Biophysical properties of the isolated spike protein binding helix of human ACE2.

The entry of the severe acute respiratory syndrome coronavirus 2 virus in human cells is mediated by the binding of its surface spike protein to the human angiotensin-converting enzyme 2 (ACE2) receptor. A 23-residue long helical segment (SBP1) at the binding interface of human ACE2 interacts with viral spike protein and therefore has generated considerable interest as a recognition element for virus detection. Unfortunately, emerging reports indicate that the affinity of SBP1 to the receptor-binding domain of the spike protein is much lower than that of the ACE2 receptor itself. Here, we examine the biophysical properties of SBP1 to reveal factors leading to its low affinity for the spike protein. Whereas SBP1 shows good solubility (solubility > 0.8 mM), circular dichroism spectroscopy shows that it is mostly disordered with some antiparallel ß-sheet content and no helicity. The helicity is substantial (>20%) only upon adding high concentrations (≥20% v/v) of 2,2,2-trifluoroethanol, a helix promoter. Fluorescence correlation spectroscopy and single-molecule photobleaching studies show that the peptide oligomerizes at concentrations >50 nM. We hypothesized that mutating the hydrophobic residues (F28, F32, and F40) of SBP1, which do not directly interact with the spike protein, to alanine would reduce peptide oligomerization without affecting its spike binding affinity. Whereas the mutant peptide (SBP1mod) shows substantially reduced oligomerization propensity, it does not show improved helicity. Our study shows that the failure of efforts, so far, to produce a short SBP1 mimic with a high affinity for the spike protein is not only due to the lack of helicity but is also due to the heretofore unrecognized problem of oligomerization.

Rational Design of Protein-Specific Folding Modifiers.

Protein-folding can go wrong in vivo and in vitro, with significant consequences for the living organism and the pharmaceutical industry, respectively. Here we propose a design principle for small-peptide-based protein-specific folding modifiers. The principle is based on constructing a "xenonucleus", which is a prefolded peptide that mimics the folding nucleus of a protein. Using stopped-flow kinetics, NMR spectroscopy, Förster resonance energy transfer, single-molecule force measurements, and molecular dynamics simulations, we demonstrate that a xenonucleus can make the refolding of ubiquitin faster by 33 ± 5%, while variants of the same peptide have little or no effect. Our approach provides a novel method for constructing specific, genetically encodable folding catalysts for suitable proteins that have a well-defined contiguous folding nucleus.

Spontaneous Fluctuations Can Guide Drug Design Strategies for Structurally Disordered Proteins.

Structure-based "rational" drug design strategies fail for diseases associated with intrinsically disordered proteins (IDPs). However, structural disorder allows large-amplitude spontaneous intramolecular dynamics in a protein. We demonstrate a method that exploits this dynamics to provide quantitative information about the degree of interaction of an IDP with other molecules. A candidate ligand molecule may not bind strongly, but even momentary interactions can be expected to perturb the fluctuations. We measure the amplitude and frequency of the equilibrium fluctuations of fluorescently labeled small oligomers of hIAPP (an IDP associated with type II diabetes) in a physiological solution, using nanosecond fluorescence cross-correlation spectroscopy. We show that the interterminal distance fluctuates at a characteristic time scale of 134 ± 10 ns, and 6.4 ± 0.2% of the population is in the "closed" (quenched) state at equilibrium. These fluctuations are affected in a dose-dependent manner by a series of small molecules known to reduce the toxicity of various amyloid peptides. The degree of interaction increases in the following order: resveratrol < epicatechin ∼ quercetin < Congo red < epigallocatechin 3-gallate. Such ordering can provide a direction for exploring the chemical space for finding stronger-binding ligands. We test the biological relevance of these measurements by measuring the effect of these molecules on the affinity of hIAPP for lipid vesicles and cell membranes. We find that the ability of a molecule to modulate intramolecular fluctuations correlates well with its ability to lower membrane affinity. We conclude that structural disorder may provide new avenues for rational drug design for IDPs.

Serotonin Promotes Vesicular Association and Fusion by Modifying Lipid Bilayers.

The primary event in chemical neurotransmission involves the fusion of a membrane-limited vesicle at the plasma membrane and the subsequent release of its chemical neurotransmitter cargo. The cargo itself is not known to have any effect on the fusion event. However, amphiphilic monoamine neurotransmitters (e.g., serotonin and dopamine) are known to strongly interact with lipid bilayers and to affect their mechanical properties, which can in principle impact membrane-mediated processes. Here, we probe whether serotonin can enhance the association and fusion of artificial lipid vesicles in vitro. We employ fluorescence correlation spectroscopy and total internal reflection fluorescence microscopy to measure the attachment and fusion of vesicles whose lipid compositions mimic the major lipid components of synaptic vesicles. We find that the association between vesicles and supported lipid bilayers is strongly enhanced in a serotonin dose-dependent manner, and this drives an increase in the rate of spontaneous fusion. Molecular dynamics simulations and fluorescence spectroscopy data show that serotonin insertion increases the water content of the hydrophobic part of the bilayer. This suggests that the enhanced membrane association is likely driven by an energetically favorable drying transition. Other monoamines, such as dopamine and norepinephrine, but not other related species, such as tryptophan, show similar effects on membrane association. Our results reveal a lipid bilayer-mediated mechanism by which monoamines can themselves modulate vesicle fusion, potentially adding to the control toolbox for the tightly regulated process of neurotransmission in vivo.

A Toxicogenic Interaction between Intracellular Amyloid-ß and Apolipoprotein-E.

Alzheimer's disease (AD) is associated with the aggregation of amyloid ß (Aß) and tau proteins. Why ApoE variants are significant genetic risk factors remains a major unsolved puzzle in understanding AD, although intracellular interactions with ApoE are suspected to play a role. Here, we show that specific changes in the fluorescence lifetime of fluorescently tagged small Aß oligomers in rat brain cells correlate with the cellular ApoE content. An inhibitor of the Aß-ApoE interaction suppresses these changes and concomitantly reduces Aß toxicity in a dose-dependent manner. Single-molecule techniques show changes both in the conformation and in the stoichiometry of the oligomers. Neural stem cells derived from hiPSCs of Alzheimer's patients also exhibit these fluorescence lifetime changes. We infer that intracellular interaction with ApoE modifies the N-terminus of the Aß oligomers, inducing changes in their stoichiometry, membrane affinity, and toxicity. These changes can be directly imaged in live cells and can potentially be used as a rapid and quantitative cellular assay for AD drug discovery.

Measuring the Size and Spontaneous Fluctuations of Amyloid Aggregates with Fluorescence Correlation Spectroscopy.

Bacterial amyloids decorate the cell surface of many bacteria by forming functional amyloid fibers. These amyloids have structural and biochemical similarities with many disease-related amyloids in eukaryotes. Amyloid aggregation starts at the individual monomer level, and the end product is the amyloid fibril. The process of amyloid aggregation involves a continuous increase of the aggregate size, and therefore size is a critical parameter to measure in aggregation experiments. Also, our understanding of the aggregation process, and our ability to design interventions, can benefit from a measurement of the conformational dynamics of proteins undergoing aggregation. Fluorescence correlation spectroscopy (FCS) is perhaps the most sensitive and rapid technique available currently for this purpose. It can measure the average size and the size distribution of molecules and aggregates down to sub-nm length scales and can also measure fast nanosecond time-scale conformational dynamics, all in an equilibrium solution. FCS achieves this by measuring the fluorescence intensity fluctuations of freely diffusing protein molecules in an optically defined microscopic probe volume in a solution. Here, we present a set of instructions for effectively measuring the size and dynamics of amyloid aggregates with FCS.

The effect of cholesterol on highly curved membranes measured by nanosecond Fluorescence Correlation Spectroscopy.

Small lipid vesicles (with diameter ≤100 nm) with their highly curved membranes comprise a special class of biological lipid bilayers. The mechanical properties of such membranes are critical for their function, e.g. exocytosis. Cholesterol is a near-universal regulator of membrane properties in animal cells. Yet measurements of the effect of cholesterol on the mechanical properties of membranes have remained challenging, and the interpretation of such measurements has remained a matter of debate. Here we show that nanosecond fluorescence correlation spectroscopy (FCS) can directly measure the ns-microsecond rotational correlation time (τr) of a lipid probe in high curvature vesicles with extraordinary sensitivity. Using a home-built 4-Pi fluorescence cross-correlation spectrometer containing polarization-modulating elements, we measure the rotational correlation time (τr) of Nile Red in neurotransmitter vesicle mimics. As the cholesterol mole fraction increases from 0 to 50%,τrincreases from 17 ± 1 to 112 ± 12 ns, indicating a viscosity change of nearly a factor of 7. These measurements are corroborated by solid-state NMR results, which show that the order parameter of the lipid acyl chains increases by about 50% for the same change in cholesterol concentration. Additionally, we measured the spectral parameters of polarity-sensitive fluorescence dyes, which provide an indirect measure of viscosity. The green/red ratio of Nile Red and the generalized polarization of Laurdan show consistent increases of 1.3× and 2.6×, respectively. Our results demonstrate that rotational FCS can directly measure the viscosity of highly curved membranes with higher sensitivity and a wider dynamic range compared to other conventional techniques. Significantly, we observe that the viscosity of neurotransmitter vesicle mimics is remarkably sensitive to their cholesterol content.

Single Molecule Measurements of the Accessibility of Molecular Surfaces.

An important measure of the conformation of protein molecules is the degree of surface exposure of its specific segments. However, this is hard to measure at the level of individual molecules. Here, we combine single molecule photobleaching (smPB, which resolves individual photobleaching steps of single molecules) and fluorescence quenching techniques to measure the accessibility of individual fluorescently labeled protein molecules to quencher molecules in solution. A quencher can reduce the time a fluorophore spends in the excited state, increasing its photostability under continuous irradiation. Consequently, the photo-bleaching step length would increase, providing a measure for the accessibility of the fluorophore to the solvent. We demonstrate the method by measuring the bleaching step-length increase in a lipid, and also in a lipid-anchored peptide (both labelled with rhodamine-B and attached to supported lipid bilayers). The fluorophores in both molecules are expected to be solvent-exposed. They show a near two-fold increase in the step length upon incubation with 5 mM tryptophan (a quencher of rhodamine-B), validating our approach. A population distribution plot of step lengths before and after addition of tryptophan show that the increase is not always homogenous. Indeed there are different species present with differential levels of exposure. We then apply this technique to determine the solvent exposure of membrane-attached N-terminus labelled amylin (h-IAPP, an amyloid associated with Type II diabetes) whose interaction with lipid bilayers is poorly understood. hIAPP shows a much smaller increase of the step length, signifying a lower level of solvent exposure of its N-terminus. Analysis of results from individual molecules and step length distribution reveal that there are at least two different conformers of amylin in the lipid bilayer. Our results show that our method ("Q-SLIP", Quenching-induced Step Length increase in Photobleaching) provides a simple route to probe the conformational states of membrane proteins at a single molecule level.