Organization of diphtheria toxin in membranes - A hydrophobic photolabeling study

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 (similar to 5.5) 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 beta-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

Analysis of protein folding using polarity-sensitive fluorescent probes

Polarity-sensitive fluorescent probes like 8-anilino-1-naphthalenesulphonate (ANS) and 1,1'-bis(4-anilino5-naphthalenesulphonic acid (bis-ANS) have been frequently used to detect equilibrium folding intermediates like the molten globule state, as the formation of the latter involves increase in hydrophobic exposure. The ability of these fluorescent probes to bind to the intermediate state thus provides a convenient method for detection of folding intermediates in a multiple-state folding transition. However, there is no convenient method available to quantitatively analyse the fluorescent changes detected as a function of denaturant concentration. Here we describe a new method for quantitative analysis that permits one to estimate free energy of folding and determine the relative population of the various states of protein in the solution

Reevaluation of the role of the Pam18:Pam16 interaction in translocation of proteins by the mitochondrial Hsp70-based import motor

Pam18, the J-protein cochaperone of the Hsp70-based mitochondrial import motor, forms a heterodimer with the structurally related protein Pam16. Genetic and biochemical studies suggest a critical role of this interaction in maintaining Pam18's association with the translocon rather than its previously proposed regulatory role

Structure and function of Tim14 and Tim16, the J and J-like components of the mitochondrial protein import motor

The import motor of the mitochondrial translocase of the inner membrane (TIM23) mediates the ATP-dependent translocation of preproteins into the mitochondrial matrix by cycles of binding to and release from mtHsp70. An essential step of this process is the stimulation of the ATPase activity of mtHsp70 performed by the J cochaperone Tim14. Tim14 forms a complex with the J-like protein Tim16. The crystal structure of this complex shows that the conserved domains of the two proteins have virtually identical folds but completely different surfaces enabling them to perform different functions. The Tim14-Tim16 dimer reveals a previously undescribed arrangement of J and J-like domains. Mutations that destroy the complex between Tim14 and Tim16 are lethal demonstrating that complex formation is an essential requirement for the viability of cells. We further demonstrate tight regulation of the cochaperone activity of Tim14 by Tim16. The first crystal structure of a J domain in complex with a regulatory protein provides new insights into the function of the mitochondrial TIM23 translocase and the Hsp70 chaperone system in general