ABSTRACT
Different models have been proposed for the nature of the potexvirus transport form that moves from cell to cell over the infected plant: (i) genomic RNA moves as native virions; or (ii) in vitro-assembled non-virion ribonucleoprotein (RNP) complexes consisting of viral RNA, coat protein (CP) and movement protein (MP), termed TGBp1, serve as the transport form in vivo. As the structure of these RNPs has not been elucidated, the products assembled in vitro from potato virus X (PVX) RNA, CP and TGBp1 were characterized. The complexes appeared as single-tailed particles (STPs) with a helical, head-like structure composed of CP subunits located at the 5'-proximal region of PVX RNA; the TGBp1 was bound to the terminal CP molecules of the head. Remarkably, no particular non-virion RNP complexes were observed. These data suggest that the CP-RNA interactions resulting in head formation prevailed over TGBp1-RNA binding upon STP assembly from RNA, CP and TGBp1. STPs could be assembled from the 5' end of PVX RNA and CP in the absence of TGBp1. The translational ability of STPs was characterized in a cell-free translation system. STPs lacking TGBp1 were entirely non-translatable; however, they were rendered translatable by binding of TGBp1 to the end of the head. It is suggested that the RNA-mediated assembly of STPs proceeds via two steps. Firstly, non-translatable CP-RNA STPs are produced, due to encapsidation of the 5'-terminal region. Secondly, the TGBp1 molecules bind to the end of a polar head, resulting in conversion of the STPs into a translatable form.
Subject(s)
Capsid Proteins/metabolism , Potexvirus/metabolism , RNA, Viral/metabolism , Viral Proteins/metabolism , Biological Transport, Active , Capsid Proteins/chemistry , Capsid Proteins/genetics , Macromolecular Substances , Microscopy, Atomic Force , Microscopy, Immunoelectron , Plant Viral Movement Proteins , Potexvirus/genetics , Protein Biosynthesis , RNA, Viral/chemistry , RNA, Viral/genetics , Viral Proteins/chemistry , Viral Proteins/geneticsABSTRACT
The structure of complexes formed in vitro by tobacco mosaic virus (TMV)-coded movement protein (MP) with TMV RNA and short (890 nt) synthetic RNA transcripts was visualized by atomic force microscopy on a mica surface. MP molecules were found to be distributed along the chain of RNA and the structure of MP-RNA complexes depended on the molar MP:RNA ratios at which the complexes were formed. A rise in the molar MP:TMV RNA ratio from 20:1 to 60-100:1 resulted in an increase in the density of the MP packaging on TMV RNA and structural conversion of complexes from RNase-sensitive 'beads-on-a-string' into a 'thick string' form that was partly resistant to RNAse. The 'thick string'-type RNase-resistant complexes were also produced by short synthetic RNA transcripts at different MP:RNA ratios. The 'thick string' complexes are suggested to represent clusters of MP molecules cooperatively bound to discrete regions of TMV RNA and separated by protein-free RNA segments.
Subject(s)
Microscopy, Atomic Force , RNA, Viral/metabolism , RNA, Viral/ultrastructure , Tobacco Mosaic Virus/chemistry , Tobacco Mosaic Virus/genetics , Viral Proteins/metabolism , Viral Proteins/ultrastructure , Aluminum Silicates , Models, Molecular , Plant Viral Movement Proteins , Plants, Toxic , Protein Binding , Protein Structure, Quaternary , RNA, Viral/chemical synthesis , RNA, Viral/genetics , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/ultrastructure , Ribonucleases/metabolism , Nicotiana/virology , Viral Proteins/chemistryABSTRACT
Escherichia coli bacterial cells of two strains JM109 and K12 J62 were imaged with atomic force microscopy (AFM) in different environmental conditions. The AFM results show that the two strains have considerable difference in the surface morphology. At the same time after rehydration both strains show the loss of the topographic features and increase in lateral and vertical dimensions. Results obtained in different AFM modes (contact, tapping, MAC) were compared. Imaging in culture medium was applied for direct observation of the surface degradation effect of lysozyme. The treatment of the cells with the enzyme in the culture medium lead to the loss of surface rigidity and eventually to dramatic changes of the bacteria shape.
Subject(s)
Escherichia coli/physiology , Escherichia coli/ultrastructure , Microscopy, Atomic Force/methods , Air Microbiology , Culture Media , Muramidase/metabolism , Surface Properties , Water , Water MicrobiologyABSTRACT
We optimized the procedure for the formation of Langmuir films of antibodies based on amphiphilic polyelectrolytes and studied the physicochemical and immunochemical properties of the films obtained. Their immunochemical properties were compared with the immunochemical activity of antibodies in Langmuir films without amphiphilic polyelectrolytes and with antibodies adsorbed on the surface of polystyrene and graphite. The efficiency of immune adsorption by the films based on amphiphilic polyelectrolytes was shown to be greater; the affinity of antibodies and surface concentration of their active conformation depended on the type of amphiphilic polyelectrolytes used to obtain the films. We investigated the structure of these films at the surface of highly oriented pyrolytic graphite using the method of atomic force microscopy. Changes in the structure of the films under study caused by the increase of surface pressure were demonstrated.
Subject(s)
Antibodies/immunology , Antibodies/metabolism , Electrolytes/metabolism , Immunosorbent Techniques , Membranes, Artificial , Polymers/metabolism , Biosensing Techniques/methodsABSTRACT
The application of the AFM technique for visualization of membrane proteins and for measuring their dimensions was demonstrated. The AFM images of the microsomal monooxygenase system components-cytochrome P450 2B4 and NADPH-cytochrome P450 reductase-were obtained by using two types of supports-hydrophobic, highly oriented pyrolytic graphite (HOPG) and hydrophilic mica. It was shown that hemo- and flavoprotein monomers and oligomers can be adsorbed to and visualized on HOPG. On the negatively charged mica matrix, flavoprotein oligomers dissociated to monomers while hemoprotein oligomers dissociated into less aggregated particles. The images of cytochrome P450 2B4 and NADPH-cytochrome P450 reductase monomers were about 3 and 5 nm high, respectively, while the images of oligomeric forms of these proteins were about 10 and 8 nm high, respectively. We were able to observe the binary complexes composed of monomeric proteins, cytochrome P450 2B4 and its reductase and to measure the heights of these complexes (7 nm). The method is applicable for visualization of not only individual proteins but also their complexes.
Subject(s)
Aryl Hydrocarbon Hydroxylases , Cytochrome P-450 Enzyme System/metabolism , Cytochrome P-450 Enzyme System/ultrastructure , NADPH-Ferrihemoprotein Reductase/metabolism , NADPH-Ferrihemoprotein Reductase/ultrastructure , Steroid Hydroxylases/metabolism , Steroid Hydroxylases/ultrastructure , Animals , Flavoproteins/chemistry , Flavoproteins/metabolism , Hemeproteins/metabolism , Hemeproteins/ultrastructure , Macromolecular Substances , Microscopy, Atomic Force/methods , Microsomes, Liver/enzymology , Oxidation-Reduction , RabbitsABSTRACT
In the present paper, the application of scanning tunneling microscopy in cytochrome P450s membrane topology is discussed. The method enables visualization of heme location in the lipid-bilayer-incorporated protein. It is supposed that the membrane-bound cytochrome P450 on the tunneling microscope substrate should behave as 'molecular diode'. A model explaining the liposome and the proteoliposome images observed is proposed.
