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Title: Structural coupling of the EF hand and C-terminal GTPase domains in the mitochondrial protein Miro

Authors:
; ; ; ; ;  [1];  [2]
  1. (DePaul)
  2. (
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
OTHERUNIVERSITYNIH
OSTI Identifier:
1104553
Resource Type:
Journal Article
Resource Relation:
Journal Name: EMBO Rep.; Journal Volume: 14; Journal Issue: (11) ; 2013
Country of Publication:
United States
Language:
ENGLISH

Citation Formats

Klosowiak, Julian L., Focia, Pamela J., Chakravarthy, Srinivas, Landahl, Eric C., Freymann, Douglas M., Rice, Sarah E., and NWU). Structural coupling of the EF hand and C-terminal GTPase domains in the mitochondrial protein Miro. United States: N. p., 2014. Web. doi:10.1038/embor.2013.151.
Klosowiak, Julian L., Focia, Pamela J., Chakravarthy, Srinivas, Landahl, Eric C., Freymann, Douglas M., Rice, Sarah E., & NWU). Structural coupling of the EF hand and C-terminal GTPase domains in the mitochondrial protein Miro. United States. doi:10.1038/embor.2013.151.
Klosowiak, Julian L., Focia, Pamela J., Chakravarthy, Srinivas, Landahl, Eric C., Freymann, Douglas M., Rice, Sarah E., and NWU). Tue . "Structural coupling of the EF hand and C-terminal GTPase domains in the mitochondrial protein Miro". United States. doi:10.1038/embor.2013.151.
@article{osti_1104553,
title = {Structural coupling of the EF hand and C-terminal GTPase domains in the mitochondrial protein Miro},
author = {Klosowiak, Julian L. and Focia, Pamela J. and Chakravarthy, Srinivas and Landahl, Eric C. and Freymann, Douglas M. and Rice, Sarah E. and NWU)},
abstractNote = {},
doi = {10.1038/embor.2013.151},
journal = {EMBO Rep.},
number = (11) ; 2013,
volume = 14,
place = {United States},
year = {Tue Feb 04 00:00:00 EST 2014},
month = {Tue Feb 04 00:00:00 EST 2014}
}
  • Replication Protein A (RPA) is the primary eukaryotic ssDNA binding protein utilized in diverse DNA transactions in the cell. RPA is a heterotrimeric protein with seven globular domains connected by flexible linkers, which enable substantial inter-domain motion that is essential to its function. Small angle X-ray scattering (SAXS) experiments on two multi-domain constructs from the N-terminus of the large subunit (RPA70) were used to examine the structural dynamics of these domains and their response to the binding of ssDNA. The SAXS data combined with molecular dynamics simulations reveal substantial interdomain flexibility for both RPA70AB (the tandem high affinity ssDNA bindingmore » domains A and B connected by a 10-residue linker) and RPA70NAB (RPA70AB extended by a 70-residue linker to the RPA70N protein interaction domain). Binding of ssDNA to RPA70NAB reduces the interdomain flexibility between the A and B domains, but has no effect on RPA70N. These studies provide the first direct measurements of changes in orientation of these three RPA domains upon binding ssDNA. The results support a model in which RPA70N remains structurally independent of RPA70AB in the DNA bound state and therefore freely available to serve as a protein recruitment module.« less
  • No abstract prepared.
  • Genes encoding the minor A component of bovine calbindins D/sub 9k/-the smallest protein known with a pair of EF-hand calcium-binding sites-with amino acid substitutions and/or deletions have been synthesized and expressed in Escherichia coli and characterized with different biophysical techniques. The mutations are confined to the N-terminal Ca/sup 2 +/-binding site and constitute Pro-20 ..-->.. Gly (M1), Pro-20 ..-->.. Gly and Asn-21 deleted (M2), Pro-20 deleted (M3), and Tyr-13 ..-->.. Phe (M4). /sup 1/H, /sup 43/Ca, and /sup 113/Cd NMR studies show that the structural changes induced are primarily localized in the modified region, with hardly any effects on themore » C-terminal Ca/sup 2 +/-binding site. The Ca/sup 2 +/ exchange rate for the N-terminal site changes from 3 s/sup -1/ in the wild-type protein (M0) and M4 to 5000 s/sup -1/ in M2 and M3, whereas there is no detectable variation in the Ca/sup 2 +/ exchange from the C-terminal site. The macroscopic Ca/sup 2 +/binding constants have been obtained from equilibration in the presence of a fluorescent chelator or by using a Ca/sup 2 +/-selective electrode. Microcalorimetric data show that the enthalpy of Ca/sup 2 +/-binding is negative for all sites except the N-terminal site in M2 and M3. The binding entropy is strongly positive in all cases. Through the observed changes in the /sup 1/H NMR spectra during Ca/sup 2 +/-titrations the authors could obtain ratios between site binding constants in M0 and M4. There is evidence (from /sup 113/ Cd NMR) for site-site interactions also in M1, M2, and M3, but the magnitude of ..delta delta..G could not be determined because of sequential Ca/sup 2 +/ binding.« less