ABSTRACT
Light-switchable proteins offer numerous opportunities as tools for manipulating biological systems with exceptional degrees of spatiotemporal control. Most designed light-switchable proteins currently in use have not been optimised using the randomisation and selection/screening approaches that are widely used in other areas of protein engineering. Here we report an approach for screening light-switchable DNA-binding proteins that relies on light-dependent repression of the transcription of a fluorescent reporter. We demonstrate that the method can be used to recover a known light-switchable DNA-binding protein from a random library.
Subject(s)
DNA-Binding Proteins/genetics , Protein Engineering , Transcription, Genetic , DNA-Binding Proteins/chemistry , Escherichia coli/genetics , LightABSTRACT
The opportunistic pathogen Pseudomonas aeruginosa causes chronic biofilm infections in cystic fibrosis patients. During colonization of the lung, P. aeruginosa converts to a mucoid phenotype characterized by overproduction of the exopolysaccharide alginate. Here we show that AlgK, a protein essential for production of high molecular weight alginate, is an outer membrane lipoprotein that contributes to the correct localization of the porin AlgE. Our 2.5 A structure shows AlgK is composed of 9.5 tetratricopeptide-like repeats, and three putative sites of protein-protein interaction have been identified. Bioinformatics analysis suggests that BcsA, PgaA, and PelB, involved in the production and export of cellulose, poly-beta-1,6-N-Acetyl-D-glucosamine, and Pel exopolysaccharide, respectively, share the same topology as AlgK/E. Together, our data suggest that AlgK plays a role in the assembly of the alginate biosynthetic complex and represents the periplasmic component of a new type of outer membrane secretin that differs from canonical bacterial capsular polysaccharide secretion systems.
Subject(s)
Bacterial Proteins/chemistry , Polysaccharides, Bacterial/chemistry , Pseudomonas aeruginosa , Alginates , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , Biofilms , Crystallography, X-Ray , Glucuronic Acid/biosynthesis , Hexuronic Acids , Humans , Models, Molecular , Molecular Sequence Data , Polysaccharides, Bacterial/metabolism , Protein Structure, Tertiary , Pseudomonas aeruginosa/metabolism , Pseudomonas aeruginosa/pathogenicity , SecretinABSTRACT
AlgK is an outer-membrane lipoprotein involved in the biosynthesis of alginate in Pseudomonads and Azotobacter vinelandii. A recombinant form of Pseudomonas fluorescens AlgK with a C-terminal polyhistidine affinity tag has been expressed and purified from the periplasm of Escherichia coli cells and diffraction-quality crystals of AlgK have been grown using the hanging-drop vapour-diffusion method. The crystals grow as flat plates with unit-cell parameters a = 79.09, b = 107.85, c = 119.15 A, beta = 96.97 degrees. The crystals exhibit the symmetry of space group P2(1) and diffract to a minimum d-spacing of 2.5 A at Station X29 of the National Synchrotron Light Source, Brookhaven National Laboratory. On the basis of the Matthews coefficient (V(M) = 2.53 A3 Da(-1)), four protein molecules are estimated to be present in the asymmetric unit.
Subject(s)
Bacterial Proteins/chemistry , Pseudomonas fluorescens/chemistry , Bacterial Proteins/isolation & purification , Base Sequence , Cloning, Molecular , Crystallization , Crystallography, X-Ray , DNA Primers , Electrophoresis, Polyacrylamide Gel , Protein ConformationABSTRACT
The in vivo labelling of viral or cellular components is usually conducted through the addition of radioactively labelled precursors to the culture medium. A limiting factor for isotope use is often the cost of isotope purchase and disposal. Therefore, significant savings can be achieved if the smallest possible volume of medium is employed. However, in the case of adherent cells growing in tissue culture dishes or multi-well plates, surface tension causes a very uneven distribution of the liquid due to the formation of a meniscus at the edge of the petri. This prevents the use of very small volumes of medium for cell growth and labelling because the cells at the center of the petri dish would dry out and die, especially after longer incubation periods. In this communication, we describe a technique whereby cells are grown in an area surrounded by a hydrophobic ring of Teflon, which greatly improves the distribution of the medium by eliminating the concave meniscus. This translates into a dramatic improvement in the condition of the cells, as well as the efficiency of labelling of phosphoproteins, such as the Simian Virus 40 large tumor antigen with 32P-orthophosphate or labelling of the cellular DNA with 3[H]thymidine. The technique is useful for any application where growth of cells in small volumes of medium is required.
