Biomedical subjects

Kathryn R Barber

Publications and source records attributed to Kathryn R Barber.

4 recordsLinked to original sources

PubMed2005

Ubiquitin manipulation by an E2 conjugating enzyme using a novel covalent intermediate.

Degradation of misfolded and damaged proteins by the 26 S proteasome requires the substrate to be tagged with a polyubiquitin chain. Assembly of polyubiquitin chains and subsequent substrate labeling potentially involves three enzymes, an E1, E2, and E3. E2 proteins are key enzymes and form a thioester intermediate through their catalytic cysteine with the C-terminal glycine (Gly76) of ubiquitin. This thioester intermediate is easily hydrolyzed in vitro and has eluded structural characterization. To overcome this, we have engineered a novel ubiquitin-E2 disulfide-linked complex by mutating Gly76 to Cys76 in ubiquitin. Reaction of Ubc1, an E2 from Saccharomyces cerevisiae, with this mutant ubiquitin resulted in an ubiquitin-E2 disulfide that could be purified and was stable for several weeks. Chemical shift perturbation analysis of the disulfide ubiquitin-Ubc1 complex by NMR spectroscopy reveals an ubiquitin-Ubc1 interface similar to that for the ubiquitin-E2 thioester. In addition to the typical E2 catalytic domain, Ubc1 contains an ubiquitin-associated (UBA) domain, and we have utilized NMR spectroscopy to demonstrate that in this disulfide complex the UBA domain is freely accessible to non-covalently bind a second molecule of ubiquitin. The ability of the Ubc1 to bind two ubiquitin molecules suggests that the UBA domain does not interact with the thioester-bound ubiquitin during polyubiquitin chain formation. Thus, construction of this novel ubiquitin-E2 disulfide provides a method to characterize structurally the first step in polyubiquitin chain assembly by Ubc1 and its related class II enzymes.

Catalytic Domain↗

PubMed2002

Interaction between ErbB-1 and ErbB-2 transmembrane domains in bilayer membranes.

The transmembrane domains of ErbB receptor tyrosine kinases are monotopic helical structures proposed to be capable of direct side-to-side contact with related receptors. Formation of the resulting homo- or hetero-oligomeric complexes is considered a key step in ligand-mediated signalling. ErbB-2, which has not been observed to form active homo-dimers in a ligand dependent manner, has been implicated as an important partner for formation of hetero-dimers with other ErbB receptors. Recent work has shown that the ErbB-2 transmembrane domain is capable of forming homo-oligomeric species in lipid bilayers, while a similar domain from ErbB-1 appears to have a lesser tendency to such interactions. Here, 2H nuclear magnetic resonance was used to investigate the role of the ErbB-2 transmembrane domain in hetero-oligomerisation with that of ErbB-1. At low total concentrations of peptide in the membrane, ErbB-2 transmembrane domains were found to decrease the mobility of corresponding ErbB-1 domains. The results are consistent with the existence of direct transmembrane domain involvement in hetero-oligomer formation within the ErbB receptor family.

Amino Acid Sequence↗

PubMed2002

Evidence of a tendency to self-association of the transmembrane domain of ErbB-2 in fluid phospholipid bilayers.

The transmembrane domains of receptor tyrosine kinases are single-span helical structures suggested to participate directly in the formation of side-to-side receptor homodimers/homooligomers that modulate signal transduction. Transmembrane peptides from the class I receptor tyrosine kinase, ErbB-2, were examined directly by 2H NMR spectroscopy as a means of following such phenomena under the dynamic conditions that characterize fluid, fully hydrated bilayers of natural phospholipids. Appropriate peptides were expressed as 50-mers, containing the transmembrane domain of ErbB-2 plus contiguous stretches of amino acids from the cytoplasmic and extracellular domains. Deuterium probes were incorporated in place of 1H at a site within the helical intramembranous portion (the -CH3 side chain of Ala657), and the peptides were assembled into bilayers of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) for study. An analogous peptide corresponding to the oncogenic variant characterized by a Val659-->Glu point mutation was also examined. At high peptide concentration, prominent spectral features could be assigned to rapidly rotating transmembrane monomers and to large oligomers rotating very slowly relative to a time scale of 10(-5) s. As peptide concentration was lowered, the latter feature was greatly reduced, and an additional population of mobile species became identifiable, consistent with the presence of homodimers and/or small oligomers. The defined nature of these latter spectral features suggests that preferred interaction sites exist on the peptides. The appearance of similar phenomena in the case of transmembrane peptides from both wild-type ErbB-2 and the transforming mutant argues for the involvement of additional factors in signal modulation, such as limitations normally imposed by the missing extramembranous portions.

Amino Acid Sequence↗

PubMed2002

Organization of model helical peptides in lipid bilayers: insight into the behavior of single-span protein transmembrane domains.

Selectively deuterated transmembrane peptides comprising alternating leucine-alanine subunits were examined in fluid bilayer membranes by solid-state nuclear magnetic resonance (NMR) spectroscopy in an effort to gain insight into the behavior of membrane proteins. Two groups of peptides were studied: 21-mers having a 17-amino-acid hydrophobic domain calculated to be close in length to the hydrophobic thickness of 1-palmitoyl-2-oleoyl phosphatidylcholine and 26-mers having a 22-amino-acid hydrophobic domain calculated to exceed the membrane hydrophobic thickness. (2)H NMR spectral features similar to ones observed for transmembrane peptides from single-span receptors of higher animal cells were identified which apparently correspond to effectively monomeric peptide. Spectral observations suggested significant distortion of the transmembrane alpha-helix, and/or potential for restriction of rotation about the tilted helix long axis for even simple peptides. Quadrupole splittings arising from the 26-mer were consistent with greater peptide "tilt" than were those of the analogous 21-mer. Quadrupole splittings associated with monomeric peptide were relatively insensitive to concentration and temperature over the range studied, indicating stable average conformations, and a well-ordered rotation axis. At high peptide concentration (6 mol% relative to phospholipid) it appeared that the peptide predicted to be longer than the membrane thickness had a particular tendency toward reversible peptide-peptide interactions occurring on a timescale comparable with or faster than approximately 10(-5) s. This interaction may be direct or lipid-mediated and was manifest as line broadening. Peptide rotational diffusion rates within the membrane, calculated from quadrupolar relaxation times, T(2e), were consistent with such interactions. In the case of the peptide predicted to be equal to the membrane thickness, at low peptide concentration spectral lineshape indicated the additional presence of a population of peptide having rotational motion that was restricted on a timescale of 10(-5) s.

Alanine↗