Torsional motion of eosin-labeled F-actin as detected in the time-resolved anisotropy decay of the probe in the sub-millisecond time range

The internal motion of F-actin in the time range from 10(-6) to 10(-3) second has been explored by measuring the transient absorption anisotropy of eosin-labeled F-actin using laser flash photolysis. The transient absorption anisotropy of eosin-F-actin at 20 degrees C has a component that decays in the submicrosecond time scale to an anisotropy of about 0.3. This anisotropy then decays with a relaxation time of about 450 microseconds to a residual anisotropy of about 0.1 after 2 ms. When the concentration of eosin-F-actin was varied in the range from 7 to 28 microM, the transient absorption anisotropy curves obtained were almost indistinguishable from each other. These results show that the anisotropy decay arises from internal motion of eosin-F-actin. Analysis of the transient absorption anisotropy curves indicates that the internal motion detected by the decay in anisotropy is primarily a twisting of actin protomers in the F-actin helix; bending of the actin filament makes a minor contribution only to the measured decay. The torsional rigidity calculated from the transient absorption anisotropy is 0.2 X 10(-17) dyn cm2 at 20 degrees C, which is about an order of magnitude smaller than the flexural rigidity determined from previous studies. Thus, we conclude that F-actin is more flexible in twisting than in bending. The calculated root-mean-square fluctuation of the torsional angle between adjacent actin protomers in the actin helix is about 4 degrees at 20 degrees C. We also found that the torsional rigidity is approximately constant in the temperature range from 5 to approximately 35 degrees C, and that the binding of phalloidin does not appreciably affect the torsional motion of F-actin.     

Spin-label studies of membrane-associated denatured hemoglobin in normal and sickle cells

A maleimide spin label (N-(1-oxyl-2,2,5,5-tetramethylpyrrolidinyl)-maleimide) was reacted with oxyhemoglobin-free cell stromata of normal and sickle cells. The EPR spectrum of spin-labeled red cell membranes showed that the spin labels are attached to at least two different binding sites. There was a major signal, A, which characterized a strongly immobilized environment and a minor signal, B, which characterized a weakly immobilized environment. Quantitative EPR measurements using equal amounts of Hb AA and Hb SS red blood cells demonstrated that Hb SS red cell membranes had an approximately four times higher EPR signal intensity than Hb AA red cell membranes ((7.98 ± 1.14) · 10⁵ and (2.2 ± 1.2) · 10⁵ spin labels/cell, respectively). Moreover, the ratio of signal intensities A and B are different in these cells. Comparative spectrophotometric studies of membrane-associated denatured hemoglobins of Hb AA and Hb SS red cell membranes suggested that the EPR signal A is derived from spin labels attached to membrane-associated denatured hemoglobin, while signal B is mainly from spin labels attached to membrane-associated denatured hemoglobin, while signal B is mainly from spin labels attached to membranes. The combination of EPR spectrum of Hb AA membranes pretreated with N-ethyl-maleimide and that of spin-labeled precipitated hemoglobin further strengthened this conclusion.     

The study of rous sarcoma virus-transformed baby hamster kidney cells using fluorescent probes

The fluorescent probes pyrene, pyrene butyric acid and $\text{N-}\text{phenyl}$ 1-naphthylamine have been used to investigate the changes that accompany in vitro transformation of a baby hamster kidney cell line using Rous sarcoma virus. The fluorescent probes which reside in the membrane were used to compare the changes in microviscosity and polarity of the membranes of normal cells with two transformed cell lines. The spectrofluorimetric data indicate that following transformation the probe $\text{N-}\text{phenyl}$ 1-naphthylamine resides in a more polar environment. However, using the probe pyrene, the yield of excimer indicates decreased mobility of this probe in the membrane of transformed cells. The data also indicate differences between the two transformed cell lines. Laser photolysis was used to study the lifetime of the pyrne probes and the quenching of the pyrene fluorescence in the membrane by several different quenching molecules. The data indicate differences between the three cell lines and suggest that transformation decreases movement within the membrane.     

Substrate selectivity of the melibiose permease (MelY) from Enterobacter cloacae

We have examined the substrate selectivity of the melibiose permease (MelY) from Enterobacter cloacae in comparison with that of the lactose permease (LacY) from Escherichia coli. Both proteins catalyze active transport of lactose or melibiose with comparable affinity and capacity. However, MelY does not transport the analogue methyl-1-thio-β,d-galactopyranoside (TMG), which is a very efficient substrate in LacY. We show that MelY binds TMG and conserves Cys148 (helix V) as a TMG binding residue but fails to transport this ligand. Based on homology modeling, organization of the putative MelY sugar binding site is the same as that in LacY and residues irreplaceable for the symport mechanism are conserved. Moreover, only 15% of the residues where a single-Cys mutant is inactivated by site-directed alkylation differ in MelY. Using site-directed mutagenesis at these positions and engineered cross-homolog chimeras, we show that Val367, at the periplasmic end of transmembrane helix XI, contributes in defining the substrate selectivity profile. Replacement of Val367 with the MelY residue (Ala) leads to impairment of TMG uptake. Exchanging domains N6 and C6 between LacY and MelY also leads to impairment of TMG uptake. TMG uptake activity is restored by the re-introduction of a Val367 in the background of chimera N6(LacY)-C6(MelY). Much less prominent effects are found with the same mutants and chimeras for the transport of lactose or melibiose.     

The reconstituted 'humanized liver' in TK-NOG mice is mature and functional

The Marasmius oreades mushroom lectin (MOA) is well known for its exquisite binding specificity for blood group B antigens. In addition to its N-terminal carbohydrate-binding domain, MOA possesses a C-terminal domain with unknown function, which structurally resembles hydrolytic enzymes. Here we show that MOA indeed has catalytic activity. It is a calcium-dependent cysteine protease resembling papain-like cysteine proteases, with Cys215 being the catalytic nucleophile. The possible importance of MOA’s proteolytic activity for mushroom defense against pathogens is discussed.     

Polarization of fluorescence from single skinned glycerinated rabbit psoas fibers in rigor and relaxation

Single skinned glycerinated muscle fibers were labelled with the fluorescent dye N-(iodoacetylamino)-1-naphthylamine-5-sulfonic acid (1,5-IAEDANS). The heavy chain of myosin (EC 3.6.1.3) was labelled predominantly when the reaction was carried out in relaxation at 0 °C. Mechanical properties of skinned fibers were little affected by labelling with the fluorophore. Rigor tension developed upon transferring native or labelled skinned fibers from relaxing to rigor solutions lacking Ca²⁺ was very small but could be enhanced by progressively increasing Ca²⁺ concentration; the rigor tension decreased with increasing sarcomere length.Polarization of fluorescence of skinned fibers reacted with 1,5-IAEDANS was measured along the line of excitation as well as at 90° to it. The mean values of parallel and perpendicular components of polarization of labelled fibers measured at 0° were close to the values obtained for native fibers irrigated with 1,5-IAEDANS-labelled heavy meromyosin, fiber “ghosts” irrigated with labelled heavy meromyosin, and oriented bundles of myofibrils reacted with the same fluorophore. Skinned fibers stretched above the rest length and then irrigated with 1,5-IAEDANS-labelled heavy meromyosin gave rise to polarized fluorescence close to the values theoretically predicted for an assembly of helically arranged fluorophores. Using 90° detection system a satisfactory fit to the theory could be obtained from single fibers labelled with 1,5-IAEDANS and measured in rigor. The angle between the fiber axis and the direction of the emission dipole of 1,5-IAEDANS attached to subfragment-1 was estimated to be near 40°.     

Helix VIII of NhaA Na(+)/H(+) antiporter participates in the periplasmic cation passage and pH regulation of the antiporter

The crystal structure of Escherichia coli NhaA determined at pH 4 has provided insights into the mechanism of activity of a pH-regulated Na⁺/H⁺ antiporter. However, because NhaA is active at physiological pH (pH 6.5-8.5), many questions related to the active state of NhaA have remained unanswered. Our Cys scanning of the highly conserved transmembrane VIII at physiological pH reveals that (1) the Cys replacement G230C significantly increases the apparent K_m of the antiporter to both Na⁺ (10-fold) and Li⁺ (6-fold). (2) Variants G223C and G230C cause a drastic alkaline shift of the pH profile of NhaA by 1 pH unit. (3) Residues Gly223-Ala226 line a periplasmic funnel at physiological pH as they do at pH 4. Both were modified by membrane-impermeant negatively charged 2-sulfonatoethyl methanethiosulfonate and positively charged 2-(trimethyl ammonium)-ethylmethanethiosulfonate sulfhydryl reagents that could reach Cys replacements from the periplasm via water-filled funnels only, whereas other Cys replacements on helix VIII were not accessible/reactive to the reagents. (4) Remarkably, the modification of variant V224C by 2-sulfonatoethyl methanethiosulfonate or 2-(trimethyl ammonium)-ethylmethanethiosulfonate totally inhibited antiporter activity, while N-ethyl maleimide modification had a very small effect on NhaA activity. Hence, the size—rather than the chemical modification or the charge—of the larger reagents interferes with the passage of ions through the periplasmic funnel. Taken together, our results at physiological pH reveal that amino acid residues in transmembrane VIII contribute to the cation passage of NhaA and its pH regulation.     

Crystal structure of the Marasmius oreades mushroom lectin in complex with a xenotransplantation epitope

MOA, a lectin from the mushroom Marasmius oreades, is one of the few reagents that specifically agglutinate blood group B erythrocytes. Further, it is the only lectin known to have exclusive specificity for Galalpha(1,3)Gal-containing sugar epitopes, which are antigens that pose a severe barrier to animal-to-human organ transplantation. We describe here the structure of MOA at 2.4 A resolution, in complex with the linear trisaccharide Galalpha(1,3)Galbeta(1,4)GlcNAc. The structure is dimeric, with two distinct domains per protomer: the N-terminal lectin module adopts a ricinB/beta-trefoil fold and contains three putative carbohydrate-binding sites, while the C-terminal domain serves as a dimerization interface. This latter domain, which has an unknown function, reveals a novel fold with intriguing conservation of an active site cleft. A number of indications suggest that MOA may have an enzymatic function in addition to the sugar-binding properties.