When second best is good enough: another probabilistic look at saturation mutagenesis.

We developed new criteria for determining the library size in a saturation mutagenesis experiment. When the number of all possible distinct variants is large, any of the top-performing variants (e.g., any of the top three) is likely to meet the design requirements, so the probability that the library contains at least one of them is a sensible criterion for determining the library size. By using a criterion of this type, one may significantly reduce the library size and thus save costs and labor while minimally compromising the quality of the best variant discovered. We present the probabilistic tools underlying these criteria and use them to compare the efficiencies of four randomization schemes: NNN, which uses all 64 codons; NNB, which uses 48 codons; NNK, which uses 32 codons; and MAX, which assigns equal probabilities to each of the 20 amino acids. MAX was found to be the most efficient randomization scheme and NNN the least efficient. TopLib, a computer program for carrying out the related calculations, is available through a user-friendly Web server.

Preferential protection of domains II and III of bacillus thuringiensis cry1Aa toxin by brush border membrane vesicles / Protección preferencial de los dominios II y III de la toxina cry1Aa de bacillus thuringiensis en vesículas de membrana de borde de cepillo

The surface exposed Leucine 371 on loop 2 of domain II, in Cry1Aa toxin, was mutated to Lysine to generate the trypsin-sensitive mutant, L371K. Upon trypsin digestion L371K is cleaved into approximately 37 and 26 kDa fragments. These are separable on SDS-PAGE, but remain as a single molecule of 65 kDa upon purification by liquid chromatography. The larger fragment is domain I and a portion of domain II (amino acid residues 1 to 371). The smaller 26-kDa polypeptide is the remainder of domain II and domain III (amino acids 372 to 609). When the mutant toxin was treated with high dose of M. sexta gut juice both fragments were degraded. However, when incubated with M. sexta BBMV, the 26 kDa fragment (domains II and III) was preferentially protected from gut juice proteases. As previously reported, wild type Cry1Aa toxin was also protected against degradation by gut juice proteases when incubated with M. sexta BBMV. On the contrary, when mouse BBMV was added to the reaction mixture neither Cry1Aa nor L371K toxins showed resistance to M. sexta gut juice proteases and were degraded. Since the whole Cry1Aa toxin and most of the domain II and domain III of L371K are protected from proteases in the presence of BBMV of the target insect, we suggest that the insertion of the toxin into the membrane is complex and involves all three domains.

La superficie de la toxina Cry1Aa, en el asa 2 del dominio II contiene expuesta la leucina 371, la cual fue modificada a lisina produciendo una mutante sensible a la tripsina, L371K. Esta mutante produce dos fragmentos de 37 y 26 kDa por acción de la tripsina que son separables por SDS-PAGE, pero que a la purificación por cromatografía líquida se mantienen como una sola molécula de 65 kDa. El fragmento grande contiene al dominio I y una parte del dominio II (aminoácidos 1 al 371). El polipéptido de 26 kDa contiene la parte restante del dominio II y dominio III (aminoácidos 372 al 609). Cuando la toxina mutante fue tratada con dosis altas de jugo intestinal de Manduca sexta, ambos fragmentos fueron degradados. Sin embargo, cuando fueron incubados en VMBC de M. sexta, el fragmento de 26 kDa fue protegido preferencialmente de las proteasas intestinales. Como se ha reportado, la toxina silvestre Cry1Aa también es protegida de la degradación de las proteasas cuando es incubada en VMBC de M. sexta. Sin embargo, cuando se adicionó VMBC de ratón a la mezcla de reacción, ni la toxina Cry1Aa ni la mutante L371K mostraron resistencia a las proteasas y fueron degradadas. Dado que la toxina completa de Cry1Aa y casi todo de los dominios II y III de L371K están protegidos de proteasas en presencia de VMBC del insecto, este estudio sugiere que la inserción de la toxina en la membrana involucra los tres dominios.

In silico mutagenesis and docking studies of Pseudomonas aeruginosa PA-IIL lectin predicting binding modes and energies.

This article is focused on the application of two types of docking software, AutoDock and DOCK. It is aimed at studying the interactions of a calcium-dependent bacterial lectin PA-IIL (from Pseudomonas aeruginosa) and its in silico mutants with saccharide ligands. The effect of different partial charges assigned to the calcium ions was tested and evaluated in terms of the best agreement with the crystal structure. The results of DOCK were further optimized by molecular dynamics and rescored using AMBER. For both software, the agreement of the docked structures and the provided binding energies were evaluated in terms of prediction accuracy. This was carried out by comparing the computed results to the crystal structures and experimentally determined binding energies, respectively. The performance of both docking software applied on a studied problem was evaluated as well. The molecular docking methods proved efficient in identifying the correct binding modes in terms of geometry and partially also in predicting the preference changes caused by mutation. Obtaining a reasonable in silico method for the prediction of lectin-saccharide interactions may be possible in the future.

A novel site-specific recombination system derived from bacteriophage phiMR11.

We report identification of a novel site-specific DNA recombination system that functions in both in vivo and in vitro, derived from lysogenic Staphylococcus aureus phage phiMR11. In silico analysis of the phiMR11 genome indicated orf1 as a putative integrase gene. Phage and bacterial attachment sites (attP and attB, respectively) and attachment junctions were determined and their nucleotide sequences decoded. Sequences of attP and attB were mostly different to each other except for a two bp common core that was the crossover point. We found several inverted repeats adjacent to the core sequence of attP as potential protein binding sites. The precise and efficient integration properties of phiMR11 integrase were shown on attP and attB in Escherichia coli and the minimum size of attP was found to be 34bp. In in vitro assays using crude or purified integrase, only buffer and substrate DNAs were required for the recombination reaction, indicating that other bacterially encoded factors are not essential for activity.