ABSTRACT
Filamentous bacteriophage virions can be engineered to display small foreign peptides in the N-terminal regions of all 2700 copies of the major coat protein (pVIII), but larger peptides can be accommodated only in hybrid virions, in which modified and wild-type coat protein subunits are interspersed. The copy number of peptides accepted in hybrid virions is generally believed to be related to peptide size: the larger the insert, the lower the number of modified coat protein subunits in the assembled virion. However, we show here that some large peptides can be displayed at a much higher copy number than smaller ones and that some relatively small peptides are poorly displayed, if at all, in hybrid virions. X-ray diffraction studies of a recombinant virion together with model building experiments with peptide and protein epitopes of known structure demonstrated that it is feasible to accommodate much larger structures, without perturbation of the capsid protein packing, than it has proved possible to generate in vivo. We show further that the insertion of certain peptides greatly slowed or even prevented the processing of the pVIII pro-coat by leader peptidase at the inner membrane of the Escherichia coli cell. A good correlation was found between the effect of the insert on the rate of the processing of the pro-coat, an essential step in virus assembly, and the number of the mature but modified proteins in the subsequently assembled hybrid virion. These results have important implications for the design of peptide display systems based on filamentous bacteriophage.
Subject(s)
Capsid/chemistry , Inovirus/chemistry , Membrane Proteins , Serine Endopeptidases , Viral Core Proteins/chemistry , Viral Matrix Proteins/metabolism , Viral Proteins/chemistry , Amino Acid Sequence , Base Sequence , Capsid/genetics , Cell Membrane/virology , Endopeptidases/metabolism , Epitopes , Escherichia coli/ultrastructure , Models, Molecular , Molecular Sequence Data , Peptides/chemistry , Peptides/immunology , Peptides/metabolism , Protein Conformation , Protein Folding , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Viral Core Proteins/immunology , Virion/chemistry , Virion/genetics , X-Ray Diffraction/methodsABSTRACT
The mechanism of inactivation of the electron-transport chain of chloroplasts within the framework of a previously described kinetic model based on two steps, i.e. activation of the electron-transport chain and its irreversible inactivation, was studied. It was found that activation results from a decrease in the transmembrane gradient of pH on the photosynthetic membrane during inactivation. The kinetics of irreversible inactivation of different parts of the electron-transport chain of chloroplasts were studied. The water decompositon system was shown to be the most labile part of the chain (at a point lying before the carrier accepting the electrons from 1,5-diphenylcarbazide). The inactivation kinetics of the electron-transport chain of chloroplasts under different environmental conditions were studied. The stabilizing effects of definite concentrations of hydrogens ions, glycerol and bovine serum albumin were demonstrated. The mechanisms of these effects are discussed. A 200-fold stabilization of the chloroplasts with respect to their irreversible inactivation due to optimization of the medium was achieved.
