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