Direct observation of phosphorylase kinase and phosphorylase b by scanning tunneling microscopy

The molecular structures of phosphorylase b and phosphorylase kinase have been visualized by scanning tunneling microscopy (STM). STM is a near field technique that can resolve structures at the nanometer level and thus can image individual molecules. Phosphorylase b can be seen in dimeric and tetrameric forms as well as linear and globular aggregates. The linear arrays consist of side by side dimers with the long axis of the dimer perpendicular to the aggregated chain. Individual molecules of phosphorylase kinase appear to be planar, bilobate structures with a 2-fold axis of symmetry and a central depression.     

The structure and properties of 27S and larger iodoproteins in the thyroid gland.

Iodoproteins larger than 19S were isolated from hog and calf thyroid glands using gel filtration on Sepharose 6B and one or two centrifugations in glycerol density gradients. Purified protein fractions were analysed in the analytical ultracentrifuge and characterized by the combined use of electrophoresis in continuous polyacrylamide gel gradients and electron microscopy. Three bands migrating more slowly than 19S could be identified in the polyacrylamide gels. Electron microscopy of the fastest of these species, having a sedimentation constant of 27S, showed pairs of 19S thyroglobulin molecules which had the same size and the same ovoid shape as normal, well-iodinated thyroglobulin molecules. The ovoids were randomly attached side to side, end to end. The more slowly migrating proteins were shown to consist of similar aggregates of three, four or more randomly attached molecules. Iodine and sialic acid determinations in 27S and 19S separated from the same pool of well iodinated protein showed no difference in iodine content but a larger amount of sialic acid in 27S than in 19S.     

Molecular Simulation and the Aggregation of the Heavy Fractions in Crude Oils

The use of Molecular Simulation in the study of aggregates of the molecules of the heavy fractions of crude oils is reviewed. Molecular Mechanics calculations of aggregates of asphaltenes having a single large aromatic region (continental type) and others having smaller aromatic regions connected by alkyl chains (archipelago type) are discussed in terms of the molecular recognition processes present in petroleum. Stacking of the aromatic regions was the most important process in the formation of aggregates of asphaltenes of the continental type with some unfavorable contributions from its saturated rings and alkyl side chains. The steric interference of these groups limits the growth of the aggregates to a small number of molecules. The asphaltenes of the archipelago type showed more complex aggregates because some molecules act as bridges and tangling between them may occur. The interaction of the asphaltene aggregates with resin molecules was analyzed and it was found that the high selectivity for some sites of the asphaltenes explains the specificity of the resins for its own crude oil.     

Alfalfa mosaic virus aggregátion forms

Extensive electron microscopy studies have been carried out on the aggregation forms of the Alfalfa mosaic virus (AMV) components occurring in vivo and obtained in vitro. Using a goniometer as a device for tilting the aggregates, it could be shown that they consist of a mixed population of the 3 major components, B, M, and Tb, in a side to side hexagonal crystalline array.     

Nucleation and decay initiation are the stiffness-sensitive phases of focal adhesion maturation

A cell plated on a two-dimensional substrate forms adhesions with that surface. These adhesions, which consist of aggregates of various proteins, are thought to be important in mechanosensation, the process by which the cell senses and responds to the mechanical properties of the substrate (e.g., stiffness). On the basis of experimental measurements, we model these proteins as idealized molecules that can bind to the substrate in a strain-dependent manner and can undergo a force-dependent state transition. The model forms molecular aggregates that are similar to adhesions. Substrate stiffness affects whether a simulated adhesion is initially formed and how long it grows, but not how that adhesion grows or shrinks. Our own experimental tests support these predictions, suggesting that the mechanosensitivity of adhesions is an emergent property of a simple molecular-mechanical system.     

Structure of branched poly-alpha-amino acids in dimethylformamide. II. Viscosity, sedimentation, and diffusion

The intrinsic viscosity, sedimentation and diffusion of a series of branched, multichain poly-α-amino acids having a poly(L -lysine) backbone and poly(γ-benzyl L -glutamate) and poly (β-benzyl L -aspartate) side chains was studied at room temperature in dimethylformamide. The molecules were found to be extremely compact structures in which the molecular backbone is either lying along the major axis in a slightly twisted configuration (the longer the side chain the smaller the twist) or is coiled up in the form of a disk with backbone and side chains coplanar. Heat treatment (to 70°C.) introduces only small changes in the hydrodynamic parameters showing that the heat-labile aggregates detected by light scattering are reversibly broken up during the hydrodynamic measurements. The above structural information concerns the initial metastable conformation of the molecules which is irreversibly destroyed by heat treatment.     

Naturally occurring protein crystals in the potato : inhibitor of papain, chymopapain, and ficin

Protein crystals isolated from potato tubers were found to consist of a proteinase inhibitor active against the cysteine proteinases papain, chymopapain, and ficin. The molecular weight as determined by gel filtration at pH 4.3 or by gel electrophoresis in the presence of dodecylsulfate was 80 kilodaltons. When the inhibitor was evaluated at pH 8.4 in a linear concentration (4-30% polyacrylamide) under nondenaturing conditions, it appeared as two bands of approximately 320 to 350 kilodaltons indicating that the inhibitor forms tetrameric aggregates in neutral or weakly alkaline media, while the monomeric form predominates under acidic conditions. Gel filtration in the presence of varying amounts of papain suggested that the monomer combines with four papain molecules. The inhibitor contains no cystine.     

The Lewy Body in Parkinson’s Disease and Related Neurodegenerative Disorders

The histopathological hallmark of Parkinson’s disease (PD) is the presence of fibrillar aggregates referred to as Lewy bodies (LBs), in which α-synuclein is a major constituent. Pale bodies, the precursors of LBs, may serve the material for that LBs continue to expand. LBs consist of a heterogeneous mixture of more than 90 molecules, including PD-linked gene products (α-synuclein, DJ-1, LRRK2, parkin, and PINK-1), mitochondria-related proteins, and molecules implicated in the ubiquitin–proteasome system, autophagy, and aggresome formation. LB formation has been considered to be a marker for neuronal degeneration because neuronal loss is found in the predilection sites for LBs. However, recent studies have indicated that nonfibrillar α-synuclein is cytotoxic and that fibrillar aggregates of α-synuclein (LBs and pale bodies) may represent a cytoprotective mechanism in PD.     

Cytoplasmic aggregates in D-galactosamine induced liver injury

D-galactosamine treatment leads to the formation of PAS-positive granules or aggregates in the cytoplasm of mouse liver cells. Ultrastructural observations show that the granules consist of particles surrounded by membranes of rough endoplasmic reticulum. Cytochemical results reveal that part of the particles is pronase-sensitive and amylase resistant, staining positively by the Thiéry silver proteinate method. The other part is stained positively by EDTA preferentional staining. According to the cyto-and histochemical results the granules consist of ribosomes and abnormal basic glycogen. The aggregates are removed from the cytoplasm mostly by lysosomal degradation.     

Interaction of histone H1 with superhelical DNA. Sedimentation and electron microscopical studies at low salt concentration

Complexes of histones H1 with superhelical SV40 DNA obtained by direct mixing were studied in 0.1 SSC buffer corresponding to 0.02 M Na+. Depending on the molar input ratio H1/DNA three classes of sedimenting species were observed: (1) a component sedimenting similar to superhelical DNA with a sedimentation coefficient s2o,w of 25 S observable up to 335 Mol H1/Mol DNA (w/w = 2); (2) a component with s2o,w = 120 S appearing at 135 Mol H1/Mol DNA and (3) growing amounts of heterogeneous aggregates greater than 1000 S. Electron micrographs revealed the 25 S component to consist of double-fibers formed from one DNA molecule and the 120 S component to consist of bundles of several such double-fibers. The aggregates represent cable-like structures. The addition of ethidium bromide to 25 S complexes induces the formation of bundles, if H1 is present in a quantity which alone is not sufficient to bring about this effect. This result indicates that ethidium bromide effects a redistribution of H1 molecules and that H1 is responsible for the bundle formation.     

Model for fluorescence quenching in light harvesting complex II in different aggregation states

Low-temperature (77 K) steady-state fluorescence emission spectroscopy and dynamic light scattering were applied to the main chlorophyll a/b protein light harvesting complex of photosystem II (LHC II) in different aggregation states to elucidate the mechanism of fluorescence quenching within LHC II oligomers. Evidences presented that LHC II oligomers are heterogeneous and consist of large and small particles with different fluorescence yield. At intermediate detergent concentrations the mean size of the small particles is similar to that of trimers, while the size of large particles is comparable to that of aggregated trimers without added detergent. It is suggested that in small particles and trimers the emitter is monomeric chlorophyll, whereas in large aggregates there is also another emitter, which is a poorly fluorescing chlorophyll associate. A model, describing populations of antenna chlorophyll molecules in small and large aggregates in their ground and first singlet excited states, is considered. The model enables us to obtain the ratio of the singlet excited-state lifetimes in small and large particles, the relative amount of chlorophyll molecules in large particles, and the amount of quenchers as a function of the degree of aggregation. These dependencies reveal that the quenching of the chl a fluorescence upon aggregation is due to the formation of large aggregates and the increasing of the amount of chlorophyll molecules forming these aggregates. As a consequence, the amount of quenchers, located in large aggregates, is increased, and their singlet excited-state lifetimes steeply decrease.     

Hsp70 chaperone machine remodels protein aggregates at the initial step of Hsp70-Hsp100-dependent disaggregation

Exposure to temperatures over a certain limit leads to massive protein aggregation in the cell. Disaggregation of such aggregates is largely dependent on the Hsp100 and Hsp70 chaperones. The exact role of the Hsp70 chaperone machine (composed of DnaK, DnaJ, and GrpE) in the Hsp100-dependent process remains unknown. In this study we focused on the Hsp70 role at the initial step of the disaggregation process. Two different aggregated model substrates, green fluorescent protein (GFP) and firefly luciferase, were incubated with the Hsp70 machine resulting in efficient fragmentation of large aggregates into smaller ones. Our data suggest that the observed fragmentation is achieved first by extraction of polypeptides from aggregates in Hsp70 chaperone machine-dependent manner and not by direct fragmentation of large aggregates. In the absence of Hsp100 (ClpB) these "extracted" polypeptides were not able to fold properly and promptly reassociated into new aggregates. The extracted GFP molecules were efficiently recognized and sequestered by a molecular trap, the mutant GroEL D87K, which binds stably to unfolded but not to native polypeptides. The binding of extracted GFP molecules to the GroEL trap prevented their reaggregation. We propose that the Hsp70 machine disentangles polypeptides from protein aggregates prior to Hsp100 action.     

Formation of α-synuclein aggregates in aqueous ethylammonium nitrate solutions

The effect of adding ethylammonium nitrate (EAN), which is an ionic liquid (IL), on the aggregate formation of α-synuclein (α-Syn) in aqueous solution has been investigated. FTIR and Raman spectroscopy were used to investigate changes in the secondary structure of α-Syn and in the states of water molecules and EAN. The results presented here show that the addition of EAN to α-Syn causes the formation of an intermolecular β-sheet structure in the following manner: native disordered state → polyproline II (PPII)-helix → intermolecular β-sheet (α-Syn amyloid-like aggregates: α-SynA). Although cations and anions of EAN play roles in masking the charged side chains and PPII-helix-forming ability involved in the formation of α-SynA, water molecules are not directly related to its formation. We conclude that EAN-induced α-Syn amyloid-like aggregates form at hydrophobic associations in the middle of the molecules after masking the charged side chains at the N- and C-terminals of α-Syn.     

Cartilage proteoglycan aggregates. Electronmicroscopic studies of native and fragmented molecules

1. Proteoglycan aggregates from bovine nasal cartilage were studied by using electron microscopy of proteoglycan/cytochrome c monolayers. 2. The aggregates contained a variably long central filament of hyaluronic acid with an average length of 1037nm. The proteoglycan monomers attached to the hyaluronic acid appeared as side chain filaments varying in length (averaging 249nm). They were distributed along the central filament at an average distance of about 36nm. 3. Chondroitin sulphate side chains were removed from the proteoglycan monomers of the aggregates by partial chondroitinase digestion. The molecules obtained had the same general appearance as intact aggregates. 4. Proteoglycan aggregates were treated with trypsin and the largest fragment, which contains the hyaluronic acid, link protein and hyaluronic acid-binding region, was recovered and studied with electron microscopy. Filaments that lacked the side chain extensions and had the same length as the central filament in the intact aggregate were observed. 5. Hyaluronic acid isolated after papain digestion of cartilage extracts gave filaments with similar length and size distribution as observed for the central filament both in the intact aggregate and in the trypsin digests. 6. Umbilical-cord hyaluronic acid was also studied and gave electron micrographs similar to those described for hyaluronic acid from cartilage. However, the length of the filament was somewhat shorter. 7. The electron micrographs of both intact and selectively degraded proteoglycans corroborate the current model of cartilage proteoglycan structure.     

Formation of double-walled microtubules and multilayered tubulin sheets by basic proteins

Some basic proteins enable microtubule protein to form special assembly products in vitro, known as double-walled microtubules. Using histones (H1, core histones) as well as the human encephalitogenic protein to induce the formation of double-walled microtubules, we made the following electron microscopic observations: (1) Double-walled microtubules consist of an "inner" microtubule which is covered by electron-dense material, apparently formed from the basic protein, and by a second tubulin wall. (2) The tubulin of the second wall seems to be arranged as protofilaments, surrounding the inner microtubule in a helical or ring-like manner. (3) The surface of double-walled microtubules lacks the projections of microtubule-associated proteins, usually found on microtubules. (4) In the case of protofilament ribbons (incomplete microtubules), H1 binds exclusively to their convex sides that correspond to the surface of microtubules. Zn2+-induced tubulin sheets, consisting in contrast to microtubules of alternately arranged protofilaments, are covered by H1 on both surfaces. Furthermore, multilayered sheet aggregates appeared. The results indicate that the basic proteins used interact only with that protofilament side which represents the microtubule surface. In accordance with this general principle, models on the structure of double-walled microtubules and multilayered tubulin sheets were derived.     

Purification and partial characterization of the type II procollagen synthesized by embryonic cartilage cells

Matrix-free cells were prepared from sternal cartilages of 17-day-old chick embryos, and procollagen synthesized and secreted by the cells was isolated by ion exchange chromatography on carboxymethyl cellulose and by gel filtration. The isolated protein was homogeneous by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and it appeared to consist of identical pro-α chains linked by interchain disulfide bonds. Amino acid analysis and cyanogen bromide peptide mapping of the purified procollagen demonstrated that it had structures similar to Type II collagen. The amino acid composition was also consistent with the conclusion that the peptide extensions on the pro-α chains of procollagen contained amino acid sequences not found in the collagen portion of the molecule. Segment-long-spacing aggregates were prepared from the procollagen, and aggregates demonstrated the same banding pattern as is found in segment-long-spacing aggregates prepared from Type II collagen. The segment-long-spacing aggregates from procollagen revealed, however, the presence of NH₂-terminal extensions of about 150 Å in length. In addition, the procollagen molecules contained irregularly shaped, large extension peptides at the COOH-terminal end of the molecule.     

Native mitochondrial creatine kinase forms octameric structures. II. Characterization of dimers and octamers by ultracentrifugation, direct mass measurements by scanning transmission electron microscopy, and image analysis of single mitochondrial creatine kinase octamers

Electron micrographs of negatively stained and metal-shadowed mitochondrial creatine kinase (Mi-CK) molecules purified as described by Schlegel et al. (Schlegel, J., Zurbriggen, B., Wegmann, E., Wyss, M., Eppenberger, H. M., and Wallimann, T. (1988) J. Biol Chem. 263, 16942-16953) revealed a homogeneous population (greater than or equal to 95%) of distinctly sized square-shaped, octameric particles with a side length of 10 nm that frequently exhibited a pronounced 4-fold axis of symmetry. The cube-like molecules consist of four dimers that are arranged around a stain-accumulating central cavity of 2.5-3 nm in diameter. This interpretation is supported by single particle averaging including correlation analysis by computer. Upon prolonged storage or high dilution, the cube-like octamers tended to dissociate into "banana-shaped" dimers. Sedimentation velocity and sedimentation equilibrium experiments yielded an s value of 12.8-13.5 S and an Mr of 328,000 +/- 25,000 for the octameric cubes. An s value of 5.0 S and a Mr of 83,000 +/- 8,000 was found under conditions which revealed banana-shaped dimers. These dimers proved to be very stable, as their dissociation into monomers of 45 kDa (s value = 2.0 S) required 6 M guanidine HCl. Thus, the oligomeric structures observed in the electron microscope are identified as Mi-CK dimers (banana-shaped structures) and cubical Mi-CK octamers assembled from four Mi-CK dimers. The octameric nature of native Mi-CK and the formation of Mi-CK dimers were confirmed by direct mass measurements of individual molecules by scanning transmission electron microscopy yielding a molecular mass of 340 +/- 55 kDa for the octamer and 89 +/- 27 kDa for the dimer. A structural model of Mi-CK octamers and the possible interaction with ATP/ADP-translocator molecules as well as with the outer mitochondrial membrane is proposed. The implications with respect to the physiological function of Mi-CK as an energy-channeling molecule at the producing side of the phosphoryl creatine shuttle are discussed.     

The cationic cell-penetrating peptide CPP(TAT) derived from the HIV-1 protein TAT is rapidly transported into living fibroblasts: optical, biophysical, and metabolic evidence

Cell-penetrating peptides (CPPs) are cationic peptides which, when linked to genes, proteins, or nanoparticles, facilitate the transport of these entities across the cell membrane. Despite their potential use for gene transfer and drug delivery, the mode of action of CPPs is still mysterious. It has even been argued that the observed transport across the cell membrane is an artifact caused by chemical fixation of the cells, a common preparation method for microscopic observation. Here we have synthesized a fluorescent derivative of the HIV-1 TAT protein transduction domain [Fg-CPP(TAT(PTD))] and have observed its uptake into nonfixated living fibroblasts with time-lapse confocal microscopy, eliminating the need for fixation. We observe that Fg-CPP(TAT(PTD)) enters the cytoplasm and nucleus of nonfixated fibroblasts within seconds, arguing against the suggested artifact of cell fixation. Using differential interference contrast microscopy, dense aggregates are detected on the cell surface. Several observations suggest that these aggregates consist of Fg-CPP(TAT(PTD)) bound to membrane-associated heparan sulfate (HS). The aggregates grow in parallel with Fg-CPP(TAT(PTD)) uptake and are detected only on fibroblasts showing Fg-CPP(TAT(PTD)) uptake. These observations resemble earlier reports of "capping" of cell surface molecules combined with a polarized endocytotic flow. Enzymatic removal of extracellular HS reduced the rate of both Fg-CPP(TAT(PTD)) uptake and aggregate formation, demonstrating that HS is involved in the uptake mechanism. The functionality of the fibroblasts during the CPP uptake was investigated with a cytosensor microphysiometer measuring the extracellular acidification rate (ECAR). Short exposures (2.5 min) to the CPP reduced the ECAR which was, however, reversible upon reperfusion with buffer only. In contrast, no recovery to baseline values was observed after repeated exposures to the CPP, suggesting that the CPP is toxic in long-term applications.     

Inactivation of monazomycin-induced voltage-dependent conductance in thin lipid membranes. II. Inactivation produced by monazomycin transport through the membrane

At sufficiently large conductances, the voltage-dependent conductance induced in thin lipid membranes by monazomycin undergoes inactivation. This is a consequence of depletion of monazomycin from the membrane solution interface, as monazomycin crosses the membrane to the opposite (trans) side from which it was added. The flux of monazomycin is directly proportional to the monazomycin-induced conductance; at a given conductance it is independent of monazomycin concentration. We conclude that when monazomycin channels break up, some or all of the molecules making up a channel are deposited on the trans side. We present a model for the monazomycin channel: approximately five molecules, each spanning the membrane with its NH3+ on the trans side and an uncharged hydrophilic (probably sugar) group anchored to the cis side, form an aqueous channel lined by--OH groups. The voltage dependence arises from the flipping by the electrical field of molecules lying parallel to the cis surface into the "spanned state;" the subsequent aggregation of these molecules into channels is, to a first approximation, voltage independent. The channel breakup that deposits monomers on the trans side involves the collapsing of the channel in such a way that the uncharged hydrophilic groups remain in contact with the water in the channel as they close the channel from behind. We also discuss the possibility that inactivation of sodium channels in nerve involves the movement from one side of the membrane to the other of the molecules (or molecule) forming the channel.     

Organization of antibiotic amphotericin B in model lipid membranes. A mini review

Amphotericin B (AmB) is a polyene antibiotic frequently applied in the treatment of fungal infections. According to the general understanding, the mode of action of AmB is directly related to the molecular organization of the drug in the lipid environment, in particular to the formation of pore-like molecular aggregates. Electronic absorption and fluorescence techniques were applied to investigate formation of molecular aggregates of AmB in the lipid environment of liposomes and monomolecular layers formed at the argon-water interface. It appears that AmB dimers, stabilized by van der Waals interactions, are present in the membrane environment along with the aggregates formed by a greater number of molecules. Linear dichroism measurements reveal that AmB is distributed between two fractions of molecules, differently oriented with respect to the bilayer. Molecules in one fraction remain parallel to the plane of the membrane and molecules in the other one are perpendicular. Scanning Force Microscopy imaging of the surface topography of the monolayers formed with AmB in the presence of lipids reveals formation of pore-like structures characterized by the external diameter close to 17 A and the internal diameter close to 6 A. All the findings are discussed in terms of importance of the molecular organization of AmB in the pharmacological action, as well as of the toxic side effects of the drug.