Topography of multiple spanning transmembrane proteins - a photochemical approach

Lala, Anil K.; Batliwala, Hoshang; Bhat, Sujata V.

doi:10.1351/pac199062071453

Cited by 5 publications

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“…We report here our results on hydrophobic photolabeling of diphtheria toxin when it is bound to membranes with diazofluorene (DAF). This reagent readily partitions into membranes and on photolysis generates a highly reactive carbene, which labels the membrane-spanning domains of transmembrane proteins (33,34). Analysis of photolabeled DT led to identification of membrane-associated segments of DT, thus permitting us to build a model of membraneassociated DT.…”

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Organization of Diphtheria Toxin in Membranes

D’Silva

Lala

2000

Journal of Biological Chemistry

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Diphtheria toxin (DT) is a disulfide linked AB-toxin consisting of a catalytic domain (C), a membrane-inserting domain (T), and a receptor-binding domain (R). It gains entry into cells by receptor-mediated endocytosis.The low pH (ϳ5.5) inside the endosomes induces a conformational change in the toxin leading to insertion of the toxin in the membrane and subsequent translocation of the C domain into the cell, where it inactivates protein synthesis ultimately leading to cell death. We have used a highly reactive hydrophobic photoactivable reagent, DAF, to identify the segments of DT that interact with the membrane at pH 5.2. This reagent readily partitions into membranes and, on photolysis, indiscriminately inserts into lipids and membrane-inserted domains of proteins. Subsequent chemical and/or enzymatic fragmentation followed by peptide sequencing allows for identification of the modified residues. Using this approach it was observed that T domain helices, TH1, TH8, and TH9 insert into the membrane. Furthermore, the disulfide link was found on the trans side leaving part of the C domain on the trans side. This domain then comes out to the cis side via a highly hydrophobic patch corresponding to residues 134 -141, originally corresponding to a ␤-strand in the solution structure of DT. It appears that the three helices of the T domain could participate in the formation of a channel from a DT-oligomer, thus providing the transport route to the C domain after the disulfide reductase separates the two chains.There are several bacterial toxins that act by modification of intracellular substrates. Despite the fact that the structure of many of these toxins is now known, the mechanism of entry of these toxins into cells is far from clear (1-3). One of the major problems associated with getting an insight into the mechanism of entry is the very limited availability of information on the structure of membrane-bound toxins. Therefore, although diphtheria toxin (DT) 1 is an extensively studied protein toxin, a detailed mechanism of entry still eludes us. DT consists of two chains, A and B, that are joined by a disulfide link and is composed of three structural domains: the NH 2 -terminal catalytic domain C (residues 1-193), transmembrane domain T (residues 205-378), and receptor-binding domain R (residues 386 -535) (4, 5). DT enters cells by moving into the endosomal compartment using receptor-mediated endocytosis. The lowering of pH in the endosomes results in insertion of the toxin into the membrane and subsequent translocation of the A-chain into the cytosol, where it ADP-ribosylates the elongation factor-2, leading to inhibition of protein synthesis and eventual cell death (6 -11). The T domain of DT is said to be primarily responsible for membrane insertion, though other domains, C and R, have also been shown to be associated with membranes (12-21). The T domain, comprised of three layers of helices with the innermost layer consisting of two hydrophobic helices TH8 and TH9, has been a subject of a number of studies inv...

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confidence: 99%

Organization of Diphtheria Toxin in Membranes

D’Silva

Lala

2000

Journal of Biological Chemistry

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“…His work on diazafluorene as a “new fluorescent photochemical reagent” provided an alternative to light activatable radio‐labels (Anjaneyulu and Lala 1982). Over the last 20 years, Lala and an extraordinarily dedicated band of graduate students developed photolabeling into an important and powerful tool for analyzing the organization of proteins in membranes using the human erythrocyte glucose transporter, Staphylococcus aureus α‐toxin and diphtheria toxin as examples, where the utility of this method was clearly demonstrated (Lala et al 1990; D'Silva and Lala 1998 D'Silva and Lala 2000). Lala sensed opportunities to exploit the techniques he developed for the characterization of protein folding intermediates (D'Silva and Lala 1999) and depth probing in phospholipids bilayers (Lala 2002).…”

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confidence: 99%