| dc.description.abstract |
Coiled coils are ubiquitous protein structural elements which support a wide range of biological functions. They can serve as molecular spacers, oligomerization motifs, mechanical levers in membrane fusion, components of cytoskeleton as well as facilitate ion transport and signal transduction. Canonical coiled coils are regular, left-handed supercoiled bundles of two or more α-helices, with a characteristic heptad repeat pattern. However, other periodicities engendering different supercoils are possible. Insertion of two (nonads) or six (hexads) residues in a heptad repeat locally breaks the helices into short β-strands which assemble as a triangular structural element we call the β-layer. In the first project, we structurally characterized two hexad repeat families. Repetitive nonads and hexads yield a new structure, the α/β coiled coil, with regularly alternating α- and β-segments. Conversion of hexads to heptads by insertion of one residue per repeat gives a canonical coiled coil. Our results support previous data that novel backbone structures are possible within the allowed regions of Ramachandran space with minor mutations to a known fold. Secondly, we characterized the human paralogs MCUR1 and CCDC90B of a novel membrane protein family conserved in prokaryotes and mitochondria. The proteins were found to exhibit a conserved head-neck-stalk-anchor architecture, where a membrane-anchored trimeric coiled-coil stalk projects the N-terminal helical head domain via a β-layer neck. Cellular localization studies showed that prokaryotic and eukaryotic proteins localize to the cytoplasmic and inner mitochondrial membranes, respectively, with an N-in C-out topology. Using MCUR1, an essential regulator of Ca(2+) uptake through mitochondrial calcium uniporter (MCU), we studied the role of individual domains and found that the conserved head interacts directly with MCU. Ca(2+) binding destabilizes MCUR1 head domain, which then accelerates its conversion to β-amyloid fibrils. Finally, we studied the effect of frameshift resistant (FSR) repeat amplification on the structure and function of existing and novel proteins. This type of repetition comprising units of n∤3 base-pairs and lacking stop codons, encodes the same protein repeat of n residues in all three frames. We focused primarily on heptad FSR repeats which conform to coiled-coil periodicity and are significantly enriched in bacteria. Using cyanobacterium Microcystis aeruginosa, we investigated the in vivo expression of FSR repeat ORFs with proteome and transcriptome analysis and found that a number of them are highly transcribed, but undetectable at the protein level. Through biophysical and biochemical methods, we showed that FSR repeat insertion products are initially unstructured and mostly non-functional; however, they can obtain beneficial mutations over evolutionary time-scales to become more structured, giving rise to novel cellular functions. |
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