Nucleotide exchange and excision technology (NExT) DNA shuffling: a robust method for DNA fragmentation and directed evolution

DNA shuffling is widely used for optimizing complex properties contained within DNA and proteins. Demonstrated here is the amplification of a gene library by PCR using uridine triphosphate (dUTP) as a fragmentation defining exchange nucleotide with thymidine, together with the three other nucleotides. The incorporated uracil bases were excised using uracil-DNA-glycosylase and the DNA backbone subsequently cleaved with piperidine. These end-point reactions required no adjustments. Polyacrylamide urea gels demonstrated adjustable fragmentation size over a wide range. The oligonucleotide pool was reassembled by internal primer extension to full length with a proofreading polymerase to improve yield over Taq. We present a computer program that accurately predicts the fragmentation pattern and yields all possible fragment sequences with their respective likelihood of occurrence, taking the guesswork out of the fragmentation. The technique has been demonstrated by shuffling chloramphenicol acetyltransferase gene libraries. A 33% dUTP PCR resulted in shuffled clones with an average parental fragment size of 86 bases even without employment of a fragment size separation, and revealed a low mutation rate (0.1%). NExT DNA fragmentation is rational, easily executed and reproducible, making it superior to other techniques. Additionally, NExT could feasibly be applied to several other nucleotide analogs

The Interaction Properties of the Human Rab GTPase Family – A Comparative Analysis Reveals Determinants of Molecular Binding Selectivity

Rab GTPases constitute the largest subfamily of the Ras protein superfamily. Rab proteins regulate organelle biogenesis and transport, and display distinct binding preferences for effector and activator proteins, many of which have not been elucidated yet. The underlying molecular recognition motifs, binding partner preferences and selectivities are not well understood.Comparative analysis of the amino acid sequences and the three-dimensional electrostatic and hydrophobic molecular interaction fields of 62 human Rab proteins revealed a wide range of binding properties with large differences between some Rab proteins. This analysis assists the functional annotation of Rab proteins 12, 14, 26, 37 and 41 and provided an explanation for the shared function of Rab3 and 27. Rab7a and 7b have very different electrostatic potentials, indicating that they may bind to different effector proteins and thus, exert different functions. The subfamily V Rab GTPases which are associated with endosome differ subtly in the interaction properties of their switch regions, and this may explain exchange factor specificity and exchange kinetics.We have analysed conservation of sequence and of molecular interaction fields to cluster and annotate the human Rab proteins. The analysis of three dimensional molecular interaction fields provides detailed insight that is not available from a sequence-based approach alone. Based on our results, we predict novel functions for some Rab proteins and provide insights into their divergent functions and the determinants of their binding partner selectivity

Dynamic Coupling of Pattern Formation and Morphogenesis in the Developing Vertebrate Retina

In this Research Article, Picker et al. show how cells in the retina get their spatial coordinates

Proposed sequential action of the guanine nucleotide exchange factor (GEF) on geranyl-geranylated Rab-GTPases.

<p>The GEF destabilizes the ternary GDP-Rab-GEF complex and GDP is released. The nucleotide-free Rab and the GEF form a stable binary complex. Subsequent GTP binding to the nucleotide-free Rab again destabilizes the ternary complex, the GEF is released and the Rab is left in its active GTP-bound state.</p

Conservation of sequence and molecular interaction fields in human Rab GTPases.

<p><b>A</b>: Rab5a (PDB entry 1R2Q) secondary structure and amino acid residues colored from variable (blue), through intermediate (green) to conserved (red) according to conservation of sequence (Seq), electrostatic potential (EP) and hydrophic interaction field (HIF). <b>B:</b> Cartoon representation of Rab5a with bound nucleotide in stick representation (pink) and the similarity of the electrostatic (EP) and hydrophobic interaction fields (HIF) mapped onto the structures. The conservation scores of the corresponding residues are represented by a color gradient from blue (variable), through green to red (conserved). Each structure is shown in two views, one focusing on switch regions I and II and one rotated 180° about the vertical axis to highlight the helices and CDRs.</p

All pairwise comparison of electrostatic potentials in human Rab GTPases.

<p>Heat-map representation of the distance matrix of electrostatic potentials computed for the entire protein skins of human Rab proteins with the color code ranging from red (identical) to blue (dissimilar). The ordering of the Rab GTPases corresponds approximately to that of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034870#pone-0034870-g002" target="_blank">Figure 2B</a>, right.</p

Rab GTPases as molecular switches.

<p><b>A</b>: Schematic figure showing the interactions of a Rab-GTPase with its effector proteins. The Rab protein is geranyl-geranylated near the C-terminus to enable membrane binding. Guanine nucleotide exchange factor proteins (GEFs) accelerate the GDP to GTP exchange and thus convert the GTPase from its inactive into its active form. Guanine nucleotide activating proteins (GAPs) deactivate the Rab GTPase by facilitating the intrinsic GTP hydrolysis. <b>B</b>: Sequence and structural mapping of characteristic segments of Rab proteins. Rab family-specific (RabF1-RabF5) sequence segments that are distinct for Rab GTPases and distinguish them from other small GTPases of the Ras superfamily (Pereira-Leal and Seabra, 2001) are mapped onto the crystal structure of the Rab5A GTPase from <i>H. sapiens</i> in an active conformation (Terzyan et al., 2004) (displayed in orange-red). Rab subfamily (RabSF1-RabSF3) specific sequence segments are characteristic for subsets of the Rab GTPases in which each subfamily displays a high sequence identity (displayed in green) (Pereira-Leal and Seabra, 2000). The nucleotide is shown in pink. The Switch I and II regions, which undergo large nucleotide dependent conformational transitions, are labeled.</p