Open Access. Powered by Scholars. Published by Universities.®
- Keyword
Articles 1 - 11 of 11
Full-Text Articles in Molecular Biology
Foldeco: A Model For Proteostasis In E. Coli, Evan T. Powers, David L. Powers, Lila Gierasch
Foldeco: A Model For Proteostasis In E. Coli, Evan T. Powers, David L. Powers, Lila Gierasch
Lila Gierasch
To gain insight into the interplay of processes and species that maintain a correctly folded, functional proteome, we have developed a computational model called FoldEco. FoldEco models the cellular proteostasis network of the E. coli cytoplasm, including protein synthesis, degradation, aggregation, chaperone systems, and the folding characteristics of protein clients. We focused on E. coli because much of the needed input information—including mechanisms, rate parameters, and equilibrium coefficients—is available, largely from in vitro experiments; however, FoldEco will shed light on proteostasis in other organisms. FoldEco can generate hypotheses to guide the design of new experiments. Hypothesis generation leads to system-wide …
Segmental Isotopic Labeling Of The Hsp70 Molecular Chaperone Dnak Using Expressed Protein Ligation, Eugenia M. Clerico, Anastasia Zhuravleva, Robert G. Smock, Lila Gierasch
Segmental Isotopic Labeling Of The Hsp70 Molecular Chaperone Dnak Using Expressed Protein Ligation, Eugenia M. Clerico, Anastasia Zhuravleva, Robert G. Smock, Lila Gierasch
Lila Gierasch
Introducing biophysical labels into specific regions of large and dynamic multidomain proteins greatly facilitates mechanistic analysis. Ligation of expressed domains that are labeled in a desired manner before assembly of the intact molecular machine provides such a strategy. We have elaborated an experimental route using expressed protein ligation (EPL) to create an Hsp70 molecular chaperone (in this case the E. coli Hsp70, DnaK) where only one of the two constituent domains was labeled, in this case with NMR active isotopes, allowing visualization of the single domain in the context of the two domain protein. Several technical obstacles were overcome, including …
An Interdomain Sector Mediating Allostery In Hsp70 Molecular Chaperones, Robert G. Smock, Olivier Rivoire, William P. Russ, Joanna F. Swain, Stanislas Leibler, Rama Ranganathan, Lila Gierasch
An Interdomain Sector Mediating Allostery In Hsp70 Molecular Chaperones, Robert G. Smock, Olivier Rivoire, William P. Russ, Joanna F. Swain, Stanislas Leibler, Rama Ranganathan, Lila Gierasch
Lila Gierasch
Allosteric coupling between protein domains is fundamental to many cellular processes. For example, Hsp70 molecular chaperones use ATP binding by their actin-like N-terminal ATPase domain to control substrate interactions in their C-terminal substrate-binding domain, a reaction that is critical for protein folding in cells. Here, we generalize the statistical coupling analysis to simultaneously evaluate co-evolution between protein residues and functional divergence between sequences in protein sub-families. Applying this method in the Hsp70/110 protein family, we identify a sparse but structurally contiguous group of co-evolving residues called a ‘sector’, which is an attribute of the allosteric Hsp70 sub-family that links the …
Role Of Hsp70 Atpase Domain Intrinsic Dynamics And Sequence Evolution In Enabling Its Functional Interactions With Nefs, Ying Liu, Lila Gierasch, Ivet Bahar
Role Of Hsp70 Atpase Domain Intrinsic Dynamics And Sequence Evolution In Enabling Its Functional Interactions With Nefs, Ying Liu, Lila Gierasch, Ivet Bahar
Lila Gierasch
12 Hide Figures Abstract Author Summary Introduction Materials and Methods Results Discussion Supporting Information Acknowledgments Author Contributions References Reader Comments (0) Figures Abstract Catalysis of ADP-ATP exchange by nucleotide exchange factors (NEFs) is central to the activity of Hsp70 molecular chaperones. Yet, the mechanism of interaction of this family of chaperones with NEFs is not well understood in the context of the sequence evolution and structural dynamics of Hsp70 ATPase domains. We studied the interactions of Hsp70 ATPase domains with four different NEFs on the basis of the evolutionary trace and co-evolution of the ATPase domain sequence, combined with elastic …
The Structure Of Escherichia Coli Signal Recognition Particle Revealed By Scanning Transmission Electron Microscopy, Lain L. Mainprize, Daniel R. Beniac, Elena Falkovskaia, Robert M. Cleverley, Lila Gierasch, F. Peter Ottensmeyer, David W. Andrews
The Structure Of Escherichia Coli Signal Recognition Particle Revealed By Scanning Transmission Electron Microscopy, Lain L. Mainprize, Daniel R. Beniac, Elena Falkovskaia, Robert M. Cleverley, Lila Gierasch, F. Peter Ottensmeyer, David W. Andrews
Lila Gierasch
Structural studies on various domains of the ribonucleoprotein signal recognition particle (SRP) have not converged on a single complete structure of bacterial SRP consistent with the biochemistry of the particle. We obtained a three-dimensional structure for Escherichia coli SRP by cryoscanning transmission electron microscopy and mapped the internal RNA by electron spectroscopic imaging. Crystallographic data were fit into the SRP reconstruction, and although the resulting model differed from previous models, they could be rationalized by movement through an interdomain linker of Ffh, the protein component of SRP. Fluorescence resonance energy transfer experiments determined interdomain distances that were consistent with our …
Dependence Of Endoplasmic Reticulum-Associated Degradation On The Peptide Binding Domain And Concentration Of Bip, Mehdi Kabani, Stephanie S. Kelley, Michael W. Morrow, Diana L. Montgomery, Renuka Sivendran, Mark D. Rose, Lila Gierasch, Jeffrey L. Brodsky
Dependence Of Endoplasmic Reticulum-Associated Degradation On The Peptide Binding Domain And Concentration Of Bip, Mehdi Kabani, Stephanie S. Kelley, Michael W. Morrow, Diana L. Montgomery, Renuka Sivendran, Mark D. Rose, Lila Gierasch, Jeffrey L. Brodsky
Lila Gierasch
ER-associated degradation (ERAD) removes defective and mis-folded proteins from the eukaryotic secretory pathway, but mutations in the ER lumenal Hsp70, BiP/Kar2p, compromise ERAD efficiency in yeast. Because attenuation of ERAD activates the UPR, we screened for kar2 mutants in which the unfolded protein response (UPR) was induced in order to better define how BiP facilitates ERAD. Among the kar2 mutants isolated we identified the ERAD-specific kar2-1 allele (Brodsky et al. J. Biol. Chem. 274, 3453–3460). The kar2-1 mutation resides in the peptide-binding domain of BiP and decreases BiP's affinity for a peptide substrate. Peptide-stimulated ATPase activity was also reduced, suggesting …
The Ldl Receptor Clustering Motif Interacts With The Clathrin Terminal Domain In A Reverse Turn Conformation., Richard G. Kibbey, Josep Rizo, Lila Gierasch, Richard G. W. Anderson
The Ldl Receptor Clustering Motif Interacts With The Clathrin Terminal Domain In A Reverse Turn Conformation., Richard G. Kibbey, Josep Rizo, Lila Gierasch, Richard G. W. Anderson
Lila Gierasch
Previously the hexapeptide motif FXNPXY807 in the cytoplasmic tail of the LDL receptor was shown to be essential for clustering in clathrin-coated pits. We used nuclear magnetic resonance line-broadening and transferred nuclear Overhauser effect measurements to identify the molecule in the clathrin lattice that interacts with this hexapeptide, and determined the structure of the bound motif. The wild-type peptide bound in a single conformation with a reverse turn at residues NPVY. Tyr807Ser, a peptide that harbors a mutation that disrupts receptor clustering, displayed markedly reduced interactions. Clustering motif peptides interacted with clathrin cages assembled in the presence or absence of …
Effect Of Charged Residue Substitutions On The Membrane-Interactive Properties Of Signal Sequences Of The Escherichia Coli Lamb Protein., Jeffrey D. Jones, Lila Gierasch
Effect Of Charged Residue Substitutions On The Membrane-Interactive Properties Of Signal Sequences Of The Escherichia Coli Lamb Protein., Jeffrey D. Jones, Lila Gierasch
Lila Gierasch
Although the central role of the signal sequence in protein export is well established, the molecular details underlying signal sequence in vivo function remain unclear. As part of our continuing effort to relate signal sequence phenotypes to specific biophysical properties, we have carried out an extensive characterization of the secondary structure and lipid interactions for a family of peptides corresponding to the wild-type E. coli LamB signal sequence, and mutants that harbor charged residue point mutations in the hydrophobic core region. We used membrane-resident fluorescence quenching according to the parallax method to determine the relative depth of insertion of tryptophan-labeled …
Effect Of Charged Residue Substitutions On The Thermodynamics Of Signal Peptide-Lipid Interactions For The Escherichia Coli Lamb Signal Sequence., Jeffrey D. Jones, Lila Gierasch
Effect Of Charged Residue Substitutions On The Thermodynamics Of Signal Peptide-Lipid Interactions For The Escherichia Coli Lamb Signal Sequence., Jeffrey D. Jones, Lila Gierasch
Lila Gierasch
We have used tryptophan fluorescence spectroscopy to characterize the binding affinities of an Escherichia coli LamB signal peptide family for lipid vesicles. These peptides harbor charged residue substitutions in the hydrophobic core region. Titrations of peptides with vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine and 1-palmitoyl-2-oleoyl-sn-3-phosphoglycerol (65:35 mol%), in conjunction with evaluation of peptide dissociation rates from these vesicles, were used to determine binding parameters quantitatively. We find that under low ionic strength conditions, point mutations introducing negatively charged aspartate residues substantially reduce peptide affinity relative to the wild-type peptide. However, the difference between wild-type and mutant peptide affinities was much lower under …
Reorganization Of Lipid Domain Structure In Membranes By A Transmembrane Peptide: An Esr Spin Label Study On The Effect Of The Escherichia Coli Outer Membrane Protein A Signal Peptide On The Fluid Lipid Domain Connectivity In Binary Mixtures Of Dimyristoyl Phosphatidylcholine And Distearoyl Phosphatidylcholine., M. B. Sankaram, D. Marsh, Lila Gierasch, T. E. Thompson
Reorganization Of Lipid Domain Structure In Membranes By A Transmembrane Peptide: An Esr Spin Label Study On The Effect Of The Escherichia Coli Outer Membrane Protein A Signal Peptide On The Fluid Lipid Domain Connectivity In Binary Mixtures Of Dimyristoyl Phosphatidylcholine And Distearoyl Phosphatidylcholine., M. B. Sankaram, D. Marsh, Lila Gierasch, T. E. Thompson
Lila Gierasch
The effect of a transmembrane peptide on the domain structure of a two-component, two-phase lipid bilayer composed of dimyristoyl phosphatidylcholine (DMPC) and distearoyl phosphatidylcholine (DSPC) was examined by spin label electron spin resonance (ESR) spectroscopy. The peptide, pOmpA, is the hydrophobic, 25-residue signal sequence of the outer membrane protein A from Escherichia coli. Nitroxide derivatives of the phospholipid DSPC, 16-DSPCSL, and of the pOmpA signal peptide, pOmpA-IASL, were used as probes. The first-derivative lineshapes of the ESR spectra were analyzed using a normalized intensity ratio, R, that gives information on the average sizes of the disconnected fluid domains and their …
Glossary For Chemists Of Terms Used In Biotechnology, B. Nagel, H. Dellweg, Lila Gierasch
Glossary For Chemists Of Terms Used In Biotechnology, B. Nagel, H. Dellweg, Lila Gierasch
Lila Gierasch
The glossary contains definitions and explanatory notes, if needed, of over 230 terms frequently used in publications in the multidisciplinary field of biotechnology. The glossary was developed as a step to help facilitate communication between chemists, chemical engineers, biologists and bioengineers and to make biotechnology and its methodologies more accessible to the chemical profession. The interrelation between chemistry, chemical engineering and the burgeoning areas of molecular biology will be especially important as chemical industrial processes begin to incorporate recombinant DNA techniques, for example. The range of terms includes microbiology, genetic engineering, biochemistry, molecular biology, biochemical engineering, bioprocessing and general concepts …