9 research outputs found
The effect of SecB on protein folding : a single molecule study
No full textIn biology, "life" is the condition which distinguishes active organisms from inorganic matter. Life of organisms is characterized by the capacity for growth, functional activity and continual changes preceding death. For all three kingdoms of life, i.e. Eukarya, Bacteria or Archaea, the process of growing involves transcription (DNA to mRNA) and translation (mRNA to protein) of genetic information (DAN) into proteins. Proteins or plypeptides fulfill diverse functions in cells. Many catalyze chemical reactions (enzymes), others are used for structural and mechanical functions (cytoskeleton) and some are used for communication between cells. Proteins are built from twenty different amino acids in various compositions and lengths. Each protein is synthesized as a linear chain of amino acids when translated from a mRNA sequence into protein and initially has no structure. In order to work properly, proteins have to fold from a random coil into a functional three-dimensional structure, the so called native state. The final three-dimensional structure is determined by the amino acid sequence of the protein. Nevertheless, many proteins cannot fold out of themselves and need help to obtain their native state, especially under stress conditions, and within the cell context with its highly crowded protein-containing cytosol. This help is provided by other proteins, so called chaperones. Many chaperones have the ability to alternate the folding pathway in favor towards the native state. However, enhancing folding and remodeling misfolded proteins are not the only functions of molecular chaperones. They also play a role in other cellular aspects, including translocation through membranes or remodelling of protein complexes. In the chapters of this thesis, I have studied the function of the chaperone SecB that plays a highly specialized role in the translocation of proteins across the cytoplasmic membrane in Escherichia coli.
SecB—A chaperone dedicated to protein translocation
No full textSecB is a molecular chaperone in Gram-negative bacteria dedicated to the post-translational translocation of proteins across the cytoplasmic membrane. The entire surface of this chaperone is used for both of its native functions in protein targeting and unfolding. Single molecule studies revealed how SecB affects the folding pathway of proteins and how it prevents the tertiary structure formation and aggregation to support protein translocation.
F1F0 ATP synthase subunit c is a substrate of the novel YidC pathway for membrane protein biogenesis
No full textThe Escherichia coli YidC protein belongs to the Oxa1 family of membrane proteins that have been suggested to facilitate the insertion and assembly of membrane proteins either in cooperation with the Sec translocase or as a separate entity. Recently, we have shown that depletion of YidC causes a specific defect in the functional assembly of F1F0 ATP synthase and cytochrome o oxidase. We now demonstrate that the insertion of in vitro–synthesized F1F0 ATP synthase subunit c (F0c) into inner membrane vesicles requires YidC. Insertion is independent of the proton motive force, and proteoliposomes containing only YidC catalyze the membrane insertion of F0c in its native transmembrane topology whereupon it assembles into large oligomers. Co-reconstituted SecYEG has no significant effect on the insertion efficiency. Remarkably, signal recognition particle and its membrane-bound receptor FtsY are not required for the membrane insertion of F0c. In conclusion, a novel membrane protein insertion pathway in E. coli is described in which YidC plays an exclusive role.
Tight Hydrophobic Contacts with the SecB Chaperone Prevent Folding of Substrate Proteins
No full textThe molecular chaperone SecB binds to hydrophobic sections of unfolded secretory proteins and thereby prevents their premature folding prior to secretion by the translocase of Escherichia coli. Here, we have investigated the effect of the single-residue mutation of leucine 42 to arginine (L42R) centrally positioned in the polypeptide binding pocket of SecB on its chaperonin function. The mutant retains its tetrameric structure and SecA targeting function but is defective in its holdase activity. Isothermal titration calorimetry and single-molecule optical tweezer studies suggest that the SecB(L42R) mutant exhibits a reduced polypeptide binding affinity allowing for partial folding of the bound polypeptide chain rendering it translocation-incompetent.
Reshaping of the conformational search of a protein by the chaperone trigger factor
No full textProtein folding is often described as a search process, in which polypeptides explore different conformations to find their native structure. Molecular chaperones are known to improve folding yields by suppressing aggregation between polypeptides before this conformational search starts, as well as by rescuing misfolds after it ends. Although chaperones have long been speculated to also affect the conformational search itself—by reshaping the underlying folding landscape along the folding trajectory—direct experimental evidence has been scarce so far. In Escherichia coli, the general chaperone trigger factor (TF) could play such a role. TF has been shown to interact with nascent chains at the ribosome, with polypeptides released from the ribosome into the cytosol, and with fully folded proteins before their assembly into larger complexes. To investigate the effect of TF from E. coli on the conformational search of polypeptides to their native state, we investigated individual maltose binding protein (MBP) molecules using optical tweezers. Here we show that TF binds folded structures smaller than one domain, which are then stable for seconds and ultimately convert to the native state. Moreover, TF stimulates native folding in constructs of repeated MBP domains. The results indicate that TF promotes correct folding by protecting partially folded states from distant interactions that produce stable misfolded states. As TF interacts with most newly synthesized proteins in E. coli, we expect these findings to be of general importance in understanding protein folding pathways.
Direct Observation of Chaperone-Induced Changes in a Protein Folding Pathway
No full textHow chaperone interactions affect protein folding pathways is a central problem in biology. With the use of optical tweezers and all-atom molecular dynamics simulations, we studied the effect of chaperone SecB on the folding and unfolding pathways of maltose binding protein (MBP) at the single-molecule level. In the absence of SecB, we find that the MBP polypeptide first collapses into a molten globulelike compacted state and then folds into a stable core structure onto which several α helices are finally wrapped. Interactions with SecB completely prevent stable tertiary contacts in the core structure but have no detectable effect on the folding of the external a helices. It appears that SecB only binds to the extended or molten globulelike structure and retains MBP in this latter state. Thus during MBP translocation, no energy is required to disrupt stable tertiary interactions.
UvA-DARE (Digital Academic Repository) Link to publication Citation for published version (APA): Inclusive search for the charmless radiative decay of the b-quark (b L3 Collaboration
No full textGeneral rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. Download date: 29 Jun 2019 Physics Letters B 317 ( 1993 ) We report on the search for the electromagnetic penguin decay b --* s7 at v~ ~ mz. We find no evidence for a signal and place an upper limit on the decay rate Br(b ~ s~,) < 1.2 × 10 -3 at 90% C.L
UvA-DARE (Digital Academic Repository) Determination of the effective electroweak mixing angle from Z decays L3 Collaboration
No full textGeneral rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. The effective electroweak mixing angle sin 20w is measured from the production and decay of the Z boson in e+e -interactions. The data sample corresponds to an integrated luminosity of 18 pb-l with about 420 000 hadronic and 40 000 leptonic Z decays. The mixing angle sin 20w is determined from several independent measurements: the leptonic and hadronic cross sections, the forward-backward asymmetries of charged leptons and b-quarks, and the z-polarization. The results are found to be in good agreement with each other. The value of sin 20w from a fit to the asymmetries in a model independent method is 0.2321-4-0.0021 and from a global fit to the data in the Standard Model framework is 0.2328+0.0013
