Rational design of protein-specific folding modifiers

Abstract

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...

Interdiction of Protein Folding for Therapeutic Drug Development in SARS CoV-2

“…For example, ceftriaxone binds specifically to the C-terminal region of the intrinsically disordered protein α-synuclein, generally understood to be involved in triggering Parkinson’s disease, and has shown therapeutic potential. Also, recent experimental results suggest that the folding kinetics of proteins with two-state transitions can be modulated by the employment of suitable peptides that mimic specific segments of the protein chain . Although the purpose of the experiment was the opposite of the one sought for here, as the goal was to speed up the folding transition, the results provide general support for the contention presented that specific peptide molecules can interfere and alter the folding kinetics of globular proteins.…”

“…Also, recent experimental results suggest that the folding kinetics of proteins with two-state transitions can be modulated by the employment of suitable peptides that mimic specific segments of the protein chain. 64 Although the purpose of the experiment was the opposite of the one sought for here, as the goal was to speed up the folding transition, the results provide general support for the contention presented that specific peptide molecules can interfere and alter the folding kinetics of globular proteins. The proposed therapeutic strategy is depicted in Figure 2 .…”

The Promise of Mutation Resistant Drugs for SARS-CoV-2 That Interdict in the Folding of the Spike Protein Receptor Binding Domain

“…The concepts presented in this paper are similar to earlier proposals to develop drugs that might be therapeutic by interfering with the folding process [21,22]. There has been a growing interest in the possibility of modulating the folding process through the employment of specific molecules, and theoretical and experimental results have been recently reported that support the view that targeting folding dynamics is a promising path for therapeutic development [23][24][25]. The main difference between the proposal presented here and other approaches is that the FITR strategy aims to interdict in the folding process while the protein is still completely unfolded [13].…”

Computational Design of Stapled Peptide Inhibitor against SARS-CoV-2 Receptor Binding Domain

“…By altering the length, attachment point, and chemical nature of the stapling agents, the design of stapled peptides can be tuned to achieve desirable affinity and target specificity. 18,24,25 Few experimental and computational reports have checked the stability of stapled ACE2 and their binding with RBD. 21,26,27 However, a rationale to combine stapling agents chemical nature and length with their binding affinity is still lacking which is crucial for the future design of stapled peptides.…”

“…The introduction of stapling agents to positions 28, 32, 36, and 40 improves the binding considerably. In the case of ACE2_al-al, the fluctuation of the N-terminal end of the peptide(21)(22)(23)(24)(25)(26)(27)(28)(29)(30) has significantly decreased compared to ACE2_wt though the C-terminal end(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45) shows deviation from strong association with RBD surface (Movie S2). This has been reflected in the RMSF (Figure5C) and fraction helicity (Figure6C) as well.…”