study of hiapp folding with ftir and 2dir yun l. ling, dave b. strasfeld, sang-hee shim, and martin...
TRANSCRIPT
Study of hIAPP folding with FTIR and 2DIR
Yun L. Ling, Dave B. Strasfeld, Sang-Hee Shim, and Martin T. Zanni
Department of Chemistry, University of Wisconsin-Madison
hIAPP type 2 diabetes
50 um
Fibril in pancreas
Motivation:More explicit insight of hIAPP folding
There is limited structural information on intermediates
Langen, R. et al., Biochemistry 2005, 44, 12113
It is not fibril but intermediate that causes pancreatic cell death.
1. FTIR and 2D IR are sensitive to peptide secondary structure2. They have the time-resolution to catch intermediates
Random coil
Beta-sheet
Method: How FTIR is sensitive to secondary structure?
1645 cm-1
Frequency (cm-1)
random coil
Frequency (cm-1) Frequency (cm-1)
alpha-helix beta-sheet
1635 cm-1
1685 cm-1
1610 cm-1
Result: FTIR spectrum of fully folded hIAPP
It is more than beta-sheet.
1620 cm-1
(beta-sheet)
1645 cm-1
(random coil)
Nd:YVO4
CW laserTi:Sapphire
Oscillator
StretcherRegenerative Amplifier
CompressorNd
:YL
Fn
s la
ser -BBO based
OPA
DifferenceFrequency
Mixingw/ AgGaS2
800 nm, 88 MHz. 2 nJ/pulse
idlerGe AOM
PulseShaper
Tunable mid-IR3-10 m, 2-5 J/pulse
800 nm, 1kHz, 1mJ/pulse
Experiment set-up:Automatic 2DIR spectrometer
signal
beam dump
optical chopper
k1
kpump-probe= k1+k1+k2
sample
k1+k1 k2
monochromatorMCT array
--+
--+
Result: 2DIR spectrum of fully folded hIAPP
1620 cm-1
(Beta-sheet)
1645 cm-1
(random coil)
1670 cm-1
(Beta-sheet)
Result: FTIR kinetics
Starting from random coil
Result: FTIR kinetics
2(1)01I c
1645 cm-1
(random coil)
1620 cm-1
(beta-sheet)t = 0 min
t = 240 min
Beta sheet structure grows in and random coil decreases.
FTIR intensity change over time
Result: 2DIR snapshots
Intensities Populations
Line shape Vibration Dynamics
t=10 min t=150 min t=300 min
Result: 2DIR kinetics
random coil (1645 cm-1)
beta sheet (1670 cm-1)
beta sheet (1620 cm-1)
Trend of random coil diagonal peak elipticity
Elipticity=diagonal width/anti-diagonal width
2DIR intensity change over time
4(3)01I c
Result: comparison
FTIR: intensity of beta-sheet peak2DIR: intensity of beta-sheet diagonal peak (1620 cm-1) 4
(3)2 0101 2 (1)
01
c IIc
FTIR
2DIR
FTIR
2DIR
beta-sheet
random coil
2(1)01I c
4(3)01I c
FTIR: intensity of random coil peak2DIR: intensity of random coil diagonal peak (1645 cm-1)
Summary:Tentative model of hIAPP folding in D2O
protofibril
newly formed fibril mature fibril
hIAPPin random coil
T1T2
T3
FacultyMartin Zanni
Eric FulmerAmber Krummel
Prabuddha Mukherjee Sang-Hee Shim
Feng (Terry) Ding Dave Strasfeld
Ann Woys Wei Xiong
Acknowledgement