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We have utilized electron paramagnetic resonance spectroscopy to study secondary structure, subunit interaction, and molecular orientation of vimentin molecules within intact intermediate filaments and assembly intermediates. Spectroscopy data prove ␣-helical coiledcoil structures at individual amino acids 316 -336 located in rod 2B. Analysis of positions 305, 309, and 312 identify this region as conforming to the helical pattern identified within 316 -336 and thus demonstrates that, contrary to some previous predictions, this region is in an ␣-helical conformation. We show that by varying the position of the spin label, we can identify both intra-and inter-dimer interactions. With a label attached to the outside of the ␣-helix, we have been able to measure interactions between positions 348 of separate dimers as they align together in intact filaments, identifying the exact point of overlap. By mixing different spin-labeled proteins, we demonstrate that the interaction at position 348 is the result of an anti-parallel arrangement of dimers. This approach provides high resolution structural information (<2 nm resolution), can be used to identify molecular arrangements between subunits in an intact intermediate filament, and should be applicable to other noncrystallizable filamentous systems as well as to the study of protein fibrils.

The lens fiber cell-specific intermediate filament protein filensin is essential for beaded filament assembly. However, although beaded filaments are not needed for normal lens fetal development or fiber cell differentiation, they appear to be necessary for the long-term maintenance of optical clarity. The mechanism by which the absence of filensin and the beaded filament affects optical clarity has yet to be defined.

Defects in mitochondrial DNA (mtDNA) are associated with several different human diseases, including the mitochondrial encephalomyopathies. The mutations include deletions but also duplications and point mutations. Individuals with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) carry a common A-to-G substitution in a highly conserved portion of the gene for transfer RNA(Leu(UUR)). Although the MELAS mutation may be comparable to the defect in the tRNA(Lys) gene associated with MERRF (myoclonus epilepsy associated with ragged-red fibres), it is also embedded in the middle of a tridecamer sequence necessary for the formation of the 3' ends of 16S ribosomal RNA in vitro. We found that the MELAS mutation results in severe impairment of 16S rRNA transcription termination, which correlates with a reduced affinity of the partially purified termination protein for the MELAS template. This suggests that the molecular defect in MELAS is the inability to produce the correct type and quantity of rRNA relative to other mitochondrial gene products.