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Daidone I.,The Interdisciplinary Center | Daidone I.,University of LAquila | Neuweiler H.,Bielefeld University | Neuweiler H.,Medical Research Council Center for Protein Engineering | And 4 more authors.
PLoS Computational Biology | Year: 2010

Characterization of the length dependence of end-to-end loop-closure kinetics in unfolded polypeptide chains provides an understanding of early steps in protein folding. Here, loop-closure in poly-glycine-serine peptides is investigated by combining single-molecule fluorescence spectroscopy with molecular dynamics simulation. For chains containing more than 10 peptide bonds loop-closing rate constants on the 20-100 nanosecond time range exhibit a power-law length dependence. However, this scaling breaks down for shorter peptides, which exhibit slower kinetics arising from a perturbation induced by the dye reporter system used in the experimental setup. The loop-closure kinetics in the longer peptides is found to be determined by the formation of intra-peptide hydrogen bonds and transient β-sheet structure, that accelerate the search for contacts among residues distant in sequence relative to the case of a polypeptide chain in which hydrogen bonds cannot form. Hydrogen-bond-driven polypeptide-chain collapse in unfolded peptides under physiological conditions found here is not only consistent with hierarchical models of protein folding, that highlights the importance of secondary structure formation early in the folding process, but is also shown to speed up the search for productive folding events. © 2010 Daidone et al. Source


Goltermann L.,Copenhagen University | Larsen M.S.Y.,Copenhagen University | Banerjee R.,Ohio State University | Joerger A.C.,Medical Research Council Center for Protein Engineering | And 2 more authors.
PLoS ONE | Year: 2010

Background: Global residue-specific amino acid mutagenesis can provide important biological insight and generate proteins with altered properties, but at the risk of protein misfolding. Further, targeted libraries are usually restricted to a handful of amino acids because there is an exponential correlation between the number of residues randomized and the size of the resulting ensemble. Using GFP as the model protein, we present a strategy, termed protein evolution via amino acid and codon elimination, through which simplified, native-like polypeptides encoded by a reduced genetic code were obtained via screening of reduced-size ensembles. Methodology/Principal Findings: The strategy involves combining a sequential mutagenesis scheme to reduce library size with structurally stabilizing mutations, chaperone complementation, and reduced temperature of gene expression. In six steps, we eliminated a common buried residue, Phe, from the green fluorescent protein (GFP), while retaining activity. A GFP variant containing 11 Phe residues was used as starting scaffold to generate 10 separate variants in which each Phe was replaced individually (in one construct two adjacent Phe residues were changed simultaneously), while retaining varying levels of activity. Combination of these substitutions to generate a Phe-free variant of GFP abolished fluorescence. Combinatorial re-introduction of five Phe residues, based on the activities of the respective single amino acid replacements, was sufficient to restore GFP activity. Successive rounds of mutagenesis generated active GFP variants containing, three, two, and zero Phe residues. These GFPs all displayed progenitor-like fluorescence spectra, temperature-sensitive folding, a reduced structural stability and, for the least stable variants, a reduced steady state abundance. Conclusions/Significance: The results provide strategies for the design of novel GFP reporters. The described approach offers a means to enable engineering of active proteins that lack certain amino acids, a key step towards expanding the functional repertoire of uniquely labeled proteins in synthetic biology. © 2010 Goltermann et al. Source


Low C.,Martin Luther University of Halle Wittenberg | Neumann P.,Martin Luther University of Halle Wittenberg | Tidow H.,Medical Research Council Center for Protein Engineering | Weininger U.,Martin Luther University of Halle Wittenberg | And 5 more authors.
Journal of Molecular Biology | Year: 2010

SlyD (sensitive to lysis D; product of the slyD gene) is a prolyl isomerase [peptidyl-prolyl cis/trans isomerase (PPIase)] of the FK506 binding protein (FKBP) type with chaperone properties. X-ray structures derived from three different crystal forms reveal that SlyD from Thermus thermophilus consists of two domains representing two functional units. PPIase activity is located in a typical FKBP domain, whereas chaperone function is associated with the autonomously folded insert-in-flap (IF) domain. The two isolated domains are stable and functional in solution, but the presence of the IF domain increases the PPIase catalytic efficiency of the FKBP domain by 2 orders of magnitude, suggesting that the two domains act synergistically to assist the folding of polypeptide chains. The substrate binding surface of SlyD from T. thermophilus was mapped by NMR chemical shift perturbations to hydrophobic residues of the IF domain, which exhibits significantly reduced thermodynamic stability according to NMR hydrogen/deuterium exchange and fluorescence equilibrium transition experiments. Based on structural homologies, we hypothesize that this is due to the absence of a stabilizing β-strand, suggesting in turn a mechanism for chaperone activity by 'donor-strand complementation.' Furthermore, we identified a conserved metal (Ni2+) binding site at the C-terminal SlyD-specific helical appendix of the FKBP domain, which may play a role in metalloprotein assembly. © 2010 Elsevier Ltd. Source


Kaar J.L.,Medical Research Council Center for Protein Engineering | Kaar J.L.,University of Colorado at Boulder | Basse N.,Medical Research Council Laboratory of Molecular Biology | Joerger A.C.,Medical Research Council Center for Protein Engineering | And 5 more authors.
Protein Science | Year: 2010

Oncogenic mutations inactivate the tumor suppressor p53 by lowering its stability or by weakening its binding to DNA. Alkylating agents that reactivate mutant p53 are currently being explored for cancer therapy. We have discovered ligands containing an α,β-unsaturated double bond, characteristic of Michael acceptors, that bind covalently to generic cysteine sites in the p53 core domain. They raised the melting temperature of the core domain of wild-type p53 and the hotspot mutants R175H, Y220C, G245S, R249S, and R282 by up to 3°C. Analysis of the relative reactivity of the cysteines in p53 by mass spectrometry found that C124 and C141 react first, followed by C135, C182, and C277, and eventually C176 and C275. Post-translational modifications of cysteines are known to be involved in regulation of other transcription factors. Modification of C277, which sits on the DNA-binding surface, may, for example, play a role in regulating p53 activity in cells in response to environmental cues. We found that the modifications progressively reduced DNA-binding activity of full-length p53. In light of these results, it is likely that the anticancer activity of the alkylating drugs works via a nontranscriptional activity of p53. Published by Wiley-Blackwell. © 2010 The Protein Society. Source


Stapf C.,University of Wurzburg | Cartwright E.,Medical Research Council Center for Protein Engineering | Bycroft M.,Medical Research Council Center for Protein Engineering | Hofmann K.,Miltenyi Biotec GmbH | Buchberger A.,University of Wurzburg
Journal of Biological Chemistry | Year: 2011

Cellular functions of the essential, ubiquitin-selective AAA ATPase p97/valosin-containing protein (VCP) are controlled by regulatory cofactors determining substrate specificity and fate. Most cofactors bind p97 through a ubiquitin regulatoryX(UBX) or UBX-like domain or linear sequence motifs, including the hitherto ill defined p97/VCP-interacting motif (VIM). Here, we present the new, minimal consensus sequence RX 5AAX 2R as a general definition of the VIM that unites a novel family of known and putative p97 cofactors, among them UBXD1 and ZNF744/ ANKZF1. We demonstrate that this minimal VIM consensus sequence is necessary and sufficient for p97 binding. Using NMR chemical shift mapping, we identified several residues of the p97 N-terminal domain (N domain) that are critical for VIM binding. Importantly, we show that cellular stress resistance conferred by the yeast VIM-containing cofactor Vms1 depends on the physical interaction between its VIM and the critical N domain residues of the yeast p97 homolog, Cdc48. Thus, the VIM-N domain interaction characterized in this study is required for the physiological function of Vms1 and most likely other members of the newly defined VIM family of cofactors. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Source

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