flexibility of nucleic acid structures investigated by ... · structure and flexibility of nucleic...

Flexibility of Nucleic Acid Structures

Investigated by Pulsed EPR

Biophysik Ringvorlesung 2017

Thomas Prisner

Structural Characterizations ofMembrane Proteins

Conformational flexibility of NA

Hyperpolarized NMR Spectroscopy

New Methods for EPR spectrocopy

CRC 807 CRC 902

SPP 1601, DFG/ANR

EU, DIP, DFG

BMRZ

DFG

DNP for MRI

Structural Characterizations ofMembrane Proteins

Conformational flexibility of NA

Hyperpolarized NMR Spectroscopy

New Methods for EPR spectrocopy

CRC 807 CRC 902

SPP 1601, DFG/ANR

EU, DIP, DFG

BMRZ

DFG

DNP for MRI

Conformational flexibility of NA

Structure and Flexibility of Nucleic Acids

Structure and Flexibility of Nucleic Acids

Nature Protocol (2007)

NAR (2007)Angewandte (2009)

Phys. Rev. E (2009)

Joachim Engels

(Uni Frankfurt) Snorri Sigurdsson

(Uni Iceland)

Magnetic Dipol-Dipol Interaction between spin-labels

B0

2 2

3 31 3cos ( ) 1 3cos ( )

eff eff

s I s Idip

SI SI

g g

r r

dip

B0

B0

Magnetic dipole-dipole interaction between two spin labels

B0

B0

Magnetic dipole-dipole interaction between two spin labels

B0

B0

Magnetic dipole-dipole interaction between two spin labels

B0

B0

Magnetic dipole-dipole interaction between two spin labels

Dipolar PakePattern

Width < MHz

Frozen Sample with random orientation of molecules

Randomly oriented powder sample

N

Ox

y

z

B0

B0

0.3 T

6.4 T

Hyperfine

dominated

G-Tensor

dominated

EPR linewidth of nitroxide spin labels

Select only small g value

Select only large g-value

t0

t1

t1

t2

t2

t3

t3

time

mic

row

ave

ECHOFID

t0

Pulsed EPRHahn Echo sequence

Refocusing technique eliminates inhomogeneous linewidth

Pulsed EPRHahn Echo sequence

Refocusing technique eliminates inhomogeneous linewidth

Pulsed EPRHahn Echo sequence

Pulse on coupled spin brakesthe symmetry – less refocusing!

PELDOR Time Trace oscillates with dipolar frequency

B (Pump Spin)

A (Probe Spin)

rAB

B0

0 1000 2000 3000 4000 50000.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1 2 3 4 5 6Distance [nm]

No

rma

lize

d E

ch

o A

mp

litu

de

t [ns]

1

3

2cos31~

cos)cos(~)(

AB

dip

dip

r

dttS

/2 A

B

t

/2 A

B

t

2 Dimensional Experimental Procedure

2D Dataset

JMR 2016

Unique pattern for a given distance / geometry

PELDOR Database

PCCP 2013

Simulated Multi-Frequency / Multi-Field Datasets for each orientation and distance (conformer)

PCCP 2013

Unique solution found with single conformerDistance & Orientation

Test of method with rigid planar biradical

2D SIFTER experiment using broadband pulses

4 pulse PELDOR/DEERwith several probe frequencies

Broadband SIFTER with FT of Echo signal

11 samples with variable distance between both spinlabelshave been synthesized and measured

Investigation of dynamics of dsDNA

Stretching

Bending

Twisting

Dx2 ~ L

Simple modes of dynamics of dsDNA

Bending Twisting Stretching (Δα) Δr Δh

Models for conformational dynamics of dsDNA

2D-PELDOR datasets and best model fits at 0.3 T

Single molecule fluoreszenz

Gore et al. Nature (2006)

SAXSI

Mathew-Fenn et al. Science (2008)

Modeling

Becker et al. Science (2009)

Models for conformational dynamics of dsDNA

The radius changing modefits best our experimental data but with a 10 timessofter streching module

JACS 2011

Comparison with MD simulations of dsDNA

Established Force Field parmbsc0

New Force Fields Parmbsc1 and OL15

Collaboration withG. Hummer(MPI Biophysics, Frankfurt)

JACS 2017

Microinjection Spin label concentration: 2 mM Sample size for EPR: 50 oocytes

Collaboration with Volker Dötsch (Uni Frankfurt)Angewandte 2011

Stacking of dsDNA in cells observed

In-cell application is possible

PELDOR Experiments at Room Temperature

40 K experiment

MD ‚powder‘

MD ‚averaged‘

RT PELDOR data

NMR structure from NOE‘sDornberger et al. Biochem. 38, 12860 (1999)

Investigation of DNA with A-buldge

Investigation of DNA with A-buldge

Investigation of DNA with A-buldge

PCCP 2017

Investigation of cocaine binding aptamer

cocaine binding

PCCP 2015

Burkhard Endeward, Vasyl Denysenkov, Andriy Marko, Claudia Grytz, Nicole Erlenbach, Markus Gränz, Thilo Hetzke

Dominik Margraf , Ivan Krstic, Olav Schiemann

Snorry Sigurdsson (Iceland)Lukas Stelzl, Gerhard Hummer (MPI Biophysik, Frankfurt)Beatrice Suess (Darmstadt)Peter Günthert (Biophysical Chemistry, Frankfurt)

Acknowledgements / Coworkers

FCI

SFB902