Large protein complexes assemble spontaneously, yet their subunits do not prematurely form unwanted aggregates. This paradox is epitomized in the bacterial flagellar motor, a sophisticated rotary motor and sensory switch consisting of hundreds of subunits. Here we demonstrate that Escherichia coli FliG, one of the earliest-assembling flagellar motor proteins, forms ordered ring structures via domain-swap polymerization, which in other proteins has been associated with uncontrolled and deleterious protein aggregation. Solution structural data, in combination with in vivo biochemical cross-linking experiments and evolutionary covariance analysis, revealed that FliG exists predominantly as a monomer in solution but only as domain-swapped polymers in assembled flagellar motors. We propose a general structural and thermodynamic model for self-assembly, in which a structural template controls assembly and shapes polymer formation into rings.
We present spectroscopic evidence consistent with the presence of a stable tyrosyl radical in partially reduced human monoamine oxidase (MAO) A. The radical forms following single electron donation to MAO A and exists in equilibrium with the FAD flavosemiquinone. Oxidative formation of the tyrosyl radical in MAO is not reliant on neighboring metal centers and uniquely requires reduction of the active site flavin to facilitate oxidation of a tyrosyl side chain. The identified tyrosyl radical provides the key missing link in support of the single electron transfer mechanism for amine oxidation by MAO enzymes.The mammalian monoamine oxidases (MAO) 1 A and B are flavoproteins localized to the outer mitochondrial membrane. MAO catalyzes the oxidative deamination of neurotransmitters and exogenous alkylamines. The human enzymes are important pharmaceutical targets for antidepressants, and inhibitors of MAO B are used synergistically with L-DOPA in the treatment of Parkinson disease (1). Elevated levels of MAO B induce apoptosis in kidney (2) and neuronal cells (3) and are also associated with plaque astrocytes in the brains of Alzheimer patients (4). The anti-apoptotic action of a MAO B inhibitor is important in novel Alzheimer treatments (5). MAO is also implicated in the onset of Parkinson syndrome through bioactivation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, an impurity in many sources of synthetic heroin (6).Despite the recent crystal structures of MAO B (7) and MAO A (8) and extensive literature on substrate and inhibitor specificities, the mechanism of substrate oxidation remains obscure (9). Much of the debate has centered on the possible existence of radical species, direct evidence for which has not been forthcoming. An early proposal invoked a polar nucleophilic mechanism involving attack of the deprotonated amine substrate at the flavin C4a to form a substrate-flavin C4a adduct and proton abstraction from the ␣-carbon of the adduct by an active site base (10). Support for this mechanism came from chemical model studies in reactions of amines with lumiflavins (11,12). Studies of quantitative structure-activity relationships with MAO B have been used to support a second mechanism in which substrate ␣-C-H bond cleavage is via direct hydrogen atom transfer to a protein-based non-flavin radical followed by electron transfer to the flavin (13,14). An organic radical species was originally reported in EPR spectra of resting bovine liver MAO B (15) but later was attributed to an artifact of purification of MAO B from bovine liver following EPR studies of highly purified recombinant sources of MAO A and MAO B (16). Edmondson and Miller (16) have proposed a concerted polar nucleophilic mechanism for MAO A involving a substrate-flavin C4a adduct and proton abstraction by the highly basic N-5 atom of the flavin. This mechanism is consistent with studies of quantitative structure activity relationships and kinetic isotope effects and with the apparent lack of an organic protein-based radical in EPR spectra of t...
The rational design of complementary DNA sequences can be used to create nanostructures that self-assemble with nanometer precision. DNA nanostructures have been imaged by atomic force microscopy and electron microscopy. Small-angle X-ray scattering (SAXS) provides complementary structural information on the ensemble-averaged state of DNA nanostructures in solution. Here we demonstrate that SAXS can distinguish between different single-layer DNA origami tiles that look identical when immobilized on a mica surface and imaged with atomic force microscopy. We use SAXS to quantify the magnitude of global twist of DNA origami tiles with different crossover periodicities: these measurements highlight the extreme structural sensitivity of single-layer origami to the location of strand crossovers. We also use SAXS to quantify the distance between pairs of gold nanoparticles tethered to specific locations on a DNA origami tile and use this method to measure the overall dimensions and geometry of the DNA nanostructure in solution. Finally, we use indirect Fourier methods, which have long been used for the interpretation of SAXS data from biomolecules, to measure the distance between DNA helix pairs in a DNA origami nanotube. Together, these results provide important methodological advances in the use of SAXS to analyze DNA nanostructures in solution and insights into the structures of single-layer DNA origami.
Mechanisms for transcription factor recognition of specific DNA base sequences are well characterized and recent studies demonstrate that the shape of these cognate binding sites is also important. Here, we uncover a new mechanism where the transcription factor GabR simultaneously recognizes two cognate binding sites and the shape of a 29 bp DNA sequence that bridges these sites. Small-angle X-ray scattering and multi-angle laser light scattering are consistent with a model where the DNA undergoes a conformational change to bend around GabR during binding. In silico predictions suggest that the bridging DNA sequence is likely to be bendable in one direction and kinetic analysis of mutant DNA sequences with biolayer interferometry, allowed the independent quantification of the relative contribution of DNA base and shape recognition in the GabR–DNA interaction. These indicate that the two cognate binding sites as well as the bendability of the DNA sequence in between these sites are required to form a stable complex. The mechanism of GabR–DNA interaction provides an example where the correct shape of DNA, at a clearly distinct location from the cognate binding site, is required for transcription factor binding and has implications for bioinformatics searches for novel binding sites.
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