Innovation with Integrity

NMR Relaxation Methods in Biological SystemsDaniel MathieuNMR ApplikationBruker NMR BenutzertagungFrankfurt am Main, 5.11.2018

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