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