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Innovation with Integrity
NMR Relaxation Methods in Biological SystemsDaniel MathieuNMR ApplikationBruker NMR BenutzertagungFrankfurt am Main, 5.11.2018
Proteins aren‘t rock solid…
5.11.218 2
Timescale of dynamics
ps ns µs ms s min hours days
R1, R2, hetNOE R1ρ HD-exchange
real-time NMRCT-CPMG
zz-exch.
RDCs
chemical exchange
5.11.218 3
Exchanging Proteins
E
state A
state B�� � ���
������� ≫ � � ����������
A B
5.11.218 4
Kay et al 2008
J. Biomol. NMR, 41, 113–120
CPMG relaxation dispersion
The HEROINE experiment
Ban, Lee, Griesinger et al 2013
J. Biomol. NMR, 57, 73-82
5.11.218 5
Relaxation dispersionWhat and Why?
• The dependency of effective transverse relaxation on an applied spin-lock field which averages exchange contributions
• Relaxation dispersion is a method to characterize exchange processes by NMR
• Can be used to characterize invisible states
• Can yield dynamic and thermodynamic parameters
5.11.218 6
Measurement of CT CPMG relaxation dispersion
• One reference experiment is recorded without a CPMG train
• Multiple CPMG fields for a constant relaxation period T are recorded
• Relaxation rates are determined by comparison to the reference
• Saves a lot of time compared to individual R1ρ
relaxation measurements at every field.
• CPMG pulse trains deposit a lot of power into the sample which leads to heating
• Scan wise interleaved measurement
• Dummy pulses in every scan to achieve temperature compensation
5.11.218 7
Outcome in the presence of exchange
• Exchange rates Rex contribute tothe effective rate R2,eff
• Exchange contributions can bechanged by variation of the CPMGfield
• In case of a two-site exchange,this can be described analytically
• The lowest possible field strengthis limited by the length of T(longer means lower fields)
• The highest possible fieldstrength is limited by thehardware capabilities for thesame time T
Kay et al J. Biomol. NMR 2008, 41,113–120
5.11.218 8
Sounds simple, what could go wrong?
• Data fully made up
• In this case 12 CPMG field strengths, data at 200 and 1250Hz using threereplicates
• Exchange occuring in the slow to medium time regime
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Error case 1:
• At a first look some data points seem to be completely off
• Any given constant time delays only allows for certain field strengths
• Any field strength that can not be realized is rounded to the closestpossible value, leaving the constant time as it‘s supposed to be
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Error case 2:
• Effective relaxation rates seem to go up towards higher field strengths
• Occuring only for off-resonance peaks? Might be an off-resonance effect, on newer probes try shorter CPMG pulses (e.g. 80 µs 180° pulses) orphase cycled CPMG pulses
• Is it occuring for all peaks?
• Possibly due to miscalibration or detuning of the respective channel
5.11.218 11
Error case 3:
• The replicate measurements do not match, however the general shape ofthe curve does not indicate poor signal to noise.
• Heating effects: the intensity of one measurement depends on the amountof power used in the previous scan.
• Use temperature compensation instead of scan wise interleavedmeasurements
5.11.218 12
(Error) case 4:
• The measured R2 rates do not reach a plateau but instead decay even upto the highest utilized CPMG field
• If this is the case for all peaks that do show exchange, reduce the constanttime in favour of higher CPMG fields (or combine with the HEROINE experiment)
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Error case 5:
• Basically everything happens in between point one and two
• Increase the constant time to be able to user smaller steps for possible RF fields (and reduce RFmax)
5.11.218 14
Scan wised interleaved measurement
��
� ���
� ���
� ���
� �
/nbl
• Important: nbl = td1 = # of list entries
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Temperature compensation
• Dummy pulses to ensure every scan applies the same amount of power
• Increases the overall dutycycle
• Usually scan wised interleaved acquisition is no longer needed
5.11.218 16
Scan wised interleaved acquisition
define list<delay> RF_field=<$VDLIST>
1 ze
d11 pl16:f3 st0
2 6m do:f3
3 6m
[…]
goscnp ph31 cpd3:f3
3m do:f3
3m st RF_field.inc
lo to 3 times nbl
3m RF_field.res
3m ipp3 ipp4 ipp5 ipp6 ipp7 ipp8 ipp9 ipp11 ipp12 ipp31
lo to 4 times ns
d1 mc #0 to 4
F1QF()
F2EA(cal…
5.11.218 17
Temperature compensation
"l3=td1"
aqseq 312
1 ze
d11 pl16:f3
"RF_max=0“
9 20u if "RF_field > RF_max“
{
20u "RF_max=RF_field“
}
3m RF_field.inc
lo to 9 times l3
3m RF_field.res
"d31=RF_field[l11]"
[…]
if "RF_max > d31„
{
20u "cnst31=sqrt((RF_max*RF_max) - (d31*d31))„
20u "TAU1=(1 / (cnst31*4) ) - p30/2000000„
20u "COUNTER2=d21*cnst31*4„
}
[…]
if "abs(RF_max-d31) < 0.1“
{
d21*2
}
else
{
8 TAU1
(p30 ph2):f3
TAU1
lo to 8 times COUNTER2
}
[…]
go=2 ph31 cpd3:f3
d11 do:f3 mc #0 to 2
F1QF(calclc(l11, 1))
F2EA(cal
5.11.218 18
Relaxation dispersionusing Proton decoupling
• Proton decoupling improves sensitivity and relaxational properties of15N
• Proton CW field strength is varied in order to suppress NH magnetization transfer: ��� � 2� · �����
Hansen, Vallurupalli & Kay, J. Phys. Chem. B 2008, 112, 5898-5904
5.11.218 19
Compensating off-resonance effects
• 160µs ϖ-pulse
• 15N projection @ 800MHz
5.11.218 20
Compensating off-resonance effects
• Phase cycled CPMG
• Improves Offset dependency
• Minimum CPMG field for a given constant time increased by a factor of 2
• Can be used as “single train CPMG” (recuces minmum field by a factor of 2)
Jiang, Yu, Zhang, Liu & Yang, J. Magn. Reson. 2015, 257, 1-7
5.11.218 21
Kay et al 2008
J. Biomol. NMR, 41, 113–120
CPMG relaxation dispersion
The HEROINE experiment
Ban, Lee, Griesinger et al 2013
J. Biomol. NMR, 57, 73-82
5.11.218 22
Why even higher power?
• Higher power for the individual pulses reduces off-resonance effects
• One of the fitting parameters is R20 (exchange-free R2)
• This parameter is extracted from the plateau towards higher field strength
• In a lot of cases the plateau is not yet reached
R20?R20?R20?R20?R20?R20?
Really High power!Really High power!Really High power!
R20!R20!
• So why not just measure the end point?
R20!
• So why not just measure the end point?
5.11.218 23
Experimental conditions
• All measurements were performed on u-13C,15N Ubiquitin (1.5 mM in 90% H2O 10% D2O)
• 500 MHz CP-TCI
• 293 K
• 80 µs π-pulses during CPMG trains
5.11.218 24
The HEROINE experiment
• Heteronuclear Rotating Overhauser Invaded Exchange
• High power T1ρ measurement
• Recorded for different spinlock offsets to get as close to the on resonance condition as possible
• The corresponding CPMG experiment with lower power is temperature compensated with respect to the HEROINE experiment
5.11.218 25
Setup
• Both experiments in one sequence
• Flag decides whether the HEROINE or CT CPMG experiment is executed
• Two lists (one for CPMG frequencies, one for T1ρ spin lock periods)
• All calculations e.g. temperature compensation are done in the pulse program
-DLABEL_R20
5.11.218 26
Results: 5 kHz spin-lock up to 125 ms
• T1 type analysis (e.g. when using PDC)
• Clear dependency on the offset (even @5kHz !)
5.11.218 27
Temperature compensated CT-CPMG measurements
• Spin-lock fields up to 1 kHz for 80 ms (using 6.25 kHz π-pulses)
• Temperature compensated to match the HEROINE experiment
5.11.218 28
Chemical Exchange Saturation Transfer
Vallurupalli et al.
J. Am. Chem. Soc. 2012, 134, 8148
5.11.218 29
How CEST works
A
B
5.11.218 30
Pulse program
• (pseudo)3D Experiment with a varying 15N B1 field offset
Vallurupalli et al.
J. Am. Chem. Soc. 2012, 134, 8148
5.11.218 31
What does this look like?
• Basically CW absorption spectra
0
0,2
0,4
0,6
0,8
1
1051151251350
0,2
0,4
0,6
0,8
1
105115125135
5.11.218 32
Experimental setup
5.11.218 33
Experimental parameters
5.11.218 34
Processed result
5.11.218 35
CEST Analysis
5.11.218 36
Sample Information
5.11.218 37
Data Selection
5.11.218 38
Data Analysis
5.11.218 39
Result
5.11.218 40
Lipari-Szabo type order parameters
TROSY based T1, T1ρ and hetNOE measurements(for perdeuterated proteins)
Lakomek N.A., Ying J. & Bax A.
J. Biomol. NMR, 2012, 53, 209–221.
5.11.218 41
What’s wrong with the current approach?
• Backbone amide detected Relaxation experiments are very sensitive using current hardware
but…
• Systematic are errors often much larger than random errors due to signal-to-noise limitations. (Especially when using a TROSY read-out)
• Cross correlated relaxation (H-N Dipole - 15N CSA)
• Water cross relaxation (due to poor water saturation or radiation damping)
• Water exchange
5.11.218 42
New in Topspin 4.0.x / 3.6.x
T1
T1ρ hetNOE
5.11.218 43
• New sequences:• trt1etf3gpsitc3d.3
• trtretf3gpsitc3d.3
• trnoeetf3gpsi3d.3
• T1 temperature compensated with respect to T1ρ
• No parametersets (yet), use non .3 ones, nbl = 1
• No integrated analysis in DynamicsCenter (yet)
New in Topspin 4.0.x / 3.6.x
5.11.218 44
Thanks…
• Donghan Lee
• Frank Löhr
• Wolfgang Bermel
• Peter Neidig
• Helena Kovacs
• Maxim Mayzel
• You for your attention
5.11.218 45
Innovation with Integrity
Innovation with Integrity