Quantum Defect Sensitization via Phase-Changing Supercharged Antibody Fragments.

Quantum defects in single-walled carbon nanotubes promote exciton localization, which enables potential applications in biodevices and quantum light sources. However, the effects of local electric fields on the emissive energy states of quantum defects and how they can be controlled are unexplored. Here, we investigate quantum defect sensitization by engineering an intrinsically disordered protein to undergo a phase change at a quantum defect site. We designed a supercharged single-chain antibody fragment (scFv) to enable a full ligand-induced folding transition from an intrinsically disordered state to a compact folded state in the presence of a cytokine. The supercharged scFv was conjugated to a quantum defect to induce a substantial local electric change upon ligand binding. Employing the detection of a proinflammatory biomarker, interleukin-6, as a representative model system, supercharged scFv-coupled quantum defects exhibited robust fluorescence wavelength shifts concomitant with the protein folding transition. Quantum chemical simulations suggest that the quantum defects amplify the optical response to the localization of charges produced upon the antigen-induced folding of the proteins, which is difficult to achieve in unmodified nanotubes. These findings portend new approaches to modulate quantum defect emission for biomarker sensing and protein biophysics and to engineer proteins to modulate binding signal transduction.

Publisher Correction: Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.

This paper was originally published under standard Springer Nature copyright. As of the date of this correction, the Analysis is available online as an open-access paper with a CC-BY license. No other part of the paper has been changed.

Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.

Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods.

Supertertiary structure of the synaptic MAGuK scaffold proteins is conserved.

Scaffold proteins form a framework to organize signal transduction by binding multiple partners within a signaling pathway. This shapes the output of signal responses as well as providing specificity and localization. The Membrane Associated Guanylate Kinases (MAGuKs) are scaffold proteins at cellular junctions that localize cell surface receptors and link them to downstream signaling enzymes. Scaffold proteins often contain protein-binding domains that are connected in series by disordered linkers. The tertiary structure of the folded domains is well understood, but describing the dynamic inter-domain interactions (the superteritary structure) of such multidomain proteins remains a challenge to structural biology. We used 65 distance restraints from single-molecule fluorescence resonance energy transfer (smFRET) to describe the superteritary structure of the canonical MAGuK scaffold protein PSD-95. By combining multiple fluorescence techniques, the conformational dynamics of PSD-95 could be characterized across the biologically relevant timescales for protein domain motions. Relying only on a qualitative interpretation of FRET data, we were able to distinguish stable interdomain interactions from freely orienting domains. This revealed that the five domains in PSD-95 partitioned into two independent supramodules: PDZ1-PDZ2 and PDZ3-SH3-GuK. We used our smFRET data for hybrid structural refinement to model the PDZ3-SH3-GuK supramodule and include explicit dye simulations to provide complete characterization of potential uncertainties inherent to quantitative interpretation of FRET as distance. Comparative structural analysis of synaptic MAGuK homologues showed a conservation of this supertertiary structure. Our approach represents a general solution to describing the supertertiary structure of multidomain proteins.

Design of a minimal di-nickel hydrogenase peptide.

Ancestral metabolic processes involve the reversible oxidation of molecular hydrogen by hydrogenase. Extant hydrogenase enzymes are complex, comprising hundreds of amino acids and multiple cofactors. We designed a 13-amino acid nickel-binding peptide capable of robustly producing molecular hydrogen from protons under a wide variety of conditions. The peptide forms a di-nickel cluster structurally analogous to a Ni-Fe cluster in [NiFe] hydrogenase and the Ni-Ni cluster in acetyl-CoA synthase, two ancient, extant proteins central to metabolism. These experimental results demonstrate that modern enzymes, despite their enormous complexity, likely evolved from simple peptide precursors on early Earth.

Computational design of a sensitive, selective phase-changing sensor protein for the VX nerve agent.

The VX nerve agent is one of the deadliest chemical warfare agents. Specific, sensitive, real-time detection methods for this neurotoxin have not been reported. The creation of proteins that use biological recognition to fulfill these requirements using directed evolution or library screening methods has been hampered because its toxicity makes laboratory experimentation extraordinarily expensive. A pair of VX-binding proteins were designed using a supercharged scaffold that couples a large-scale phase change from unstructured to folded upon ligand binding, enabling fully internal binding sites that present the maximum surface area possible for high affinity and specificity in target recognition. Binding site residues were chosen using a new distributed evolutionary algorithm implementation in protCAD. Both designs detect VX at parts per billion concentrations with high specificity. Computational design of fully buried molecular recognition sites, in combination with supercharged phase-changing chassis proteins, enables the ready development of a new generation of small-molecule biosensors.

Optimizing methods to recover absolute FRET efficiency from immobilized single molecules.

Microscopy-based fluorescence resonance energy transfer (FRET) experiments measure donor and acceptor intensities by isolating these signals with a series of optical elements. Because this filtering discards portions of the spectrum, the observed FRET efficiency is dependent on the set of filters in use. Similarly, observed FRET efficiency is also affected by differences in fluorophore quantum yield. Recovering the absolute FRET efficiency requires normalization for these effects to account for differences between the donor and acceptor fluorophores in their quantum yield and detection efficiency. Without this correction, FRET is consistent across multiple experiments only if the photophysical and instrument properties remain unchanged. Here we present what is, to our knowledge, the first systematic study of methods to recover the true FRET efficiency using DNA rulers with known fluorophore separations. We varied optical elements to purposefully alter observed FRET and examined protein samples to achieve quantum yields distinct from those in the DNA samples. Correction for calculated instrument transmission reduced FRET deviations, which can facilitate comparison of results from different instruments. Empirical normalization was more effective but required significant effort. Normalization based on single-molecule photobleaching was the most effective depending on how it is applied. Surprisingly, per-molecule gamma-normalization reduced the peak width in the DNA FRET distribution because anomalous gamma-values correspond to FRET outliers. Thus, molecule-to-molecule variation in gamma has an unrecognized effect on the FRET distribution that must be considered to extract information on sample dynamics from the distribution width.

Reconstitution of multivalent PDZ domain binding to the scaffold protein PSD-95 reveals ternary-complex specificity of combinatorial inhibition.

Multidomain scaffold proteins serve as hubs in the signal transduction network. By physically colocalizing sequential steps in a transduction pathway, scaffolds catalyze and direct incoming signals. Much is known about binary interactions with individual domains, but it is unknown whether "scaffolding activity" is predictable from pairwise affinities. Here, we characterized multivalent binding to PSD-95, a scaffold protein containing three PDZ domains connected in series by disordered linkers. We used single molecule fluorescence to watch soluble PSD-95 recruit diffusing proteins to a surface-attached receptor cytoplasmic domain. Different ternary complexes showed unique concentration dependence for scaffolding despite similar pairwise affinity. The concentration dependence of scaffolding activity was not predictable based on binary interactions. PSD-95 did not stabilize specific complexes, but rather increased the frequency of transient binding events. Our results suggest that PSD-95 maintains a loosely connected pleomorphic ensemble rather than forming a stereospecific complex containing all components.

Domain orientation in the N-Terminal PDZ tandem from PSD-95 is maintained in the full-length protein.

Tandem PDZ domains have been suggested to form structurally independent supramodules. However, dissimilarity between crystallography and NMR models emphasize their malleable conformation. Studies in full-length scaffold proteins are needed to examine the effect of tertiary interactions within their native context. Using single-molecule fluorescence to characterize the N-terminal PDZ tandem in PSD-95, we provide the first direct evidence that PDZ tandems can be structurally independent within a full-length scaffold protein. Molecular refinement using our data converged on a single structure with an antiparallel alignment of the ligand-binding sites. Devoid of interaction partners, single-molecule conditions captured PSD-95 in its unbound, ground state. Interactions between PDZ domains could not be detected while fluctuation correlation spectroscopy showed that other conformations are dynamically sampled. We conclude that ultra-weak interactions stabilize the conformation providing a "low-relief" energy landscape that allows the domain orientation to be flipped by environmental interactions.

Beyond the random coil: stochastic conformational switching in intrinsically disordered proteins.

Intrinsically disordered proteins (IDPs) participate in critical cellular functions that exploit the flexibility and rapid conformational fluctuations of their native state. Limited information about the native state of IDPs can be gained by the averaging over many heterogeneous molecules that is unavoidable in ensemble approaches. We used single molecule fluorescence to characterize native state conformational dynamics in five synaptic proteins confirmed to be disordered by other techniques. For three of the proteins, SNAP-25, synaptobrevin and complexin, their conformational dynamics could be described with a simple semiflexible polymer model. Surprisingly, two proteins, neuroligin and the NMDAR-2B glutamate receptor, were observed to stochastically switch among distinct conformational states despite the fact that they appeared intrinsically disordered by other measures. The hop-like intramolecular diffusion found in these proteins is suggested to define a class of functionality previously unrecognized for IDPs.