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Tischer A.,Mayo Medical School | Pultke H.,Scil Proteins | Topf A.,Institute For Biophysikalische Chemie | Auton M.,Mayo Medical School | And 2 more authors.
FEBS Journal

The ionic liquid N-ethyl-N′-methyl imidazolium chloride (EMIMCl) has been described as being very efficient in promoting refolding of the recombinant plasminogen activator rPA. Our study reveals that molar concentrations of EMIMCl increase the solubility of native and unfolded proteins due to favorable interactions with amino acid side chains rather than favorably interacting with the peptide backbone. This delicate balance of favorable interactions with side chains and unfavorable interactions with the peptide backbone provides a molecular explanation of how EMIMCl suppresses protein aggregation and simultaneously promotes refolding. By contrast, high concentrations of EMIMCl denature proteins because of a reduced water content and strong favorable interactions with amino acid side chains. This denatured species is not soluble and aggregates because, in contrast to the classical denaturants, guanidine hydrochloride and urea, EMIMCl does not solubilize the peptide backbone. Structured digital abstract PNP and PNP bind by molecular sieving (1, 2, 3, 4) The ionic liquid EMIMCl is efficient in promoting the refolding of rPA. Molar concentrations of EMIMCl increase the solubility of native and unfolded proteins due to favorable interactions with amino acid side chains, while the interaction with the peptide backbone is unfavorable. Although a refolding enhancer on one side, high concentrations of EMIMCl result in denaturation and aggregation of proteins. © 2014 FEBS. Source

Tsiavaliaris G.,Institute For Biophysikalische Chemie | Itel F.,University of Basel | Hedfalk K.,Gothenburg University | Al-Samir S.,AG Vegetative Physiologie 4220 | And 3 more authors.
FASEB Journal

Here we ask the following: 1) what is the CO2 permeability (Pco2) of unilamellar liposomes composed of l-α-phosphatidylcholine (PC)/l-α-phosphatidylserine (PS) =4:1 and containing cholesterol (Chol) at levels often occurring in biologic membranes (50 mol%), and 2) does incorporation of the CO2 channel aquaporin (AQP)1 cause a significant increase in membrane Pco2? Presently, a drastic discrepancy exists between the answers to these two questions obtained from mass-spectrometric 18O-exchange measurements (Chol reduces Pco2100-fold, AQP1 increases Pco2 10-fold) vs.from stopped-flow approaches observing CO2 uptake (no effects of either Chol or AQP1). A novel theory of CO2 uptake by vesicles predicts that in a stopped-flow apparatus this fast process can only be resolved temporally and interpreted quantitatively, if 1) a very low CO2 partial pressure (pCO2) is used (e.g., 18 mmHg), and 2) intravesicular carbonic anhydrase (CA) activity is precisely known. With these prerequisites fulfilled, we find by stopped-flow that 1) Chol-containing vesicles possess a Pco2 = 0.01cm/s, and Chol-free vesicles exhibit ∼1 cm/s, and 2) the Pco2 of 0.01 cm/s is increased ≥ 10-fold by AQP1. Both results agree with previous mass-spectrometric results and thus resolve the apparent discrepancy between the two techniques. We confirm that biologic membranes have an intrinsically low Pco2 that can be raised when functionally necessary by incorporating protein-gas channels such as AQP1. © FASEB. Source

Fonfara I.,Justus Liebig University | Curth U.,Institute For Biophysikalische Chemie | Pingoud A.,Justus Liebig University | Wende W.,Justus Liebig University
Nucleic Acids Research

Zinc-finger nucleases and TALE nucleases are produced by combining a specific DNA-binding module and a non-specific DNA-cleavage module, resulting in nucleases able to cleave DNA at a unique sequence. Here a new approach for creating highly specific nucleases was pursued by fusing a catalytically inactive variant of the homing endonuclease I-SceI, as DNA binding-module, to the type IIP restriction enzyme PvuII, as cleavage module. The fusion enzymes were designed to recognize a composite site comprising the recognition site of PvuII flanked by the recognition site of I-SceI. In order to reduce activity on PvuII sites lacking the flanking I-SceI sites, the enzymes were optimized so that the binding of I-SceI to its sites positions PvuII for cleavage of the composite site. This was achieved by optimization of the linker and by introducing amino acid substitutions in PvuII which decrease its activity or disturb its dimer interface. The most specific variant showed a more than 1000-fold preference for the addressed composite site over an unaddressed PvuII site. These results indicate that using a specific restriction enzyme, such as PvuII, as cleavage module, offers an alternative to the otherwise often used catalytic domain of FokI, which by itself does not contribute to the specificity of the engineered nuclease. © The Author(s) 2011. Published by Oxford University Press. Source

Brinkmann K.,University of Munster | Winterhoff M.,Institute For Biophysikalische Chemie | Onel S.-F.,University of Marburg | Schultz J.,University of Wurzburg | And 2 more authors.
Journal of Cell Science

Wiskott-Aldrich syndrome proteins (WASPs) are nucleationpromoting factors (NPF) that differentially control the Arp2/3 complex. In Drosophila, three different family members, SCAR (also known asWAVE),WASP andWASH (also known as CG13176), have been analyzed so far. Here, we characterized WHAMY, the fourth Drosophila WASP family member. whamy originated from a wasp gene duplication and underwent a sub-neofunctionalization. Unlike WASP, we found that WHAMY specifically interacted with activated Rac1 through its two CRIB domains, which were sufficient for targeting WHAMY to lamellipodial and filopodial tips. Biochemical analyses showed that WHAMY promoted exceptionally fast actin filament elongation, although it did not activate the Arp2/3 complex. Loss- and gain-of-function studies revealed an important function of WHAMY in membrane protrusions and cell migration in macrophages. Genetic data further implied synergistic functions between WHAMY and WASP during morphogenesis. Double mutants were late-embryonic lethal and showed severe defects in myoblast fusion. Trans-heterozygous mutant animals showed strongly increased defects in sensory cell fate specification. Thus, WHAMY is a novel actin polymerase with an initial partitioning of ancestral WASP functions in development and subsequent acquisition of a new function in cell motility during evolution. Source

Heissler S.M.,Institute For Biophysikalische Chemie | Manstein D.J.,Institute For Biophysikalische Chemie
Cellular and Molecular Life Sciences

Myosin-7a participates in auditory and visual processes. Defects in MYO7A, the gene encoding the myosin-7a heavy chain, are causative for Usher syndrome 1B, the most frequent cause of deaf-blindness in humans. In the present study, we performed a detailed kinetic and functional characterization of the isolated human myosin-7a motor domain to elucidate the details of chemomechanical coupling and the regulation of motor function. A rate-limiting, slow ADP release step causes long lifetimes of strong actin-binding intermediates and results in a high duty ratio. Moreover, our results reveal a Mg 2+-sensitive regulatory mechanism tuning the kinetic and mechanical properties of the myosin-7a motor domain. We obtained direct evidence that changes in the concentration of free Mg 2+ ions affect the motor properties of human myosin-7a using an in vitro motility assay system. Our results suggest that in a cellular environment, compartment-specific fluctuations in free Mg 2+ ions can mediate the conditional switching of myosin-7a between cargo moving and tension bearing modes. © 2011 The Author(s). Source

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