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NMR Relaxation Methods in Biological Systems - Bruker · 2018-11-12 · NMR Relaxation Methods in...
Transcript of NMR Relaxation Methods in Biological Systems - Bruker · 2018-11-12 · NMR Relaxation Methods in...
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Innovation with Integrity
NMR Relaxation Methods in Biological SystemsDaniel MathieuNMR ApplikationBruker NMR BenutzertagungFrankfurt am Main, 5.11.2018
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Proteins aren‘t rock solid…
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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
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Exchanging Proteins
E
state A
state B�� � ���
������� ≫ � � ����������
A B
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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
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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
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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
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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
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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
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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
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(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)
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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
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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…
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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
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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
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Compensating off-resonance effects
• 160µs ϖ-pulse
• 15N projection @ 800MHz
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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
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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
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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?
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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
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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
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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
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Results: 5 kHz spin-lock up to 125 ms
• T1 type analysis (e.g. when using PDC)
• Clear dependency on the offset (even @5kHz !)
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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
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Chemical Exchange Saturation Transfer
Vallurupalli et al.
J. Am. Chem. Soc. 2012, 134, 8148
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How CEST works
A
B
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Pulse program
• (pseudo)3D Experiment with a varying 15N B1 field offset
Vallurupalli et al.
J. Am. Chem. Soc. 2012, 134, 8148
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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
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Experimental setup
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Experimental parameters
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Processed result
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CEST Analysis
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Sample Information
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Data Selection
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Data Analysis
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Result
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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.
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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
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New in Topspin 4.0.x / 3.6.x
T1
T1ρ hetNOE
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• 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
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Thanks…
• Donghan Lee
• Frank Löhr
• Wolfgang Bermel
• Peter Neidig
• Helena Kovacs
• Maxim Mayzel
• You for your attention
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Innovation with Integrity
Innovation with Integrity