High-throughput mutagenesis using a two-fragment PCR approach.

Site-directed scanning mutagenesis is a powerful protein engineering technique which allows studies of protein functionality at single amino acid resolution and design of stabilized proteins for structural and biophysical work. However, creating libraries of hundreds of mutants remains a challenging, expensive and time-consuming process. The efficiency of the mutagenesis step is the key for fast and economical generation of such libraries.

Short Linear Sequence Motif LxxPTPh Targets Diverse Proteins to Growing Microtubule Ends.

Microtubule plus-end tracking proteins (+TIPs) are involved in virtually all microtubule-based processes. End-binding (EB) proteins are considered master regulators of +TIP interaction networks, since they autonomously track growing microtubule ends and recruit a plethora of proteins to this location. Two major EB-interacting elements have been described: CAP-Gly domains and linear SxIP sequence motifs.

Structural basis for misregulation of kinesin KIF21A autoinhibition by CFEOM1 disease mutations.

Tight regulation of kinesin activity is crucial and malfunction is linked to neurological diseases. Point mutations in the KIF21A gene cause congenital fibrosis of the extraocular muscles type 1 (CFEOM1) by disrupting the autoinhibitory interaction between the motor domain and a regulatory region in the stalk. However, the molecular mechanism underlying the misregulation of KIF21A activity in CFEOM1 is not understood.

Centriolar CPAP/SAS-4 Imparts Slow Processive Microtubule Growth.

Centrioles are fundamental and evolutionarily conserved microtubule-based organelles whose assembly is characterized by microtubule growth rates that are orders of magnitude slower than those of cytoplasmic microtubules. Several centriolar proteins can interact with tubulin or microtubules, but how they ensure the exceptionally slow growth of centriolar microtubules has remained mysterious. Here, we bring together crystallographic, biophysical, and reconstitution assays to demonstrate that the human centriolar protein CPAP (SAS-4 in worms and flies) binds and "caps" microtubule plus ends by associating with a site of β-tubulin engaged in longitudinal tubulin-tubulin interactions.

SAS-6 engineering reveals interdependence between cartwheel and microtubules in determining centriole architecture.

Centrioles are critical for the formation of centrosomes, cilia and flagella in eukaryotes. They are thought to assemble around a nine-fold symmetric cartwheel structure established by SAS-6 proteins. Here, we have engineered Chlamydomonas reinhardtii SAS-6-based oligomers with symmetries ranging from five- to ten-fold.

Structure of the BoNT/A1--receptor complex.

Botulinum neurotoxin A causes botulism but is also used for medical and cosmetic applications. A detailed molecular understanding of BoNT/A--host receptor interactions is therefore fundamental for improving current clinical applications and for developing new medical strategies targeting human disorders. Towards this end, we recently solved an X-ray crystal structure of BoNT/A1 in complex with its neuronal protein receptor SV2C.

Coronin 1 regulates cognition and behavior through modulation of cAMP/protein kinase A signaling.

Cognitive and behavioral disorders are thought to be a result of neuronal dysfunction, but the underlying molecular defects remain largely unknown. An important signaling pathway involved in the regulation of neuronal function is the cyclic AMP/Protein kinase A pathway. We here show an essential role for coronin 1, which is encoded in a genomic region associated with neurobehavioral dysfunction, in the modulation of cyclic AMP/PKA signaling.

Functional map of arrestin-1 at single amino acid resolution.

Arrestins function as adapter proteins that mediate G protein-coupled receptor (GPCR) desensitization, internalization, and additional rounds of signaling. Here we have compared binding of the GPCR rhodopsin to 403 mutants of arrestin-1 covering its complete sequence. This comprehensive and unbiased mutagenesis approach provides a functional dimension to the crystal structures of inactive, preactivated p44 and phosphopeptide-bound arrestins and will guide our understanding of arrestin-GPCR complexes.

Structural basis for recognition of synaptic vesicle protein 2C by botulinum neurotoxin A.

Botulinum neurotoxin A (BoNT/A) belongs to the most dangerous class of bioweapons. Despite this, BoNT/A is used to treat a wide range of common medical conditions such as migraines and a variety of ocular motility and movement disorders. BoNT/A is probably best known for its use as an antiwrinkle agent in cosmetic applications (including Botox and Dysport).

AAscan, PCRdesign and MutantChecker: a suite of programs for primer design and sequence analysis for high-throughput scanning mutagenesis.

Scanning mutagenesis is a powerful protein engineering technique used to study protein structure-function relationship, map binding sites and design more stable proteins or proteins with altered properties. One of the time-consuming tasks encountered in application of this technique is the design of primers for site-directed mutagenesis. Here we present an open-source multi-platform software AAscan developed to design primers for this task according to a set of empirical rules such as melting temperature, overall length, length of overlap regions, and presence of GC clamps at the 3' end, for any desired substitution.

High-level secretion of recombinant full-length streptavidin in Pichia pastoris and its application to enantioselective catalysis.

Artificial metalloenzymes result from the incorporation of a catalytically competent biotinylated organometallic moiety into full-length (i.e. mature) streptavidin.

Structural basis for the oligomerization-state switch from a dimer to a trimer of an engineered cortexillin-1 coiled-coil variant.

Coiled coils are well suited to drive subunit oligomerization and are widely used in applications ranging from basic research to medicine. The optimization of these applications requires a detailed understanding of the molecular determinants that control of coiled-coil formation. Although many of these determinants have been identified and characterized in great detail, a puzzling observation is that their presence does not necessarily correlate with the oligomerization state of a given coiled-coil structure.

Synthesis and evaluation of biphenyl compounds as kinesin spindle protein inhibitors.

Kinesin spindle protein (KSP), an ATP-dependent motor protein, plays an essential role in bipolar spindle formation during the mitotic phase (M phase) of the normal cell cycle. KSP has emerged as a novel target for antimitotic anticancer drug development. In this work, we synthesized a range of new biphenyl compounds and investigated their properties in vitro as potential antimitotic agents targeting KSP expression.

Structural basis of tubulin tyrosination by tubulin tyrosine ligase.

Tubulin tyrosine ligase (TTL) catalyzes the post-translational retyrosination of detyrosinated α-tubulin. Despite the indispensable role of TTL in cell and organism development, its molecular mechanism of action is poorly understood. By solving crystal structures of TTL in complex with tubulin, we here demonstrate that TTL binds to the α and β subunits of tubulin and recognizes the curved conformation of the dimer.

Outcome, general, and symptom-specific quality of life after various types of parotid resection.

Objectives/hypothesis: To document the outcome and impact on general and symptom-specific quality of life (QOL) after various types of parotid resection.

Study Design: General and symptom-specific QOL assessment at least 1 year after performed surgery. Retrospective data and outcome analysis of patients.

Interaction of mammalian end binding proteins with CAP-Gly domains of CLIP-170 and p150(glued).

End binding proteins (EBs) track growing microtubule ends and play a master role in organizing dynamic protein networks. Mammalian cells express up to three different EBs (EB1, EB2, and EB3). Besides forming homodimers, EB1 and EB3 also assemble into heterodimers.

Expression analysis of proline rich 15 (Prr15) in mouse and human gastrointestinal tumors.

Proline rich 15 (Prr15), which encodes a protein of unknown function, is expressed almost exclusively in postmitotic cells both during fetal development and in adult tissues, such as the intestinal epithelium and the testis. To determine if this specific expression is lost in intestinal neoplasias, we examined Prr15 expression by in situ hybridization (ISH) on mouse intestinal tumors caused by different gene mutations, and on human colorectal cancer (CRC) samples. Prr15/PRR15 expression was consistently observed in mouse gastrointestinal (GI) tumors caused by mutations in the Apc gene, as well as in several advanced stage human CRCs.

Automated seamless DNA co-transformation cloning with direct expression vectors applying positive or negative insert selection.

Background: Molecular DNA cloning is crucial to many experiments and with the trend to higher throughput of modern approaches automated techniques are urgently required. We have established an automated, fast and flexible low-cost expression cloning approach requiring only vector and insert amplification by PCR and co-transformation of the products.

Results: Our vectors apply positive selection for the insert or negative selection against empty vector molecules and drive strong expression of target proteins in E.

The effect of perfluorocarbon-based artificial oxygen carriers on tissue-engineered trachea.

The biological effect of the perfluorocarbon-based artificial oxygen carrier (Oxygent) was investigated in tissue-engineered trachea (TET) construction. Media supplemented with and without 10% Oxygent were compared in all assessments. Partial tissue oxygen tension (PtO(2)) was measured with polarographic microprobes; epithelial metabolism was monitored by microdialysis inside the TET epithelium perfused with the medium underneath.

Display of E. coli Alkaline Phosphatase pIII or pVIII Fusions on Phagemid Surfaces Reveals Monovalent Decoration with Active Molecules.

Active alkaline phosphatase of Escherichia coli (PhoA, EC 3.1.3.

Orf virus VEGF-E NZ2 promotes paracellular NRP-1/VEGFR-2 coreceptor assembly via the peptide RPPR.

Vascular endothelial growth factors (VEGFs) interact with the receptor tyrosine kinases (RTKs) VEGFR-1, -2, and -3; neuropilins (NRPs); and heparan sulfate (HS) proteoglycans. VEGF RTKs signal to downstream targets upon ligand-induced tyrosine phosphorylation, while NRPs and HS act as coreceptors that lack enzymatic activity yet modulate signal output by VEGF RTKs. VEGFs exist in various isoforms with distinct receptor specificity and biological activity.

Structure-function relationship of CAP-Gly domains.

In all eukaryotes, CAP-Gly proteins control important cellular processes. The molecular mechanisms underlying the functions of CAP-Gly domains, however, are still poorly understood. Here we use the complex formed between the CAP-Gly domain of p150(glued) and the C-terminal zinc knuckle of CLIP170 as a model system to explore the structure-function relationship of CAP-Gly-mediated protein interactions.

Intracellular signal transduction in mouse oocytes and irradiated early embryos.

In order to determine the effect of X-irradiation on intracellular signal transduction in mouse oocytes and embryos, JNK, ERK and p38 kinase activities were measured by the state of phosphorylation of their respective substrates (c-Jun, Elk-1 and ATF-2, respectively) in two mouse strains differing in radiation sensitivity, namely C57BL and BALB/c. In a first step, control oocytes and embryos were compared for their respective kinase activities at various stages of oocyte maturation (germinal vesicle and metaphases of 1st and 2nd meiosis stages) and early embryonic development (1-, 2-, 4-, 8- and 16-cell, morula and blastula stages). Levels of p38, ERK or JNK kinase activities were shown to vary with the stage of oocyte maturation and embryo development.