A microfluidic chip-compatible bioassay based on single-molecule detection with high sensitivity and multiplexing.

Many applications in pharmaceutical development, clinical diagnostics, and biological research demand rapid detection of multiple analytes (multiplexed detection) in a minimal volume. This need has led to the development of several novel array-based sensors. The most successful of these so far have been suspension arrays based on polystyrene beads. However, the 5 microm beads used for these assays are incompatible with most microfluidic chip technologies, mostly due to clogging problems. The challenge, then, is to design a detection particle that has high information content (for multiplexed detection), is compatible with miniaturization, and can be manufactured easily at low cost. DNA is a solid molecular wire that is easily produced and manipulated, which makes it a useful material for nanoparticles. DNA molecules are very information-rich, readily deformable, and easily propagated. We exploit these attributes in a suspension array sensor built from specialized recombinant DNA, Digital DNA, that carries both specific analyte-recognition units, and a geometrically encoded identification pattern. Here we show that this sensor combines high multiplexing with high sensitivity, is biocompatible, and has sufficiently small particle size to be used within microfluidic chips that are only 1 microm deep. We expect this technology will be the foundation of a broadly applicable technique to identify and quantitate proteins, nucleic acids, viruses, and toxins simultaneously in a minimal volume.

Human-murine transthyretin heterotetramers are kinetically stable and non-amyloidogenic. A lesson in the generation of transgenic models of diseases involving oligomeric proteins.

The transthyretin amyloidoses appear to be caused by rate-limiting tetramer dissociation and partial monomer unfolding of the human serum protein transthyretin, resulting in aggregation and extracellular deposition of amorphous aggregates and amyloid fibrils. Mice transgenic for few copies of amyloid-prone human transthyretin variants, including the aggressive L55P mutant, failed to develop deposits. Silencing the murine transthyretin gene in the presence of the L55P human gene resulted in enhanced tissue deposition. To test the hypothesis that the murine protein interacted with human transthyretin, preventing the dissociation and partial unfolding required for amyloidogenesis, we produced recombinant murine transthyretin and human/murine transthyretin heterotetramers and compared their structures and biophysical properties to recombinant human transthyretin. We found no significant differences between the crystal structures of murine and human homotetramers. Murine transthyretin is not amyloidogenic because the native homotetramer is kinetically stable under physiologic conditions and cannot dissociate into partially unfolded monomers, the misfolding and aggregation precursor. Heterotetramers composed of murine and human subunits are also kinetically stable. These observations explain the lack of transthyretin deposition in transgenics carrying a low copy number of human transthyretin genes. The incorporation of mouse subunits into tetramers otherwise composed of human amyloid-prone transthyretin subunits imposes kinetic stability, preventing dissociation and subsequent amyloidogenesis.

The dynamin middle domain is critical for tetramerization and higher-order self-assembly.

The large multidomain GTPase dynamin self-assembles around the necks of deeply invaginated coated pits at the plasma membrane and catalyzes vesicle scission by mechanisms that are not yet completely understood. Although a structural role for the 'middle' domain in dynamin function has been suggested, it has not been experimentally established. Furthermore, it is not clear whether this putative function pertains to dynamin structure in the unassembled state or to its higher-order self-assembly or both. Here, we demonstrate that two mutations in this domain, R361S and R399A, disrupt the tetrameric structure of dynamin in the unassembled state and impair its ability to stably bind to and nucleate higher-order self-assembly on membranes. Consequently, these mutations also impair dynamin's assembly-dependent stimulated GTPase activity.

Structure of the Sec13/31 COPII coat cage.

Endomembranes of eukaryotic cells are dynamic structures that are in continuous communication through the activity of specialized cellular machineries, such as the coat protein complex II (COPII), which mediates cargo export from the endoplasmic reticulum (ER). COPII consists of the Sar1 GTPase, Sec23 and Sec24 (Sec23/24), where Sec23 is a Sar1-specific GTPase-activating protein and Sec24 functions in cargo selection, and Sec13 and Sec31 (Sec13/31), which has a structural role. Whereas recent results have shown that Sec23/24 and Sec13/31 can self-assemble to form COPII cage-like particles, we now show that Sec13/31 can self-assemble to form minimal cages in the absence of Sec23/24. We present a three-dimensional reconstruction of these Sec13/31 cages at 30 A resolution using cryo-electron microscopy and single particle analysis. These results reveal a novel cuboctahedron geometry with the potential to form a flexible lattice and to generate a diverse range of containers. Our data are consistent with a model for COPII coat complex assembly in which Sec23/24 has a non-structural role as a multivalent ligand localizing the self-assembly of Sec13/31 to form a cage lattice driving ER cargo export.

The pathway by which the tetrameric protein transthyretin dissociates.

The homotetrameric protein transthyretin (TTR) must undergo rate-limiting dissociation to its constituent monomers in order to enable partial denaturation that allows the process of amyloidogenesis associated with human pathology to ensue. The TTR quaternary structure contains two distinct dimer interfaces, one of which creates the two binding sites for the natural ligand thyroxine. Tetramer dissociation could proceed through three distinct pathways; scission into dimers along either of the two unique quaternary interfaces followed by dimer dissociation represents two possibilities. Alternatively, the tetramer could lose monomers sequentially. To elucidate the TTR dissociation pathway, we employed two different TTR constructs, each featuring covalent attachment of proximal subunits. We demonstrate that tethering the A and B subunits of TTR with a disulfide bond (as well as the symmetrically disposed C and D subunits) allows urea-mediated dissociation of the resulting (TTR-S-S-TTR)(2) construct, affording (TTR-S-S-TTR)(1) retaining a stable 16-stranded beta-sheet structure that is equivalent to the dimer not possessing a thyroid binding site. In contrast, linking the A and C subunits employing a peptide tether (TTR-L-TTR)(2) affords a kinetically stable quaternary structure that does not dissociate or denature in urea. Both tethered constructs and wild-type TTR exhibit analogous stability based on guanidine hydrochloride denaturation curves. The latter denaturant can denature the tetramer, unlike urea, which can only denature monomeric TTR; hence urea requires dissociation to monomers to function. Under native conditions, the (TTR-S-S-TTR)(2) construct is able to dissociate and incorporate subunits from labeled WT TTR homotetramers at a rate equivalent to that exhibited by WT TTR. In contrast, the (TTR-L-TTR)(2) construct is unable to exchange any subunits, even after 180 h. All of the data presented herein and elsewhere demonstrate that the pathway of TTR tetramer dissociation occurs by scission of the tetramer along the crystallographic C(2) axis affording AB and CD dimers that rapidly dissociate into monomers. Determination of the mechanism of dissociation provides an explanation for why small molecules that bind at the AB/CD dimer-dimer interface impose kinetic stabilization upon TTR and disease-associated variants thereof.

Genistein, a natural product from soy, is a potent inhibitor of transthyretin amyloidosis.

The misfolding of transthyretin (TTR), including rate-limiting tetramer dissociation and partial monomer denaturation, is sufficient for TTR misassembly into amyloid and other abnormal quaternary structures associated with three amyloid diseases: senile systemic amyloidosis, familial amyloid polyneuropathy, and familial amyloid cardiomyopathy. Small molecules can bind to one or both of the unoccupied TTR thyroid hormone-binding sites, stabilizing the native tetramer more than the dissociative transition state, thereby raising the kinetic barrier for tetramer dissociation. Herein we demonstrate that genistein, the major isoflavone natural product in soy, works in this fashion and is an excellent inhibitor of transthyretin tetramer dissociation and amyloidogenesis, reducing acid-mediated fibril formation to <10% of that exhibited by TTR alone. Genistein also inhibits the amyloidogenesis of the most common familial amyloid polyneuropathy and familial amyloid cardiomyopathy mutations in TTR: V30M and V122I, respectively. Genistein additionally inhibits tetramer dissociation under physiological conditions thought to lead to slow amyloidogenesis in humans. Furthermore, this natural product exhibits highly selective binding to TTR in plasma over all of the other plasma proteins. Isothermal titration calorimetry shows that genistein binds to TTR with negative cooperativity (K(d1) = 40 nM, K(d2) = 1.4 microM). The benefits of using a nutraceutical such as genistein to treat orphan diseases such as the TTR amyloidoses include known oral bioavailability and safety data. It is conceivable that some patients could benefit from simply increasing their intake of soy products or supplements.

Kinetic stabilization of an oligomeric protein by a single ligand binding event.

Protein native state stabilization imposed by small molecule binding is an attractive strategy to prevent the misfolding and misassembly processes associated with amyloid diseases. Transthyretin (TTR) amyloidogenesis requires rate-limiting tetramer dissociation before misassembly of a partially denatured monomer ensues. Selective stabilization of the native TTR tetramer over the dissociative transition state by small molecule binding to both thyroxine binding sites raises the kinetic barrier of tetramer dissociation, preventing amyloidogenesis. Assessing the amyloidogenicity of a TTR tetramer having only one amyloidogenesis inhibitor (I) bound is challenging because the two small molecule binding constants are generally not distinct enough to allow for the exclusive formation of TTR.I in solution to the exclusion of TTR.I(2) and unliganded TTR. Herein, we report a method to tether one fibril formation inhibitor to TTR by disulfide bond formation. Occupancy of only one of the two thyroxine binding sites is sufficient to inhibit tetramer dissociation in 6.0 M urea and amyloidogenesis under acidic conditions by imposing kinetic stabilization on the entire tetramer. The sufficiency of single occupancy for stabilizing the native state of TTR provides the incentive to search for compounds displaying striking negative binding cooperativity (e.g., K(d1) in nanomolar range and K(d2) in the micromolar to millimolar range), enabling lower doses of inhibitor to be employed in the clinic, mitigating potential side effects.

Kinetic stabilization of the native state by protein engineering: implications for inhibition of transthyretin amyloidogenesis.

The amyloidogenic homotetrameric protein transthyretin (TTR) must undergo rate-limiting dissociation to partially denatured monomers in order to aggregate. TTR contains two distinct quaternary interfaces, one of which defines the binding sites for thyroxine and small-molecule amyloidogenesis inhibitors. Kinetic stabilization of the tetramer can be accomplished either by the binding of amyloidogenesis inhibitors selectively to the native state over the dissociative transition state or by the introduction of trans-suppressor subunits (T119M) into heterotetramers to destabilize the dissociative transition state. In each case, increasing the dissociation activation barrier prevents tetramer dissociation. Herein, we demonstrate that tethering two subunits whose quaternary interface defines the thyroxine binding site also dramatically increases the barrier for tetramer dissociation, apparently by destabilization of the dissociative transition state. The tethered construct (TTR-L-TTR)2 is structurally and functionally equivalent to wild-type TTR. Urea is unable to denature (TTR-L-TTR)2, yet it is able to maintain the denatured state once denaturation is achieved by GdnHCl treatment, suggesting that (TTR-L-TTR)2 is kinetically rather than thermodynamically stabilized, consistent with the identical wild-type TTR and (TTR-L-TTR)2 GdnHCl denaturation curves. Studies focused on a construct containing a single TTR-L-TTR chain and two normal monomer subunits establish that alteration of only one quaternary structural interface is sufficient to impose kinetic stabilization on the entire quaternary structure.