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...
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