Rapid Kinetics with IR Protein folding examplesRapid Kinetics with IR Protein folding examples. Time...

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Rapid Kinetics with IR Protein folding examples

Transcript of Rapid Kinetics with IR Protein folding examplesRapid Kinetics with IR Protein folding examples. Time...

Page 1: Rapid Kinetics with IR Protein folding examplesRapid Kinetics with IR Protein folding examples. Time dependent data with FTIR Stop-flow methods - msec limits so far Continuous, micro-flow

Rapid Kinetics with IRProtein folding examples

Page 2: Rapid Kinetics with IR Protein folding examplesRapid Kinetics with IR Protein folding examples. Time dependent data with FTIR Stop-flow methods - msec limits so far Continuous, micro-flow

Time dependent data with FTIR

Stop-flow methods - msec limits so far

Continuous, micro-flow methods - < 100 µsec

Rapid scan FT-IR - msec

Multichannel laser Raman, faster - µsec

T-jump and Flash photolysis -nsec time scalesusing step scan methods

Page 3: Rapid Kinetics with IR Protein folding examplesRapid Kinetics with IR Protein folding examples. Time dependent data with FTIR Stop-flow methods - msec limits so far Continuous, micro-flow

6 β β sheet

, 2 )

Tyr97

Tyr25

Tyr92

H1

H3H2

Tyr76

Tyr115

Tyr73

• 124 amino acid residues, 1 domain, MW= 13.7 KDa

• 3 α-helices

• 6 β-strands in an AP β-sheet

• 6 Tyr residues (no Trp), 4 Pro residues (2 cis, 2 trans)

Ribonuclease A combined uv-CD and FTIR study

Simona Stelea, Prot Sci 2001

Page 4: Rapid Kinetics with IR Protein folding examplesRapid Kinetics with IR Protein folding examples. Time dependent data with FTIR Stop-flow methods - msec limits so far Continuous, micro-flow

Scheme of Stop Flow

Spacer

Mixer

GasketCell Window

Cell WindowFront Plate

Cell nest

Luer Plug

To Cell

Rea

gent

Prot

ein

Cell and mixer blowout

Syringe drive system

Mix protein and perturbant rapidly to get new state, follow spectra

Backplate

Page 5: Rapid Kinetics with IR Protein folding examplesRapid Kinetics with IR Protein folding examples. Time dependent data with FTIR Stop-flow methods - msec limits so far Continuous, micro-flow

Refolding of Ribonuclease A by FTIR

Wavenumber (cm-1)

15501600165017001750

log

(Si/S

f)

-0.03

-0.02

-0.01

0.00

0.01

0.021660 cm-1

loss of random coil

1630 cm-1

gain of sheet

time (s)

0 5 10 15 20

Peak

Inte

nsity

-0.03

-0.02

-0.01

0.00

0.01

1632 cm-1 (sheet)k = 0.156 s-1

1660 cm-1 (random coil)k = 0.342 s-1

Inverse T-jump: Refolding initiated by injecting Ribo A stored in syringe at 80 °C into IR cell at 25 °C

Sheet refolding 2x slower than loss of coil

One single beam spectrum (IF scan) is collected for each time point.

Time resolution = 50 ms. IR resolution separate coil decrease sheet fold.

Page 6: Rapid Kinetics with IR Protein folding examplesRapid Kinetics with IR Protein folding examples. Time dependent data with FTIR Stop-flow methods - msec limits so far Continuous, micro-flow

Austin, GerwertPNAS 98 2001, 6646

%TFE

Side view: 2D Fluid dynamics simulation

Top view: green: inlet channels, red: 8µm deep outlet channel

Continuous flow mixer

Page 7: Rapid Kinetics with IR Protein folding examplesRapid Kinetics with IR Protein folding examples. Time dependent data with FTIR Stop-flow methods - msec limits so far Continuous, micro-flow

Lifetimes of intermediates in the β-sheet to α−helixtransition of β-lactoglobulinusing diffusional IR mixer

E. Kauffmann, N. C. Darntont, R. H. Austin, C. Batts, and K. Gerwert

PNAS 2001 98 6646-6649

a) Spectra along channel: 1.1,3.4,5.7,10.2,21.6,103 ms

b) 2nd deriv. & 3-state fitc) 3 basic spectra derivedd) Time course of 3 states

Page 8: Rapid Kinetics with IR Protein folding examplesRapid Kinetics with IR Protein folding examples. Time dependent data with FTIR Stop-flow methods - msec limits so far Continuous, micro-flow

Lipid-induced Conformational Transition of β-Lactoglobulin:

Equilibrium and Kinetic StudiesGlobular protein with 9-stranded sheet

(flattened β-barrel) and one helical segmentTerminal segments have high helical propensity

Good model for β-to-α conversion

Binding to lipid vesicle acts as perturbation—cell modelToo complex to waste on a single technique!

Xiuqi Zhang, Ning Ge,TAK Biochemistry 2006/2007

Page 9: Rapid Kinetics with IR Protein folding examplesRapid Kinetics with IR Protein folding examples. Time dependent data with FTIR Stop-flow methods - msec limits so far Continuous, micro-flow

Lipid-induced Conformational Transition of β-LactoglobulinIntroduction β→α transition Driving force Membrane insertion

Native state: β-sheet dominant, but high helical propensity.Model: intramolecular β↔α transition pathway as opposed to folding pathways from a denatured state.

β-lactoglobulin:

Page 10: Rapid Kinetics with IR Protein folding examplesRapid Kinetics with IR Protein folding examples. Time dependent data with FTIR Stop-flow methods - msec limits so far Continuous, micro-flow

Lipid-induced Conformational Transition of β-LactoglobulinIntroduction β→α transition Driving force Membrane insertion

1. DMPG-dependent β→α transition at pH 6.8

190 200 210 220 230 240 250-15

-10

-5

0

5

10

15

20

25

DMPG/mM

[θ]×

10-3

/deg

.cm

2 .dm

ol-1

Wavelength/nm

0 2 4-12

-10

-8

-6

-4

A

[θ] 22

2nm×1

0-3/d

eg.c

m2 .d

mol

-1

0.0 0.1 0.2 0.3 0.4-11

-10

-9

-8

-7

-6

-5

0 1 2 3 4 5

0.1

0.2

0.3

0.4

0.5

β-Sheet

α-Helix

Unordered

Frac

tiona

l sec

onda

ry st

ruct

ure

DMPG / mM

Page 11: Rapid Kinetics with IR Protein folding examplesRapid Kinetics with IR Protein folding examples. Time dependent data with FTIR Stop-flow methods - msec limits so far Continuous, micro-flow

Introduction β→α transition Driving force Membrane insertion

1. DMPG-dependent β→α transition at pH 6.8

2. Tertiary structure change

Lipid-induced Conformational Transition of β-Lactoglobulin

300 330 360 390 420 4500

2

4

6

8

10

DMPG/mM

Fluo

resc

ence

Wavelength/nm

0.0 0.5 1.0 1.5 2.01.0

1.1

1.2

1.3

1.4

R

elat

ive

fluor

esce

nce

260 280 300 320 340

-80

-60

-40

-20

0

Near-UV CD

0mM DMPG1mM DMPG3mM DMPG9mM DMPG6M GndCl

292nm

284nm[θ

]/deg

.cm

2 .dm

ol-1

Wavelength/nm

Page 12: Rapid Kinetics with IR Protein folding examplesRapid Kinetics with IR Protein folding examples. Time dependent data with FTIR Stop-flow methods - msec limits so far Continuous, micro-flow

Dynamics--Scheme of Stopped-flow System

protein solution

Lipid vesicle solution

-add dynamics to experiment

Page 13: Rapid Kinetics with IR Protein folding examplesRapid Kinetics with IR Protein folding examples. Time dependent data with FTIR Stop-flow methods - msec limits so far Continuous, micro-flow

Kinetics for βLG in Membrane

FluorescenceCircular Dichroism

Time/s

0 5 10 15 20

Rel

ativ

e In

tens

ity

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2.0

0.15mM DOPG

0.25mM DOPG

0.5mM DOPG

1mM DOPG

2mM DOPG

Time/s

0 5 10 15 20

Ellip

ticity

(mde

g)

-50

-40

-30

-20

-10

0.15mM DMPG

0.5mM DMPG

1mM DMPG2mM DMPG5mM DMPG

N

0.5mM DMPG

CD fits single exponential, fluorescence (Fl) fits two. Rate constant for CD is slower than fast Fl kinetic

Page 14: Rapid Kinetics with IR Protein folding examplesRapid Kinetics with IR Protein folding examples. Time dependent data with FTIR Stop-flow methods - msec limits so far Continuous, micro-flow

Summary

Nw Ns Unfolding Us Insertion UmBinding

Page 15: Rapid Kinetics with IR Protein folding examplesRapid Kinetics with IR Protein folding examples. Time dependent data with FTIR Stop-flow methods - msec limits so far Continuous, micro-flow

Laser induced Temperature jump

IR pulse heats the solvent ( Raman shifted YAG to 1.9 µ for D2O)

Probe heated spot with tunable IR laser (Pb-salt diode, FTIR experiments proposed)

Fast MCT needed for ns responseRepetition rate limited by cooling back initial stateAnalysis is relaxation kinetics, krel = kf + kr

Signal average thousands of shots, single frequency (diode laser) normal method

Page 16: Rapid Kinetics with IR Protein folding examplesRapid Kinetics with IR Protein folding examples. Time dependent data with FTIR Stop-flow methods - msec limits so far Continuous, micro-flow

Callender/Dyer general T-jump setup

Generic design for T-jump, IR diode laser detection transmit to MCT

Fluorescence use cavity doubled, lots cw power

180o back scatter geom.

H2 gives 1.9 µ for D2O, CH4 ~1.5 µ H2O

Diode laser

Fast MCT

Page 17: Rapid Kinetics with IR Protein folding examplesRapid Kinetics with IR Protein folding examples. Time dependent data with FTIR Stop-flow methods - msec limits so far Continuous, micro-flow

Character of Temperature jump--timing

D2O - - -Sample ……Difference:a-1655 cm-1

b-1644 cm-1

c-1637 cm-1

d-1632 cm-1

Fit to biexpon.<10 ns160+/-60 ns

Helix example

D2O

10nsPump ∆T at 2µm focus to 300µm, 110 µm path use split cell

fast (50MHz) MCT detector, avg. 9000 shots, 10 Hz

3.0x10-5 to 4.0x10-5 (OD)/°C.µm for 1700 and 1632 cm-1

T-jump calibrated by change of D2O absorption with temperature

Page 18: Rapid Kinetics with IR Protein folding examplesRapid Kinetics with IR Protein folding examples. Time dependent data with FTIR Stop-flow methods - msec limits so far Continuous, micro-flow

Apo-Mb kinetics, T-jump Fluorescence & IR

Fluorescence

IR

Follow different processes, µs response

Fluorescence – tertiary structure unfoldIR – secondary structure - helices

Page 19: Rapid Kinetics with IR Protein folding examplesRapid Kinetics with IR Protein folding examples. Time dependent data with FTIR Stop-flow methods - msec limits so far Continuous, micro-flow

Kinetic IR response to T-jump (45-60 C) - apo Mb

Solvated helix (1632 cm-1) lost very fast, ~100 ns, as is 1664 (turns?)protected helices (1655 cm-1) slower. Laser pulse heat water in 10’s ns

Gilmanshin, et al. PNAS 1997

Page 20: Rapid Kinetics with IR Protein folding examplesRapid Kinetics with IR Protein folding examples. Time dependent data with FTIR Stop-flow methods - msec limits so far Continuous, micro-flow

Vilin head-piece – very fast folder

Page 21: Rapid Kinetics with IR Protein folding examplesRapid Kinetics with IR Protein folding examples. Time dependent data with FTIR Stop-flow methods - msec limits so far Continuous, micro-flow

A57 13C labeled Vilin Head-piece Results (IR/T-Jump)

1573 cm-1

1644 cm-1

Dyer