Atomic force microscopy to characterize the molecular size of prion protein.

The conformational transition of alpha-helix-rich cellular prion protein (PrP(C)) to an isomer with high beta-sheet content is associated with transmissible spongiform encephalopathies. With the ultimate long-term goal of using imaging techniques to study PrP aggregation, we report the results of initial experiments to determine whether PrP molecules could be visualized as single molecules, and if the observed size corresponded to the calculated size for PrP. The investigation of single molecules, and not those embedded into larger aggregates, was the key in our experimental approach. Using atomic force microscopy (AFM) as an imaging method, the immobilization of recombinant histidine (His)10-tagged PrP on mica was performed in the presence of different heavy metal ions. The addition of Cu2+ resulted in an enhanced PrP immobilization, whereas Ni2+ reduced coverage of the surface by PrP. High-resolution data from dried PrP preparations provided a first approximation to geometrical parameters of PrP precipitates, which indicated that the volume of a single PrP molecule was 30 nm3. Molecular dynamics simulations performed to complement the structural aspects of the AFM investigation yielded a calculated molecular volume of 33 nm3 for PrP. These experimentally observed and theoretically expected values provide basic knowledge for further studies on the size and composition of larger amyloidal PrP aggregates, PrP isoforms or mutants such as PrP molecules without octarepeats.

Cellular prion protein co-localizes with nAChR beta4 subunit in brain and gastrointestinal tract.

PrP(C), the cellular isoform of prion protein, is widely expressed in most tissues, including brain, muscle and gastrointestinal tract. Despite its involvement in several bioprocesses, PrP has still no apparent physiological role. During propagation of transmissible spongiform encephalopathies (TSE), prion protein is converted to the pathological isoform, PrP(Sc), in a process believed to be mediated by unknown host factors. The identification of proteins associated with PrP may provide information about both the biology of prions and the pathogenesis of TSE. Thus far, PrP(C) has been shown to interact with synaptic proteins, components of the cytoskeleton and intracellular proteins involved in signalling pathways. Here, we describe the association of PrP with the beta4 subunit of nicotinic acetylcholine receptor (nAChR), as indicated by co-immunoprecipitation assays and double-label immunofluorescence. The interaction between prion protein and native beta4 subunit was further studied by affinity chromatography, using immobilized and refolded recombinant PrP as a bait and brain homogenates from normal individuals. Additionally, the participation of beta4 subunit in the pathogenesis of TSE was studied by in vivo assays. beta4(-/-) and wild-type mice were challenged with the RML (Rocky Mountain Laboratories) infectious agent. Transgenic animals displayed altered incubation times but the deletion of beta4 subunit did not result in a significant change of the incubation period of the disease. Our results suggest that PrP(C) is a member of a multiprotein membrane complex participating in the formation and function of alpha3beta4 nAChR.

Antigenic profile of human recombinant PrP: generation and characterization of a versatile polyclonal antiserum.

We describe the quality of a rabbit polyclonal antiserum (Sal1) that was raised against mature human recombinant prion protein (rhuPrP). Epitope mapping demonstrated that the Sal1 antiserum recognized six to eight linear antigenic sites, depending on the animal species. The versatility of the antiserum was evident from the range of animal species and immunochemical techniques where it could be applied successfully. Antigen absorption studies revealed differences in the location and number of epitopes remaining after incubation with soluble or aggregated antigen.Our knowledge concerning immunoprophylaxis against prion diseases and the important role played by conformational changes of PrP is increasing rapidly. The findings reported here should add to this body of knowledge.

The ATP-binding cassette subunit of the maltose transporter MalK antagonizes MalT, the activator of the Escherichia coli mal regulon.

Transcription of the mal regulon of Escherichia coli K-12 is regulated by the positive activator, MalT. In the presence of ATP and maltotriose, MalT binds to decanucleotide MalT boxes that are found upstream of mal promoters and activates transcription at these sites. The earliest studies of the mal regulon, however, suggested a negative role for the MalK protein, the ATP-binding cassette subunit of the maltose transporter, in regulating mal gene expression. More recently, it was found that overexpression of the MalK protein resulted in very low levels of mal gene transcription. In this report we describe the use of tagged versions of MalT to provide evidence that it physically interacts with the MalK protein both in vitro and in vivo. In addition, we show that a novel malK mutation, malK941, results in an increased ability of MalK to down-modulate MalT activity in vivo. The fact that the MalK941 protein binds but does not hydrolyse ATP suggests that the MalK941 mutant protein mimics the inactive, ATP-bound form of the normal MalK protein. In contrast, cells with high levels of MalK ATPase show a reduced ability to down-modulate MalT and express several mal genes constitutively. These results are consistent with a model in which the inactive form of MalK down-modulates MalT and decreases transcription, whereas the active form of MalK does not. This model suggests that bacteria may be able to couple information about extracellular substrate availability to the transcriptional apparatus via the levels of ATP hydrolysis associated with transport.

Mutations that alter the transmembrane signalling pathway in an ATP binding cassette (ABC) transporter.

The maltose transport system of Escherichia coli is a well-characterized member of the ATP binding cassette transporter superfamily. Members of this family share sequence similarity surrounding two short sequences (the Walker A and B sequences) which constitute a nucleotide binding pocket. It is likely that the energy from binding and hydrolysis of ATP is used to accomplish the translocation of substrate from one location to another. Periplasmic binding protein-dependent transport systems, like the maltose transport system of E.coli, possess a water-soluble ligand binding protein that is essential for transport activity. In addition to delivering ligand to the membrane-bound components of the system on the external face of the membrane, the interaction of the binding protein with the membrane complex initiates a signal that is transmitted to the ATP binding subunit on the cytosolic side and stimulates its hydrolytic activity. Mutations that alter the membrane complex so that it transports independently of the periplasmic binding protein also result in constitutive activation of the ATPase. Genetic analysis indicates that, in general, two mutations are required for binding protein-independent transport and constitutive ATPase. The mutations alter residues that cluster to specific regions within the membrane spanning segments of the integral membrane components MalF and MalG. Individually, the mutations perturb the ability of MBP to interact productively with the membrane complex. Genetic alteration of this signalling pathway suggests that other agents might have similar effects. These could be potentially useful for modulating the activities of ABC transporters such as P-glycoprotein or CFTR, that are implicated in disease.

Tinkering with transporters: periplasmic binding protein-dependent maltose transport in E. coli.

Periplasmic binding protein-dependent transport systems represent a common mechanism for nutrient and ion uptake in bacteria. As a group, these systems are related to one another and to other transporters of both prokaryotes and eukaryotes, based on sequence similarity within an ATP-binding subunit and overall structural organization. These transporters probably all use energy derived from ATP to pump substrates across membranes. Although there is considerable information about the sequences and identity of the transporters, there is little information about how they work. That is, where do ligands bind? Where do the subunits or domains interact with one another? How is the energy of nucleotide binding and/or hydrolysis converted to conformational changes? In order to address these questions we have taken a genetic approach that involves studying mutant forms of a transporter. Rather than study mutations that result in complete loss of function, the study of mutations which perturb or alter the normal function of the transporter in a defined manner has provided a limited insight into how the answers to these questions may be obtained.

Characterization of the structural requirements for assembly and nucleotide binding of an ATP-binding cassette transporter. The maltose transport system of Escherichia coli.

The periplasmic maltose-binding protein-dependent, maltose transport system of Escherichia coli is a well studied member of the ATP-binding cassette family of transport ATPases. In addition to the water-soluble maltose-binding protein, the system comprises three membrane proteins, MalF, MalG, and MalK, which form a heterotetrameric complex (FGK2) in the cytoplasmic membrane. The purified complex exhibits transport-associated ATPase activity. To characterize the requirements for nucleotide binding and hydrolysis by the FGK2 complex, we used plasmids to express different combinations of the individual subunits as well as mutant forms of the MalK subunit. Prior to measuring nucleotide binding, we examined membrane preparations for the presence of each subunit from strains that contained all possible permutations of the three structural genes, malF, malG, and malK. We found that when all three genes were present or when malF and malK were present together, the corresponding antigens were detected easily on Western immunoblots and were soluble in the non-ionic detergent, Triton X-100. In contrast, all other permutations resulted in decreased amounts of antigen or antigen that was Triton X-100-insoluble. We relied on photocross-linking with 8-azido-[32P]ATP and ATP hydrolysis as indicators of the ability of the transport complex to interact with purine nucleotides. 8-Azido-[32P]ATP was photocross-linked to the MalK subunit. Photolabeling of MalK was inhibited by ATP, ADP, and GTP and not by other nucleotides. Photolabeling of MalK required the presence of MalF but not MalG. Mutations in malK that affect amino acid residues thought to be directly involved in nucleotide binding did indeed abolish labeling and resulted in loss of transport activity without affecting protein stability. In general, ATP hydrolysis correlated with the photocross-linking. A notable exception is the MalK941 mutant protein which retained the ability to be labeled by 8-azido-[32P]ATP but was unable to catalyze detectable levels of ATP hydrolysis. Some, but not all, of the malK mutations were dominant to wild type. To study the mechanism of dominance we devised a means of measuring the ability of different wild-type and mutant MalK proteins to interact with the MalF and MalG subunits. This assay relies on the fact that, when a bifunctional MalK-LacZ hybrid protein is associated with the MalF and MalG subunits, it is membrane-bound. Excess MalK competed with the MalK-LacZ hybrid protein for sites in the membrane and resulted in the hybrid fractionating as a soluble protein.(ABSTRACT TRUNCATED AT 400 WORDS)