Force-dependent chemical reactions

1. Force-dependent chemical reactions F

2. 10 nm The Experiment Mechanical unfolding exposes the buried disulfide to nucleophilic attack Extension Time 4 nm F

3. 10 nm The Experiment 11 nm Extension Time 15 nm F

4. 10 nm The Experiment Extension Time 15 nm F

5. 10 nm The Experiment 15 nm Extension Time 30 nm F

6. 10 nm The Experiment Extension Time 30 nm F

7. Thermocouple Piezo Heatsink Peltier Liquid Cell

8. Temperature controlled measurements of the rate of reduction by TCEP 35 C 300 pN 250 pN 45 C 25 C 200 pN 150 pN 15 C 100 pN 5 C T = 15 C F = 250 pN

9. r045 r035 r025 r015 r05 Force and temperature dependency of TCEP reduction 45 C 35 C 25 C 15 C 5 C

10. Arrhenius term describes temperature dependency of TCEP reduction Ea = 35 ± 4 kJ/mol r045 r035 r025 r015 r05 A  8 × 107 s-1

11. A force driven reactivity switch: reduction by Hydroxide

12. Hydroxide concentration controls the reduction rate Force (pN) Dx2~0.1 Å Dx1~0.5 Å [OH-] Force (pN)

13. I2724-55

14. I2728-54

15. Reactivity switch is present in all S-S constructs

16. High mechanical forces cause a shift in the ground state of the disulfide bond F χ = 180° χ = 84.9° F

17. Probing the chemistry of thioredoxin catalysis with force Chemistry: SN2 attack of thiolate anion on disulfide Arne Holmgren ; Eur. J. Biochem, 1968, 6:475-484 Arne Holmgren et al; PNAS, 1975, 72:2305–2309

18. cantilever tip polyprotein Trx exposed disulfide bond 10 nm gold substrate

19. Identifying disulfide reduction by single Trx enzymes Trx= 0 Trx= 8mM

20. The rate of reduction is both force and [Trx] dependent [Trx]= 8 mM F= 100 pN

21. Trx catalysis has a bimodal force dependency • two pathways for Trx reduction (I & II) k01 = k01(0) [Trx] k12 = k12(0) exp(FΔx/kBT)) k02 = k02(0) [Trx] exp(FΔx/kBT))

22. 35 C 25 C 15 C

23. Nucleophilic attacks are directional Reorientation of the stretched bond is required to have all three S atoms in a line

24. Rotation of the substrate bond is probabilistic Pmd()

25. The P34H groove mutation • reduced k01 unchangedΔx12 and Δx02

26. Human and E coli thioredoxinsare distinguished by path II The sequence identity between E. coli Trx and Human Trx is 25%

27. Three distinct chemical mechanisms of reduction

28. ~15 nm ~11 nm

31. Groove deepens

32. Evolution of chemical mechanisms in thioredoxin enzymes Node 205 Extant LUCA