Center for Biomembrane Research


Center for Biomembrane Research

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Soderstrom B.,Structural Cellular Biology Unit | Mirzadeh K.,Center for Biomembrane Research | Toddo S.,Center for Biomembrane Research | von Heijne G.,Stockholm University 106 91Stockholm Sweden | And 2 more authors.
Molecular Microbiology | Year: 2016

The divisome is the macromolecular complex that carries out cell division in Escherichia coli. Every generation it must be assembled, and then disassembled so that the sequestered proteins can be recycled. Whilst the assembly process has been well studied, virtually nothing is known about the disassembly process. In this study, we have used super-resolution SIM imaging to monitor pairs of fluorescently tagged divisome proteins as they depart from the division septum. These simple binary comparisons indicated that disassembly occurs in a coordinated process that consists of at least five steps: [FtsZ, ZapA] ⇒ [ZipA, FtsA] ⇒ [FtsL, FtsQ] ⇒ [FtsI, FtsN] ⇒ [FtsN]. This sequence of events is remarkably similar to the assembly process, indicating that disassembly follows a first-in, first-out principle. A secondary observation from these binary comparisons was that FtsZ and FtsN formed division rings that were spatially separated throughout the division process. Thus the data indicate that the divisome structure can be visualized as two concentric rings; a proto-ring containing FtsZ and an FtsN-ring. © 2016 John Wiley & Sons Ltd.

Massad T.,Center for Biomembrane Research | Skaar K.,Center for Biomembrane Research | Nilsson H.,University of Stockholm | Damberg P.,Center for Biomembrane Research | And 5 more authors.
Nucleic Acids Research | Year: 2010

As opposed to the vast majority of prokaryotic repressors, the immunity repressor of temperate Escherichia coli phage P2 (C) recognizes non-palindromic direct repeats of DNA rather than inverted repeats. We have determined the crystal structure of P2 C at 1.8 Å. This constitutes the first structure solved from the family of C proteins from P2-like bacteriophages. The structure reveals that the P2 C protein forms a symmetric dimer oriented to bind the major groove of two consecutive turns of the DNA. Surprisingly, P2 C has great similarities to binders of palindromic sequences. Nevertheless, the two identical DNA-binding helixes of the symmetric P2 C dimer have to bind different DNA sequences. Helix 3 is identified as the DNA-recognition motif in P2 C by alanine scanning and the importance for the individual residues in DNA recognition is defined. A truncation mutant shows that the disordered C-terminus is dispensable for repressor function. The short distance between the DNA-binding helices together with a possible interaction between two P2 C dimers are proposed to be responsible for extensive bending of the DNA. The structure provides insight into the mechanisms behind the mutants of P2 C causing dimer disruption, temperature sensitivity and insensitivity to the P4 antirepressor. © 2010 The Author(s).

Mellroth P.,Karolinska Institutet | Daniels R.,Center for Biomembrane Research | Eberhardt A.,Karolinska Institutet | Ronnlund D.,Albanova University Center | And 5 more authors.
Journal of Biological Chemistry | Year: 2012

The pneumococcal autolysin LytA is a virulence factor involved in autolysis as well as in fratricidal- and penicillin-induced lysis. In this study, we used biochemical and molecular biological approaches to elucidate which factors control the cytoplasmic translocation and lytic activation of LytA.Weshow that LytA is mainly localized intracellularly, as only a small fraction was found attached to the extracellular cell wall. By manipulating the extracellular concentration of LytA, we found that the cells were protected from lysis during exponential growth, but not in the stationary phase, and that a defined threshold concentration of extracellular LytA dictates the onset of autolysis. Stalling growth through nutrient depletion, or the specific arrest of cell wall synthesis, sensitized cells for LytA-mediated lysis. Inhibition of cell wall association via the choline binding domain of an exogenously added enzymatically inactive form of LytA revealed a potential substrate for the amidase domain within the cell wall where the formation of nascent peptidoglycan occurs. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

Mirzadeh K.,Center for Biomembrane Research | Martinez V.,Technical University of Denmark | Toddo S.,Center for Biomembrane Research | Guntur S.,Center for Biomembrane Research | And 5 more authors.
ACS Synthetic Biology | Year: 2015

Protein production in Escherichia coli is a fundamental activity for a large fraction of academic, pharmaceutical, and industrial research laboratories. Maximum production is usually sought, as this reduces costs and facilitates downstream purification steps. Frustratingly, many coding sequences are poorly expressed even when they are codon-optimized and expressed from vectors with powerful genetic elements. In this study, we show that poor expression can be caused by certain nucleotide sequences (e.g., cloning scars) at the junction between the vector and the coding sequence. Since these sequences lie between the Shine-Dalgarno sequence and the start codon, they are an integral part of the translation initiation region. To identify the most optimal sequences, we devised a simple and inexpensive PCR-based step that generates sequence variants at the vector-coding sequence junction. These sequence variants modulated expression by up to 1000-fold. FACS-seq analyses indicated that low GC content and relaxed mRNA stability (δG) in this region were important, but not the only, determinants for high expression. © 2015 American Chemical Society.

Kauko A.,Center for Biomembrane Research | Hedin L.E.,Center for Biomembrane Research | Thebaud E.,Center for Biomembrane Research | Cristobal S.,Center for Biomembrane Research | And 2 more authors.
Journal of Molecular Biology | Year: 2010

We have determined the optimal placement of individual transmembrane helices in the Pyrococcus horikoshii GltPh glutamate transporter homolog in the membrane. The results are in close agreement with theoretical predictions based on hydrophobicity, but do not, in general, match the known three-dimensional structure, suggesting that transmembrane helices can be repositioned relative to the membrane during folding and oligomerization. Theoretical analysis of a database of membrane protein structures provides additional support for this idea. These observations raise new challenges for the structure prediction of membrane proteins and suggest that the classical two-stage model often used to describe membrane protein folding needs to be modified. © 2010 Elsevier Ltd. All rights reserved.

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