in the fully phosphorylated CTD (three phosphate groups) in the presence of anionic SDS (7:1, detergent/CTD molar ratio); in these conditions, the CTD became an all-β protein, with 83% β-structure and no α-helix. The CTD in all-β conformation readily formed ribbon-like fibers. The entire H1 also formed fibers when fully phosphorylated in the CTD. Fibers were of the amyloid type, as judged by strong birefringence in the presence of Congo red and thioflavin fluorescence enhancement. Amyloid fiber formation was only observed in SDS, suggesting that it requires the joint effects of partial charge neutralization and hydrophobic interactions, together with the all-β potential provided by full phosphorylation.

Crystals of the β-subunit of bovine luteinizing hormone and indicators for the involvement of proteolysis in protein crystallization

The beta-subunit of luteinizing hormone (LH), the subunit responsible for the physiological response, has been crystallized beginning with the intact alphabeta-heterodimeric hormone purified from bovine pituitary glands. The crystals were grown at 310 K in the presence of neutral detergents along with trypsin. The tetragonal bipyramidal crystals diffract to 3 A resolution and belong to space group I4(1)22, with unit-cell parameters a = b = 57, c = 207 A. It is noted that proteins exposed to proteases sometimes yield products that crystallize better than the native molecule and that the beta-subunit of LH represents yet another example. Some indicators of when proteolysis may be a factor in crystallization, as well as some consequences, are described.

Spectroscopic studies of the C-terminal secretion signal of the Serratia marcescens haem acquisition protein (HasA) in various membrane-mimetic environments.

The structure of a peptide comprising the last 56 C-terminal residues of the Serratia marcescens haem acquisition protein (HasA) secreted by an ATP-binding cassette exporter was examined by 1H-NMR, circular dichroic and fluorescence spectroscopies. The peptide, which contains the secretion signal of HasA, is efficiently secreted by the HasA transporter. It is largely unstructured and flexible in aqueous buffer solution, but its helical content increases upon addition of trifluoroethanol, detergents and lipids. By circular dichroism, a stable helical conformation is observed between 20% and 70% (by vol.) trifluoroethanol. The 1H-NMR spectrum was analysed at these two trifluoroethanol concentrations; residues 7-15, 21-30 and 40-50 were shown to form relatively stable helices. In the presence of neutral detergent, alpha-helix is induced to a similar extent upon micelle formation; in this case, fluorescence data indicate that at least the N-terminus of the peptide interacts with the micelle. In the presence of negatively charged detergent, alpha-helix is induced before micelle formation and the N-terminus of the peptide seems not to be involved in this interaction. In the presence of negatively charged liposomes, the peptide interacts with the vesicle, again inducing a helical conformation. However, the helical content remains lower than upon addition of trifluoroethanol or neutral micelles. These results are compared to those previously obtained with the secretion signal of one of the Erwinia chrysanthemi metalloproteases which are transported efficiently by the HasA transporter. Both signals exhibit similar conformational features, despite their low sequence similarity.

The secondary structure of the ferredoxin transit sequence is modulated by its interaction with negatively charged lipids

Import of proteins into chloroplasts depends on an N-terminal transit sequence. Transit sequences contain little primary sequence similarity and therefore recognition of these sequences is thought to involve specific folding. To assess the conformational flexibility of the transit sequence, we studied the transit peptide of preferredoxin (trfd) by circular dichroism. In buffer, trfd is in a random coil conformation. A large increase in α-helix was induced in the presence of micelles or vesicles formed by anionic lipids. Less pronounced changes in secondary structure were induced by zwitterionic detergents but no changes were observed in the presence of neutral detergents or vesicles composed of phosphatidylcholine.

PHOTOOXIDATION OF CELL MEMBRANES USING EOSIN DERIVATIVES THAT LOCATE IN LIPID OR PROTEIN TO STUDY THE ROLE OF DIFFUSIBLE INTERMEDIATES

Eosin derivatives that bind primarily to lipid or protein sites in erythrocyte membranes were studied in solution and as sensitizers of erythrocyte membranes. In 50% ethanol-water mixtures eosin maleimide (EYMA) and 5-N-hexadecanoyl amino eosin (E16) had nearly identical absorption spectra. Higher ethanol concentrations did not change peak absorbances. In the presence of neutral detergent both sensitizers had equivalent absorbance at all ethanol concentrations. In water, EYMA was more effective than E16 at bleaching RNO, probably because of E16 aggregation into micelles, while in ethanol-water mixtures E16 was slightly more effective at bleaching DPBF, indicating equivalent singlet oxygen generation when the sensitizers are in monomeric form. In water with neutral detergent, azide in the 20 microM range inhibited the majority of RNO bleaching with both sensitizers; in 50% ethanol-water mixtures azide at 1 mM showed a 50% inhibition of DPBF bleaching with both sensitizers. Iodide in the 30 mM range reduced DPBF bleaching by 50% in 50% ethanol-water mixtures. When matched for amount loaded in erythrocyte membranes these sensitizers were about equally effective at sensitizing induction of cation permeability, assayed as rate of delayed photohemolysis, while E16 was slightly more effective at sensitizing loss of cholinesterase (AchE) activity. The relation of lysis rate to load was somewhat steeper for E16 than EYMA. For both sensitizers lysis rate increased at about the 1.5 power of light dose. Deoxygenation of the reaction media with argon totally blocked detectable photomodification. Ghost membranes made from sensitizer-treated cells were effective generators of singlet oxygen, assayed by RNO bleaching. However, when mixtures of EYMA-treated and untreated cells were illuminated together, only the EYMA-treated cells showed evidence of photomodification. Azide at 5 mM slowed the initial rate of AchE loss by about 75% with E16 and EYMA. Azide partially slowed photohemolysis. Azide decreased RNO bleaching by sensitizer-treated ghosts as it did in water with detergent micelles. A deuterium oxide solvent increased photohemolysis rate with E16 by 41%, but did not increase photohemolysis rate with EYMA. Deuterium oxide had a positive, but statistically insignificant effect on loss of AchE with both sensitizers. Deuterium oxide following illumination slowed lysis sensitized by both sensitizers more than 50%. Iodide exerted a modest inhibition of photohemolysis and loss of AchE sensitized by E16, but had virtually no influence on sensitization by EYMA. The results in solution indicate that EYMA and E16 have nearly identical photochemical properties when in monome

Purification of polysomes from a lysozyme- resistant desiccation-tolerant cyanobacterium

Six different techniques were compared for the extraction and purification of polysomes from cells of the desiccation-tolerant cyanobacterium Nostoc commune UTEX 584. Cells resisted treatment with lysozyme, and methods which relied upon ‘gentle lysis’ resulted in inefficient cell breakage and poor yields of polysomes. In contrast, the passage of cells through a French Pressure Cell achieved complete disruption of even the most resistant cell aggregates but only monosomes and ribosomal subunits were recovered. The grinding of cells with glass beads in the presence of neutral detergents was the most successful of all the methods tested and resulted in efficient cell lysis with high yields of polysomes. Treatment of the cells with acetone, at 0°C, prior to homogenization, also resulted in good yields of polysomes although the degree of cell breakage was less than when the cells were ground. The choice of the grinding material, and the extent of the grinding, were both critical for polysome extraction. Grinding of cells with alumina and sterile sand gave very efficient cell breakage but no polysomes were recovered. Excessive grinding with glass beads led to a progressive loss of intact polysomes and concomitant increase in 70 S monosomes and subunits in cell extracts. This study provides data on various physical treatments and buffer compositions which may be used effectively in the isolation and purification of polysomal RNA from highly resistant bacterial cells. A method which relies upon the grinding of cells in the presence of neutral detergents will permit further studies of gene expression in cells which resist methods of ‘gentle lysis’.

Purification of mammalian tumor (L1210) thymidylate synthetase by affinity chromatography on stable biospecific adsorbent. Stabilization of the enzyme with neutral detergents.

Thymidylate synthetase from mouse leukemic L1210 cells was purified to electrophoretic homogeneity with 70% yield as a result of an affinity chromatography procedure based on reversible deoxyuridylate-dependent binding of the enzyme to a stable biospecific adsorbent, 10-formyl-5,8-dideazafolate, immobilized on aminoethyl-Sepharose. The presence of neutral detergents, Triton X-100, or Nonidet P40 stabilized thymidylate synthetase during purification. Analytical electrophoresis of the enzyme treated with an excess of 5-fluorodeoxyuridylate and 5,10-methylenetetrahydrofolate showed the presence of two forms of thymidylate synthetase--5-fluorodeoxyuridylate.5,10-methylenetetrahydrofolate complex, indicating that there are two binding sites for 5-fluorodeoxyuridylate present on the enzyme molecule. Molecular weight of native thymidylate synthetase was found to be 75,000, whereas that for the monomer was 38,500.

The effects of detergents and phospholipids on a glycolipid galactosyltransferase

1. 1. phospholipids activated the enzyme, lactosylceramide: UDP-galactose α-galactosyltransferase in hamster cells (NIL 2E clone 8) when assayed in the presence of neutral detergents. 2. 2. Phosphatidylserine and phosphatidylinositol were the most effective phospholipids in activating the enzyme. Other phospholipids were also effective, but sphingomyelin and lysophosphatidylcholine were inhibitory. 3. 3. Considerable enzyme activity was obtained in the absence of any detergent. Most of this activity was due to glycosylation of endogenous acceptors. 4. 4. There was a complex effect of detergents on the enzyme activity. Very low concentrations were sharply inhibitory, but higher concentrations, above the critical micelle concentration for detergent, caused regeneration of activity. 5. 5. The phospholipids, in the absence of a detergent, are required to maintain the lipid substrate, lactosylceramide, in a suitable dispersion where it can be acted upon by the enzyme. In the presence of detergents, it is proposed that the phospholipids also act by affecting the state of the lipid substrate.

Preparation and Rates of Hydrolysis of Perfluoro- Esters of Cellulose

Partial esters of cellulose varying in degree of substitution from 0.02 to 0.58 were prepared by reacting cellulose immersed in dimethylformamide with either perfluoro butyryl chloride or perfluorooctanoyl chloride in the presence of a tertiary aromatic amine. The use of essentially homogeneous solutions of acid chlorides resulted in prod ucts which retained properties of the original cotton and showed in addition oil and water repellency which was durable to dry cleanings and aqueous launderings in the presence of neutral detergents. Rates of uncatalyzed hydrolysis of the perfluorooctanoyl cellulose esters were determined at 25°, 35°, and 45° C., and the rate of the alkaline catalyzed hydrolysis was estimated at 25° C. The energy and entropy of activation for the uncatalyzed hydrolysis were determined.