Upload
others
View
4
Download
0
Embed Size (px)
Citation preview
Rapid Kinetics with IRProtein folding examples
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
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
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
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.
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
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
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
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:
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
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
Dynamics--Scheme of Stopped-flow System
protein solution
Lipid vesicle solution
-add dynamics to experiment
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
Summary
Nw Ns Unfolding Us Insertion UmBinding
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
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
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
Apo-Mb kinetics, T-jump Fluorescence & IR
Fluorescence
IR
Follow different processes, µs response
Fluorescence – tertiary structure unfoldIR – secondary structure - helices
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
Vilin head-piece – very fast folder
A57 13C labeled Vilin Head-piece Results (IR/T-Jump)
1573 cm-1
1644 cm-1
Dyer