The Agrobacterium tumefaciens virB7 gene product contains a typical signal sequence ending with a consensus signal peptidase II cleavage site characteristic of bacterial lipoproteins. VirB7 was shown ...to be processed as a lipoprotein by (i) in vivo labeling of native VirB7 and a VirB7::PhoA fusion with 3Hpalmitic acid and (ii) inhibition of VirB7 processing by globomycin, a known inhibitor of signal peptidase II. A VirB7 derivative sustaining a Ser substitution for the invariant Cys-15 residue within the signal peptidase II cleavage site could not be visualized immunologically and failed to complement a (delta)virB7 mutation, establishing the importance of this putative lipid attachment site for VirB7 maturation and function. VirB7 partitioned predominantly with outer membrane fractions from wild-type A348 cells as well as a (delta)virB operon derivative transformed with a virB7 expression plasmid. Expression of virB7 fused to phoA, the alkaline phosphatase gene of Escherichia coli, gave rise to high alkaline phosphatase activities in E. coli and A. tumefaciens cells, providing genetic evidence for the export of VirB7 in these hosts. VirB7 was shown to be intrinsically resistant to proteinase K; by contrast, a VirB7::PhoA derivative was degraded by proteinase K treatment of A. tumefaciens spheroplasts and remained intact upon treatment of whole cells. Together, the results of these studies favor a model in which VirB7 is topologically configured as a monotopic protein with its amino terminus anchored predominantly to the outer membrane and with its hydrophilic carboxyl domain located in the periplasmic space. Parallel studies of VirB5, VirB8, VirB9, and VirB10 established that each of these membrane-associated proteins also contains a large periplasmic domain whereas VirB11 resides predominantly or exclusively within the interior of the cell
Experimental studies of protein stability often rely on the determination of an “m value”, which describes the denaturant dependence of the free energy change between two states (ΔG = ΔG H 2 O − ...mdenaturant). Changes in the m value accompanying site specific mutations are usually attributed to structural alterations in the native or unfolded ensemble. Here, we provide an example of significant reduction in the m value resulting from a subtle deviation in two-state behavior not detected by traditional methods. The protein that is studied is a variant of Escherchia coli RNase H* in which three residues predicted to be involved in a partially buried salt bridge network were mutated to alanine (R46A, D102A, and D148A). Equilibrium denaturant profiles monitored by both fluorescence and circular dichroism appeared to be cooperative, and a two-state analysis yielded a ΔG UN of approximately −3 kcal/mol with an m value of 1.4 kcal mol-1 M-1 (vs 2.3 for RNase H*). Analysis of kinetic refolding experiments suggests that the system is actually three-state at equilibrium with an appreciable concentration of an intermediate state under low denaturant concentrations. The stability of the native state determined from a fit of these kinetic data is −6.7 kcal/mol, suggesting that the stability determined by traditional two-state equilibrium analysis is a gross underestimate. The only hint to this loss of two-state behavior was a decrease in the apparent m value, and the presence of the equilibrium intermediate was only identified by a kinetic analysis. Our work serves as a cautionary note; the possibility of a three-state system should be closely addressed before interpreting a change in the m value as a change in the native or unfolded state.
A point mutation (I53A) in the core of Escherichia coli RNase H* is known to destabilize both the native conformation (ΔGUN) and the kinetic intermediate (ΔGUI) by 2 kcal/mole. Here, we have used ...native‐state hydrogen deuterium exchange to ask how this destabilization is propagated throughout the molecule. Stability parameters were obtained for individual residues in I53A and compared with those from the wild‐type protein. A destabilization of 2 kcal/mole was observed in residues in the core but was not detected in the periphery of the molecule. These results are consistent with the localized destabilization of the core observed in the early intermediate of the kinetic folding pathway, supporting the resemblance of this kinetic intermediate to the partially unfolded form detected in the native state at equilibrium. A thermodynamic cycle also shows no interaction between Ile 53 and a residue in the periphery. There is, however, an increase in the number of denaturant‐independent exchange events in the periphery of I53A, showing that effects of the point mutation are communicated to regions outside the core, although perhaps not through changes in stability. In sum, this work shows that localized regions within a protein can be destabilized independently. Furthermore, it implies a correspondence between the kinetic intermediate and the equilibrium PUF, as the magnitude and localization of the destabilization are the same in both.
Vesicle transport is essential for the movement of proteins, lipids and other molecules between membrane compartments within the cell. The role of the class VI myosins in vesicular transport is ...especially intriguing because they are the only class that has been shown to move “backwards” towards the minus end of actin filaments
1
. Myosin VI is found in distinct intracellular locations and implicated in processes such as endocytosis
2
,
3
, exocytosis, maintenance of Golgi morphology
4
,
5
and cell movement
6
. We have shown that the C-terminal tail is the key targeting region and have identified three binding sites: a WWY motif for Dab2 binding, a RRL motif for GIPC/Optineurin binding and a site that binds specifically and with high affinity (
K
d
0.3 μM) to PIP
2
-containing liposomes. This is the first demonstration that myosin VI binds lipid membranes. Lipid binding induces a large structural change in the tail (31% increase in helicity) and when associated with lipid vesicles it can dimerise.
In vivo
targeting and recruitment of myosin VI to clathrin-coated structures (CCSs) at the plasma membrane is mediated by Dab2 and PIP
2
binding.
Genome browsing with Ensembl: a practical overview Spudich, Giulietta; Fernández-Suárez, Xosé M; Birney, Ewan
Briefings in functional genomics & proteomics,
09/2007, Letnik:
6, Številka:
3
Journal Article
Odprti dostop
A wealth of gene information is accruing in public databases. Genome browsers such as Ensembl are needed to organize and depict this information in the context of the genome. Ensembl provides an open ...source gene set based on experimental evidence for over 30 species, the majority of which are vertebrates. Genes and annotation are accessible through the Ensembl browser (http://www.ensembl.org), and through direct queries of its databases using the Perl API (Application Programme Interface), MySQL or BioMart.
Some kinetic intermediates appear to be subsets of the native state, supporting a hierarchical model of folding. How similar are these intermediates to the native state ensemble? This dissertation ...focuses on specific interactions in the early folding intermediate of E. coli RNase H, and how they compare to the native state. I address the role of electrostatic interactions in the kinetic intermediate by mutating a partially buried, salt bridge network. In this variant, the native state is selectively destabilized, causing the kinetic intermediate, usually only transiently populated, to be present at equilibrium. The sole indicator of this deviation from two-state behavior was the change in the denaturant dependence of the free energy of unfolding, or “m value”. These results suggest electrostatic interactions may not be formed in the kinetic intermediate. I also compare interactions at the packing interface of two core helices (A and D) in both the kinetic intermediate and the native state core by characterizing a series of hydrophobic mutations at a residue at this interface, Ile 53. Comparisons to the native state using “ value analysis” demonstrate that although the kinetic intermediate is more loosely-packed than the native state, steric changes can be destabilizing, supporting a role of packing interactions in the intermediate. Characterization of one of these core mutations (Ile 53 to Ala) by native state hydrogen exchange strengthens the resemblance of the kinetic intermediate to a partially unfolded form (PUF) in the native state ensemble, as both forms are destabilized in the variant. This supports a hierarchical model of folding, as the PUF is a subset of the native state. These experiments show a localized destabilization of the core upon the point mutation, however, an increase is seen in the denaturant-independent, “local fluctuations” in the periphery. The studies in this dissertation characterize specific tertiary interactions in the kinetic intermediate, and further tighten the link to the PUF found in the native state ensemble. This work supports a hierarchical model of folding, in which early interactions in the kinetic intermediate are built upon in the high-energy transition state in order to achieve the native state.
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