F

Force-dependent Force-dependent chemical reactionschemical reactions

F

4 nm

Time

The Experiment

10 nm

Extension

Mechanical unfolding exposes the buried disulfide to nucleophilic attack

F

15 nm

Time

The Experiment

10 nm

Extension 11 nm

F

15 nm

Time

The Experiment

10 nm

Extension

F

30 nm

Time

The Experiment

10 nm

Extension15 nm

F

30 nm

Time

The Experiment

10 nm

Extension

Thermocouple

Piezo

Heatsink

LiquidCell

Peltier

1.0

0.8

0.6

0.4

0.2

0.0

10s86420Time (sec)

Pr(t

)

100 pN

150 pN

200 pN

250 pN300 pN

T = 15 C

1.0

0.8

0.6

0.4

0.2

0.0

3.0s2.52.01.51.00.50.0Time (sec)

Pr(t

)

5 C

15 C

25 C

35 C

45 C

F = 250 pN

Temperature controlled measurements of the rate of Temperature controlled measurements of the rate of reduction by TCEPreduction by TCEP

2

4

68

0.1

2

4

68

1

2

4

300250200150100500

Force (pN)

Rat

e (s

ec-1

)

45 C35 C25 C15 C 5 C

r05

r015

r025

r035

r045

Tk

xFE

B

ra

eTCEPAr

][

Tk

E

B

a

eTCEPAr

][0

0F

Force and temperature dependency of TCEP reductionForce and temperature dependency of TCEP reduction

0.18

0.16

0.14

0.12

0.10

0.08

0.06

0.04

0.02

320310300290280

Temperature (K)

r 0 (sec

-1)

-4.5

-4.0

-3.5

-3.0

-2.5

-2.0

-1.5

-440x10-6

-430 -420 -410 -400 -390 -380 -370

-1/RT (J-1

)

ln (r

0)

r045

r035

r025

r015

r05

Tk

E

B

a

eTCEPAr

][0

Tk

ETCEPAr

B

a ])[ln(ln 0

Ea = 35 ± 4 kJ/mol

A 8 × 107 s-1

Arrhenius term describes temperature dependency of Arrhenius term describes temperature dependency of TCEP reductionTCEP reduction

A force driven reactivity switch: reduction by HydroxideA force driven reactivity switch: reduction by Hydroxide

[OH-]

Force (pN)

Force (pN)

x1~0.5 Å

x2~0.1 Å

Hydroxide concentration controls the reduction rateHydroxide concentration controls the reduction rate

Reactivity switch is present in all S-S constructsReactivity switch is present in all S-S constructs

F

F

χ = 84.9°χ = 180°

High mechanical forces cause a shift in the ground High mechanical forces cause a shift in the ground state state

of the disulfide bondof the disulfide bond

Chemistry: SN2 attack of thiolate anion on disulfide

Probing the chemistry of thioredoxin catalysis Probing the chemistry of thioredoxin catalysis with forcewith force

Arne Holmgren et al; PNAS, 1975, 72:2305–2309

Arne Holmgren ; Eur. J. Biochem, 1968, 6:475-484

cantilever tip

polyprotein

exposed disulfide

bond

gold substrate10 nm

Trx

Trx= 0

Trx= 8M

Identifying disulfide reduction by single Trx Identifying disulfide reduction by single Trx enzymesenzymes

The rate of reduction is both force and [Trx] dependent

[Trx]= 8 M F= 100 pN

• two pathways for Trx reduction (I & II)

Trx catalysis has a bimodal force dependencyTrx catalysis has a bimodal force dependency

k01 = k01(0) [Trx]k12 = k12(0) exp(FΔx/kBT))k02 = k02(0) [Trx] exp(FΔx/kBT))

0.8

0.6

0.4

0.2

6005004003002001000

Force (pN)

Ra

te (

sec-1

)

35 C25 C15 C

Nucleophilic attacks

are directional

Reorientation of the

stretched bond is required to have all three S atoms in a

line

Pmd()

Rotation of the substrate bond is probabilistic

The P34H groove The P34H groove mutation mutation

• reduced k01

unchanged Δx12 and Δx02

Human and E coli thioredoxinsare distinguished by path II

6

5

4

3

2

1

r (s

-1)

6005004003002001000

Force (pN)

[hTrx] = 10 uM [E.coli Trx] = 8 uM

The sequence identity between E. coli Trx and Human Trx is 25%

Three distinct chemical mechanisms of reduction

~11 nm~15 nm

Groove deepens

Evolution of chemical mechanisms in thioredoxin enzymes

LUCA Extant

Node 205