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.
16Protein folding can go wrong in vivo and in vitro, with significant consequences for the living cell and 17 the pharmaceutical industry, respectively. Here we propose a general design principle for constructing 18 small peptide-based protein-specific folding modifiers. We construct a 'xenonucleus', which is a pre-19folded peptide that resembles the folding nucleus of a protein, and demonstrate its activity on the 20 folding of ubiquitin. Using stopped-flow kinetics, NMR spectroscopy, Förster Resonance Energy 21 transfer, single-molecule force measurements, and molecular dynamics simulations, we show that the 22 ubiquitin xenonucleus can act as an effective decoy for the native folding nucleus. It can make the 23 refolding faster by 33 ± 5% at 3 M GdnHCl. In principle, our approach provides a general method for 24 constructing specific, genetically encodable, folding modifiers for any protein which has a well-defined 25 contiguous folding nucleus. 26 27 flow kinetics, time resolved Forster Resonance Energy transfer, NMR, and coarse grain simulation 70 studies show that kinetics of ubiquitin folding can indeed be made faster by a suitable xenonucleus 71 interacting at the appropriate site. 72 73 Results: 74 75Refolding kinetics: Ubiquitin has been widely studied for its thermodynamic and mechanical stability. 76 Previous mutational studies on Ubiquitin, especially using the F45W mutant, has established 77 tryptophan fluorescence as a valuable probe for studying its folding. Here, we measured the refolding 78 kinetics of Ubiquitin (F45W) in presence and in absence of a 19 residue nucleus mimic. It has the amino 79 acid sequence C-MQIFVKTLTGKTITLEV-C, which is the same as residues 1 to 17 of Ubiquitin, except for 80 the terminal cysteines. It has a disulphide bridge between the termini, and will henceforth be called 81 the 'stapled xenonucleus'. We use a three syringe stopped-flow fluorescence instrument (SFM300, 82Biologic, see Methods) to change the GdnHCl concentration from 4.25 M to a series of lower 83 concentrations in less than 6 ms. We measure the change in fluorescence intensity of the Trp residue 84as a function of time as a reporter for the progress of folding (or unfolding). The unfolded state of 85 ubiquitin F45W has a higher quantum yield and has an emission maximum at 360 nm, but when it 86 goes to the folded form, the emission blue-shifts to 340 nm. The quantum yield of fluorescence also 87 goes down (it is speculated that a backbone carboxyl oxygen may be quenching the fluorescence) 57,58 . 88Hence, as folding proceeds, the overall fluorescence signal at 360 nm decreases. We have plotted the 89 change in the fluorescence intensity as a function of time for ubiquitin F45W, with (Figure 1 (A), red) 90 and without (Figure 1 (A), blue) the xenonucleus. The data are fitted with a two-component 91 exponential decay function: 92 102 Refolding kinetics as a function of the xenonucleus concentration: We also perform the refolding 103 experiment as a function of the concentration of the xenonucleus peptide...
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.
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.
Small lipid vesicles (with diameter ≤ 100nm) 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 %, τr increases 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 1.3x and 2.6x, respectively. Our results demonstrate that rotational FCS can directly measure the viscosity of highly curved membranes with higher sensitivity and 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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.