Amyloid diagnostics: probing protein aggregation and conformation with ultrasensitive fluorescence detection

Abhyankar, Rajiv; Sahoo, Bankanidhi; Singh, Niraj Kumar; Meijer, Linda M.; Sarkar, Bidyut; Das, Anand Kant; Nag, Suman; Chandrakesan, Muralidharan; Bhowmik, Debanjan; Dandekar, Sucheta; Maiti, Sudipta

doi:10.1117/12.909829

Cited by 4 publications

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“…We used FCS for measuring the binding affinity of the peptides with the RBD of the SARS-CoV-2 virus as well as for measuring the hydrodynamic radii (R h ) of the peptides. These measurements were performed using a home-built FCS instrument (17,18). Briefly, a 488-nm laser beam was expanded and collimated using a 1:4 telescope set up before focusing into the sample using an apochromatic 60Â water immersion objective with the numerical aperture of 1.2 (Olympus, Center Valley, PA).…”

Section: Fcs Measurementsmentioning

confidence: 99%

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

Das

Vishvakarma

Dey

et al. 2021

Biophysical Journal

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The entry of the SARS-CoV2 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 residues 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 (RBD) 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. While SBP1 shows good solubility (solubility > 0.8 mM), CD spectroscopy shows that it is mostly disordered with some anti-parallel beta-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. While the mutant peptide (SBP1 mod ) 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 also due to the heretofore unrecognized problem of oligomerization.

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Section: Fcs Measurementsmentioning

confidence: 99%

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

Das

Vishvakarma

Dey

et al. 2021

Biophysical Journal

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“…FCS is a proven technique for measuring equilibrium fluctuations. − It is capable of following fast dynamics on the nanosecond to microsecond time scale even at very low concentrations. − Spontaneous molecular fluctuations of unfolded proteins and IDPs have been measured using FCS. − This temporal regime is difficult to access by the single-molecule Förster resonance energy transfer (smFRET) technique and falls in the nuclear magnetic resonance (NMR)-inaccessible time window. , We note that the changes in the donor lifetime may reflect ligand interactions, but only if the average distance between the FRET pairs changes because of such interactions. , This is not a requirement for fluctuation-based assays, as explained below. Other techniques, such as triplet–triplet energy transfer or tryptophan quenching, can also monitor the dynamics on a submicrosecond time scale but have relatively low sensitivity. , The sensitivity of FCS allows it to probe low concentrations of the peptides, which makes it rather advantageous especially for IDPs, as they are typically aggregation prone even in the micromolar concentration range .…”

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confidence: 99%

“…Instrumental Setup. We have constructed the crosscorrelation FCS instrument in house, 31 and details are given in the Supporting Information. Briefly, a 532 nm laser beam excites the sample in a confocal geometry.…”

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Spontaneous Fluctuations Can Guide Drug Design Strategies for Structurally Disordered Proteins

et al. 2018

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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.

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Biophotonics in Disease Diagnosis and Therapy

Biswas

Gavra

Das

et al. 2019

Biomedical Engineering and Its Applications in Healthcare

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Amyloid diagnostics: probing protein aggregation and conformation with ultrasensitive fluorescence detection

Cited by 4 publications

References 0 publications

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

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

Spontaneous Fluctuations Can Guide Drug Design Strategies for Structurally Disordered Proteins

Biophotonics in Disease Diagnosis and Therapy

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