Time resolved biological SAXS at SPring-8 Naoto Yagi Japan - - PowerPoint PPT Presentation

Time resolved biological SAXS at SPring-8

Naoto Yagi Japan Synchrotron Radiation Research Institute / SPring-8

SPring-8

 The biggest (circumference 1.4km)

synchrotron radiation facility

 The highest ring energy (8 GeV)  53 beamlines in operation

Methods of time-resolved experiments

• Continuous recording

X-ray Photon Correlation Spectroscopy

• Pump-probe

X-ray Free Electron Laser pump by laser probe by X-ray

τ time time 1 2 3

Time resolution is limited by the detector.

Repeat the experiment changing τ. The experiment need to be repeatable on the same sample.

Classification of detectors

• Photon counting

Geiger counter, Pilatus, solid state detector

• Integrating

film, image plate, CCD, ionization chamber

BL40XU High flux beamline

Beamline: Helical undulator + KB-mirrors (“pink beam”: bandwidth ~2%) Flux: 1×1015 cps@12.4keV Beam size: 200(H) ×40(V) µm Energy: 8 - 16 keV Camera length: up to 3 m Detectors: High-speed X-II + CCD/CMOS etc. Applications: Time-resolved studies on muscle, microbeam diffraction, protein dynamics, XPCS

size : 200 µm (H)×40 µm (V) flux : 6.5×1014 photons/sec (@8 keV) 9.0×1014 photons/sec (@10 keV) 3.0×1015 photons/sec (@11.5 keV) 1.0×1015 photons/sec (@12.4 keV) 6.0×1014 photons/sec (@15 keV) flux density : ~1×1017 photons/sec/mm2 front end slit : 0.5mm (H)×0.165mm (V)

Beam profile

Blue light

Burning a paper

Boiling water

Muscle contraction is regulated by intracellular calcium. Ca2+ binding to troponin molecule

• n the thin filament allows interaction

between actin and myosin, and then muscle contraction takes place. In this study, Ca2+ binding to troponin in live frog muscle was monitored by Small-Angle X-ray Diffraction (SAXD). SAXD is a unique method measuring the structural changes of contractile and regulatory proteins in live muscle. The troponin-related reflection intensity is measured in the SAXD patterns.

Ca-regulation of contraction

Experimental Setup for Muscle Diffraction Experiment at BL40XU@SPring-8

motor for muscle length control cooling water IN cooling water OUT electrical stimulation force transducer vacuum path X-ray detector solenoid shutter windows for X-rays skeletal muscle X-rays

vertical translation: 100mm/sec

Radiation damage in SAXS

“Radiation damage to a protein solution, detected by synchrotron X-ray small-angle scattering: dose-related considerations and suppression by cryoprotectants”

• S. Kuwamoto, S. Akiyama and T. Fujisawa
• J. Synchrotron Rad. (2004). 11, 462-468
• Serious problem at 3rd generation synchrotrons
• Flux density matters!
• Due to oxidation of protein molecules by radicals
• Depends on the protein
• Aggregation --- increase of scatter in small-angles
• DTT, glycerol, sucrose may help
• Moving the sample during exposure helps

Control of caged-ATP experiment

Wakayama, J., T. Tamura, N. Yagi and H. Iwamoto.

• Biophys. J. 87, 430-441 (2004)

Fast CCD camera

• 290 frames/sec (90 M-pixels/sec)
• 640×480 pixels
• 10-bit ADC
• 4 GB frame memory

Yagi, N., K. Inoue and

• T. Oka. J.

Synchrotron Rad., 11, 456-461 (2004)

Muscle diffraction experiment at BL40XU using an X-ray image intensifier and a fast CCD camera

1 104 2 104 3 104 4 104 5 104 10 20 30 40 50 60 70

sfrog10a_143 sfrog10a_143

Integrated intensity Frame number

3.4 msec (640×480 pixels) 0.52 msec (640×72 pixels)

High-speed CMOS camera

Skeletal muscle contraction @1ms

X-ray diffraction from skeletal muscle

actin layer-lines

1st 2nd

resting contracting

myosin meridional reflections

6th 5th 4th 3rd 2nd

3rd myosin meridional reflection (at d=14.3nm) Troponin reflection at 38.5nm and C-protein reflection at 44.1nm

2 4 6 5

troponin reflections 3rd 2nd 1st

Troponin reflections

Comparison of time courses

０℃ 38.5nm electrical stimulus

Experiments were made at BL40XU in SPring-8 (Hyogo, Japan).

Experimental Conditions Specimen: Semitendinosus muscle (SL:2.8µm) of bullfrog Temperature: 16ºC Time resolution: 1msec (Small-angle X-ray diffraction), 0.5ms (fluorescence) Condition: Single pulse or double pulse (interval of 15msec) Ca-indicator: fluo3-AM The SAXD and fluorescence from fluo3 were recorded in the same specimen alternately.

X-ray 1st slit 2nd slit specimen X-II CCD vacuum path (~2800mm) detector specimen Hg lamp Photomultiplier PC Fluo 3-AM

SAXD measurements Fluo3 measurements X-ray specimen

Measurement of Ca2+

Calcium signals

Comparison of the time courses

16 ºC stimulus

• Troponin intensity increases

more slowly than Ca- binding ==> Cooperativity in the thin filament

• Troponin intensity decays

when tension begins to develop ==> Myosin binding to actin affects troponin conformation.

Comparison of the time courses

Matsuo, T., H. Iwamoto and N. Yagi.

• Biophys. J. 99, 193-200 (2010)

Bacteriorhodopsin

“Structural Transition of Bacteriorhodopsin Is Preceded by Deprotonation of Schiff Base: Microsecond Time-Resolved X-Ray Diffraction Study of Purple Membrane“

• T. Oka, K. Inoue, M. Kataoka and N. Yagi
• Biophys. J. 88, 436-442 (2005).

Bacteriorhodopsin

Proton pumping

Diffraction from purple membrane

Shutter system

Intensity changes

Comparison of time courses

deprotonation of the Schiff base precedes the structural change around the G-helix. SVD (Singular Value Decomposition)

Projected electron density map of BR

A

A A B C D E F G

Akiyama et al. PNAS 99,1329-1334 (2002)

CTF (continuous flow) 0.5-15msec STF (stopped- flow)

BL45XU

folding of cytochrome c

160us

analysis of intermediates

Kratky plot Unfolded ↔ I → II → Native Reconstructed scattering profiles for the three kinetic components observed during the folding of cyt c.

I II N → 12500/s ← 2000/s 2400/s 68/s

Pair distribution functions

I N II

Single-molecule tracking

Motion of KcsA potassium channel revealed by single molecule tracking (Shimizu et al., Cell 2008)

BL44B2

white or pink

pH 7.5 not gating pH 4.0 gating

Time ranges of protein motions

msec nsec µsec psec local changes larger changes (diffusion) domain movement sec chain conformation size scale crystallography SAXS

Thank you for your attention.