Dung M. Vu

A Fluorescence Light-Up Ag Nanocluster Probe that Discriminates Single-Nucleotide Variants by Emission Color

Rapid and precise screening of small genetic variations, such as single-nucleotide polymorphisms (SNPs), among an individuals genome is still an unmet challenge at point-of-care settings. One crucial step toward this goal is the development of discrimination probes that require no enzymatic reaction and are easy to use. Here we report a new type of fluorescent molecular probe, termed a chameleon NanoCluster Beacon (cNCB), that lights up into different colors upon binding SNP targets. NanoCluster Beacons (NCBs) are collections of a small number of Ag atoms templated on single-stranded DNA that fluoresce strongly when placed in proximity to particular DNA sequences, termed enhancers. Here we show the fluorescence emission color of a NCB can change substantially (a shift of 60-70 nm in the emission maximum) depending upon the alignment between the silver nanocluster and the DNA enhancer sequence. Chameleon NCBs exploit this color shift to directly detect SNPs, based on the fact that different SNPs produce a different alignment between the Ag nanocluster and the enhancer. This SNP detection method has been validated on all single-nucleotide substitution scenarios in three synthetic DNA targets, in six disease-related SNP targets, and in two clinical samples taken from patients with ovarian serous borderline tumors. Samples with single-nucleotide variations can be easily identified by the naked eye under UV excitation, making this method a reliable and low-cost assay with a simple readout format.

A DNA-templated fluorescent silver nanocluster with enhanced stability

We report the discovery of a DNA sequence that templates a highly stable fluorescent silver nanocluster. In contrast to other DNA templated silver nanoclusters that have a relatively short shelf-life, the fluorescent species templated in this new DNA sequence retains significant fluorescence for at least a year. Moreover, this new silver nanocluster possesses low cellular toxicity and enhanced thermal, oxidative, and chemical stability.

The helix–turn–helix motif as an ultrafast independently folding domain: The pathway of folding of Engrailed homeodomain

Helices 2 and 3 of Engrailed homeodomain (EnHD) form a helix–turn–helix (HTH) motif. This common motif is believed not to fold independently, which is the characteristic feature of a motif rather than a domain. But we found that the EnHD HTH motif is monomeric and folded in solution, having essentially the same structure as in full-length protein. It had a sigmoidal thermal denaturation transition. Both native backbone and local tertiary interactions were formed concurrently at 4 × 105 s−1 at 25°C, monitored by IR and fluorescence T-jump kinetics, respectively, the same rate constant as for the fast phase in the folding of EnHD. The HTH motif, thus, is an ultrafast-folding, natural protein domain. Its independent stability and appropriate folding kinetics account for the stepwise folding of EnHD, satisfy fully the criteria for an on-pathway intermediate, and explain the changes in mechanism of folding across the homeodomain family. Experiments on mutated and engineered fragments of the parent protein with different probes allowed the assignment of the observed kinetic phases to specific events to show that EnHD is not an example of one-state downhill folding.

NMR and Temperature-jump Measurements of de Novo Designed Proteins Demonstrate Rapid Folding in the Absence of Explicit Selection for Kinetics

We address the importance of natural selection in the origin and maintenance of rapid protein folding by experimentally characterizing the folding kinetics of two de novo designed proteins, NC3-NCAP and ENH-FSM1. These 51 residue proteins, which adopt the helix-turn-helix homeodomain fold, share as few as 12 residues in common with their most closely related natural analog. Despite the replacement of up to 3/4 of their residues by a computer algorithm optimizing only thermodynamic properties, the designed proteins fold as fast or faster than the 35,000 s(-1) observed for the closest natural analog. Thus these de novo designed proteins, which were produced in the complete absence of selective pressures or design constraints explicitly aimed at ensuring rapid folding, are among the most rapidly folding proteins reported to date.

Early turn formation and chain collapse drive fast folding of the major cold shock protein CspA of Escherichia coli.

The folding mechanism of the β-sheet protein CspA, the major cold shock protein of Escherichia coli, was previously reported to be a concerted, two-state process. We have reexamined the folding of CspA using multiple spectroscopic probes of the equilibrium transition and laser-induced temperature jump (T-jump) to achieve better time resolution of the kinetics. Equilibrium temperature-dependent Fourier transform infrared (1634 cm(-1)) and tryptophan fluorescence measurements reveal probe-dependent thermal transitions with midpoints (T(m)) of 66 ± 1 and 61 ± 1 °C, respectively. Singular-value decomposition analysis with global fitting of the temperature-dependent infrared (IR) difference spectra reveals two spectral components with distinct melting transitions with different midpoints. T-jump relaxation measurements of CspA probed by IR and fluorescence spectroscopy show probe-dependent multiexponential kinetics characteristic of non-two-state folding. The frequency-dependent IR transients all show biphasic relaxation with average time constants of 50 ± 7 and 225 ± 25 μs at a T(f) of 77 °C and almost equal amplitudes. Similar biphasic kinetics are observed using Trp fluorescence of the wild-type protein and the Y42W and T68W mutants, with comparable lifetimes. All of these observations support a model for the folding of CspA through a compact intermediate state. The transient IR and fluorescence spectra are consistent with a diffuse intermediate having β-turns and substantial β-sheet structure. The loop β3-β4 structure is likely not folded in the intermediate state, allowing substantial solvent penetration into the barrel structure.

Identification of Minimally Interacting Modules in an Intrinsically Disordered Protein

The conformational characterization of intrinsically disordered proteins (IDPs) is complicated by their conformational heterogeneity and flexibility. If an IDP could somehow be divided into smaller fragments and reconstructed later, theoretical and spectroscopic studies could probe its conformational variability in detail. Here, we used replica molecular-dynamics simulations and network theory to explore whether such a divide-and-conquer strategy is feasible for α-synuclein, a prototypical IDP. We characterized the conformational variability of α-synuclein by conducting >100 unbiased all-atom molecular-dynamics simulations, for a total of >10 μs of trajectories. In these simulations, α-synuclein formed a heterogeneous ensemble of collapsed coil states in an aqueous environment. These states were stabilized by heterogeneous contacts between sequentially distant regions. We find that α-synuclein contains residual secondary structures in the collapsed states, and the heterogeneity in the collapsed state makes it feasible to split α-synuclein into sequentially contiguous minimally interacting fragments. This study reveals previously unknown characteristics of α-synuclein and provides a new (to our knowledge) approach for studying other IDPs.

Characterization of a Disordered Protein During Micellation: Interactions of α-Synuclein with Sodium Dodecyl Sulfate

To better understand the interaction of α-synuclein (αSyn) with lipid membranes, we carried out self-assembly molecular dynamics simulations of αSyn with monomeric and micellar sodium dodecyl sulfate (SDS), a widely used membrane mimic. We find that both electrostatic and hydrophobic forces contribute to the interactions of αSyn with SDS. In the presence of αSyn, our simulations suggest that SDS aggregates along the protein chain and forms small-size micelles at very early times. Aggregation is followed by formation of a collapsed protein-SDS micelle complex, which is consistent with experimental results. Finally, interaction of αSyn with preformed micelles induces alterations in the shape of the micelle, and the N-terminal helix (residues 3 through 37) tends to associate with micelles. Overall, our simulations provide an atomistic description of the early time scale αSyn-SDS interaction during the self-assembly of SDS into micelles.

Deducing conformational variability of intrinsically disordered proteins from infrared spectroscopy with Bayesian statistics.

As it remains practically impossible to generate ergodic ensembles for large intrinsically disordered proteins (IDP) with molecular dynamics (MD) simulations, it becomes critical to compare spectroscopic characteristics of the theoretically generated ensembles to corresponding measurements. We develop a Bayesian framework to infer the ensemble properties of an IDP using a combination of conformations generated by MD simulations and its measured infrared spectrum. We performed 100 different MD simulations totaling more than 10 µs to characterize the conformational ensemble of αsynuclein, a prototypical IDP, in water. These conformations are clustered based on solvent accessibility and helical content. We compute the amide-I band for these clusters and predict the thermodynamic weights of each cluster given the measured amide-I band. Bayesian analysis produces a reproducible and non-redundant set of thermodynamic weights for each cluster, which can then be used to calculate the ensemble properties. In a rigorous validation, these weights reproduce measured chemical shifts.

Detection of Lipomannan in Cattle Infected with Bovine Tuberculosis

Early and rapid detection of bovine tuberculosis (bTB) is critical to controlling the spread of this disease in cattle and other animals. In this study, we demonstrate the development of an immunoassay for the direct detection of the bovine bTB biomarker, lipomannan (LM) in serum using a waveguide-based optical biosensor. We apply an ultra-sensitive detection strategy developed by our team, termed lipoprotein capture, that exploits the pull-down of high-density lipoprotein (HDL) nanodiscs from cattle blood that allows for the recovery and detection of associated LM. We also profile the change in the expression of these TB biomarkers as a function of time from a small set of samples collected from studies of bovine TB-infected cattle. We demonstrate for the first time the direct detection of bovine LM in serum, and clearly show that the biomarker is expressed in detectable concentrations during the entire course of the infection.

Detection of Lipid and Amphiphilic Biomarkers for Disease Diagnostics

Rapid diagnosis is crucial to effectively treating any disease. Biological markers, or biomarkers, have been widely used to diagnose a variety of infectious and non-infectious diseases. The detection of biomarkers in patient samples can also provide valuable information regarding progression and prognosis. Interestingly, many such biomarkers are composed of lipids, and are amphiphilic in biochemistry, which leads them to be often sequestered by host carriers. Such sequestration enhances the difficulty of developing sensitive and accurate sensors for these targets. Many of the physiologically relevant molecules involved in pathogenesis and disease are indeed amphiphilic. This chemical property is likely essential for their biological function, but also makes them challenging to detect and quantify in vitro. In order to understand pathogenesis and disease progression while developing effective diagnostics, it is important to account for the biochemistry of lipid and amphiphilic biomarkers when creating novel techniques for the quantitative measurement of these targets. Here, we review techniques and methods used to detect lipid and amphiphilic biomarkers associated with disease, as well as their feasibility for use as diagnostic targets, highlighting the significance of their biochemical properties in the design and execution of laboratory and diagnostic strategies. The biochemistry of biological molecules is clearly relevant to their physiological function, and calling out the need for consideration of this feature in their study, and use as vaccine, diagnostic and therapeutic targets is the overarching motivation for this review.