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PLoS One. 2012; 7(4): e35910 The BH3-only protein Bid plays a key role in the induction of mitochondrial apoptosis, but its mechanism of action is still not completely understood. Here we studied the two main activation events of Bid: Caspase-8 cleavage and interaction with the membrane bilayer. We found a striking reversible behaviour of the dissociation-association events between the Bid fragments p15 and p7. Caspase-8 cleavage does not induce per se separation of the two Bid fragments, which remain in a stable complex resembling the full length Bid. Detergents trigger a complete dissociation, which can be fully reversed by detergent removal in a range of protein concentrations from 100 µM down to 500 nM. Incubation of cBid with cardiolipin-containing liposomes leads to partial dissociation of the complex. Only p15 (tBid) fragments are found at the membrane, while p7 shows no tendency to interact with the bilayer, but complete removal of p7 strongly increases the propensity of tBid to become membrane-associated. Despite the striking structural similarities of inactive Bid and Bax, Bid does not form oligomers and reacts differently in the presence of detergents and membranes, highlighting clear differences in the modes of action of the two proteins. The partial dissociation of cBid triggered by the membrane is suggested to depend on the strong and specific interaction between p15 and p7. The reversible disassembly and re-assembly of the cBid molecules at the membrane was as well proven by EPR using spin labeled cBid in the presence of isolated mitochondria. The observed dynamic dissociation of the two Bid fragments could allow the assistance to the pore-forming Bax to occur repeatedly and may explain the proposed “hit-and-run” mode of action of Bid at the bilayer. Continue reading here:
PLoS One. 2012; 7(4): e34923 Multivalent glycolipid binding toxins such as cholera toxin have the capacity to cluster glycolipids, a process thought to be important for their functional uptake into cells. In contrast to the highly dynamic properties of lipid probes and many lipid-anchored proteins, the B-subunit of cholera toxin (CTxB) diffuses extremely slowly when bound to its glycolipid receptor GM(1) in the plasma membrane of living cells. In the current study, we used confocal FRAP to examine the origins of this slow diffusion of the CTxB/GM(1) complex at the cell surface, relative to the behavior of a representative GPI-anchored protein, transmembrane protein, and fluorescent lipid analog. We show that the diffusion of CTxB is impeded by actin- and ATP-dependent processes, but is unaffected by caveolae. At physiological temperature, the diffusion of several cell surface markers is unchanged in the presence of CTxB, suggesting that binding of CTxB to membranes does not alter the organization of the plasma membrane in a way that influences the diffusion of other molecules. Furthermore, diffusion of the B-subunit of another glycolipid-binding toxin, Shiga toxin, is significantly faster than that of CTxB, indicating that the confined diffusion of CTxB is not a simple function of its ability to cluster glycolipids. By identifying underlying mechanisms that control CTxB dynamics at the cell surface, these findings help to delineate the fundamental properties of toxin-receptor complexes in intact cell membranes. See the original post here:
Biopolymers. 2012 Jul; 97(7): 558-67 Membrane transporters catalyze the transport of small solute molecules across biological barriers such as lipid bilayer membranes. As the experimental annotation of which proteins transport which substrates is incomplete it is highly desirable to develop computational methods that can assist in the classification and substrate annotation of putative membrane transport proteins. Here, we determined the similarity of membrane transporter sequences annotated in the Transport Classification Database (Saier et al., Nucleic Acids Res 2006, 34, D181-D186) and Arabidopsis thaliana membrane transporters annotated in the database Aramemnon (Schwacke et al., Plant Physiol 2003, 131, 16-26). The similarity measure was based on the amino acid composition either considering the full sequences or separately in the transmembrane (TM) and external parts of the sequences. We considered four different substrate sets and three different subfamilies and tried to classify the given proteins into these classes. Family or substrate prediction based on the simple amino acid frequency had an average accuracy of 76%. The differentiation between TM and non-TM regions led to an improved accuracy of 80% on average. © 2012 Wiley Periodicals, Inc. Biopolymers 97:558-567, 2012. Read the rest here:
Biopolymers. 2012 Jul; 97(7): 558-67 Membrane transporters catalyze the transport of small solute molecules across biological barriers such as lipid bilayer membranes. As the experimental annotation of which proteins transport which substrates is incomplete it is highly desirable to develop computational methods that can assist in the classification and substrate annotation of putative membrane transport proteins. Here, we determined the similarity of membrane transporter sequences annotated in the Transport Classification Database (Saier et al., Nucleic Acids Res 2006, 34, D181-D186) and Arabidopsis thaliana membrane transporters annotated in the database Aramemnon (Schwacke et al., Plant Physiol 2003, 131, 16-26). The similarity measure was based on the amino acid composition either considering the full sequences or separately in the transmembrane (TM) and external parts of the sequences. We considered four different substrate sets and three different subfamilies and tried to classify the given proteins into these classes. Family or substrate prediction based on the simple amino acid frequency had an average accuracy of 76%. The differentiation between TM and non-TM regions led to an improved accuracy of 80% on average. © 2012 Wiley Periodicals, Inc. Biopolymers 97:558-567, 2012. Read the original: |
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