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The transport and targeting of a number of periplasmic proteins is carried out by the Sec‐independent Mtt (also referred to as Tat) protein translocase. Proteins using this translocase have a distinct twin‐arginine‐containing leader. We hypothesized that specific leader‐binding proteins exist to escort proteins to the translocase complex. A fusion was constructed with the twin‐arginine leader from dimethyl sulphoxide (DMSO) reductase, subunit DmsA, to the N‐terminus of glutathione‐S‐transferase. This leader fusion was bound to a glutathione affinity column through which an Escherichia coli anaerobic cell‐free extract was passed. Proteins that bound to the leader were then separated and identified by N‐terminal sequencing, which identified DnaK and a protein originating from the uncharacterized reading frame ynfI. This gene has been designated dmsD based on the findings presented in this paper. DmsD was purified as a His6 fusion and was shown to interact with preprotein forms of DmsA and TorA (trimethyl amine N‐oxide reductase). A strain carrying a dmsD knock‐out mutation showed a loss of anaerobic growth on glycerol–DMSO medium and reduced growth on glycerol–fumarate medium. This work suggests that DmsD is a twin‐arginine leader‐binding protein.

β2-microglobulin (β2m) is a 99-residue protein with an immunoglobulin fold that forms β-sheet-rich amyloid fibrils in dialysis-related amyloidosis. Here the environment and accessibility of side chains within amyloid fibrils formed in vitro from β2m with a long straight morphology are probed by site-directed spin labeling and accessibility to modification with N-ethyl maleimide using 19 site-specific cysteine variants. Continuous wave electron paramagnetic resonance spectroscopy of these fibrils reveals a core predominantly organized in a parallel, in-register arrangement, by contrast with other β2m aggregates. A continuous array of parallel, in-register β-strands involving most of the polypeptide sequence is inconsistent with the cryoelectron microscopy structure, which reveals an architecture based on subunit repeats. To reconcile these data, the number of spins in close proximity required to give rise to spin exchange was determined. Systematic studies of a model protein system indicated that juxtaposition of four spin labels is sufficient to generate exchange narrowing. Combined with information about side-chain mobility and accessibility, we propose that the amyloid fibrils of β2m consist of about six β2m monomers organized in stacks with a parallel, in-register array. The results suggest an organization more complex than the accordion-like β-sandwich structure commonly proposed for amyloid fibrils.

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