- Basic understandings: - Mapping interactions:

Transcription

1 NMR-lecture April 6th, 2009, FMP Berlin Outline: Christian Freund - Basic understandings: Relaxation Chemical exchange - Mapping interactions: -Chemical shift mapping (fast exchange) Linewidth analysis (slow exchange) -Cross saturation transfer -Transfer NOE/STD NMR -Half filter exepriments/isotope labeling

2 Monitoring protein:protein interactions: Capitalizing on chemical shift and relaxation rate changes In the absence of of an applied rf field, the Bloch equations are defined as: dm x (t) dt = Ω M y (t) R 2 M x (t) In the case of chemical exchange it is : dm y (t) dt dm z (t) dt = Ω M x (t) R 2 M y (t) = Ω{M z (t) M 0 } dm xy (t) dt dm z (t) dt = (Ω R 2 + K)M xy (t) = (Ω + K) M z (t) Formal solution : M z (t) = e -Rt M z (0) R = ρ I σ IS [ ] σ IS ρ S

3 Transitions between states in a two-spin system W I (1) ββ W S (2) αβ W 0 βα W S (1) W 2 W I (2) αα σ = W 2 -W 0 σ ij ~ 1/r ij6 * [6J(2ω 0 ) J(0)]

4 Relaxation times depend on the overall tumbling time of molecules which is proportional to molecular weight Biochemistry, Vol. 28, No. 23, 1989 τ = c 4πηr 3kT 3

5 T 1 relaxation z T 1 Bo y x Measuring T1 from inversion recovery pulse sequence z z z π π/2 y x y x y x

6 Measuring T1 relaxation times z z Inversion Recovery π/2 x y y x τ z 90 π/2 y x τ z z 90 π/2 y x y x

7 Inversion Recovery - Measure NMR Intensity as a function of the delay time τ and fit to an exponential function τ Mz Mz = Mo (1-2e -τ/t1 ) 0 τ Adopted from Roy Hofmann, Hebrew University

8 The inversion recovery experiment yields T1 values for different signals that may have different relaxation times residual H 2 O CH 3 group Example: Ethylbenzene in CDCl 3

9 T 2 relaxation leads to line-width broadening Current amplitude frequency time Current amplitude frequency time

10 Spin-echo pulse sequence for measuring T 2 Example: Ethylbenzene in CDCl 3

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12 Chemical exchange complicates matters K ex Conformational equilibrium K B Chemical equilibrium Fast exchange - one sharp average resonance temp Intermediate exchange - one broad resonance Slow exchange - two distinct resonances

13 Linewidth simulations for slow exchange interactions I(ω) ~ Re {W *A -1 *1} A = i(ω ωe) + K + R Changing R 2 of the bound state: 500, 250,100,50 and 23 s- 1. No chemical shift difference of free and bound state 500 Hz Same parameters used as above. R 2 (free) is 23 s -1 and k off = 200 s -1. The fraction of free protein is 0.5. R 2 (bound) is set to 250 s -1 and chemical shifts are varied : 500, 250, 100, 50 and 23 Hz Published in: Hiroshi Matsuo; Kylie J. Walters; Kenta Teruya; Takeyuki Tanaka; George T. Gassner; Stephen J. Lippard; Yoshimasa Kyogoku; Gerhard Wagner; J. Am. Chem. Soc. 1999, 121,

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15 Mapping protein interactions:

16 Chemical shift as a measure of chemical environment : Reference against: 2,2-dimethyl-2-silapentane-5-sulfonic acid (DSS)

17 Predicting protein chemical shifts via CSI Structure refinement via CSI Berjanskii & Wishart, Nature Protocols 1, (2006)

18 Chemical shift changes : a single non-disruptive mutation Wt(rot)/Y33A(grün) G32 GYF domain F34 V29 S30 Y39 Y33 W8 W28

19 Chemical shift changes : two non-disruptive mutations Wt(rot)/Y33AW8R(blau) G32 W8 K7 E5 S30 D36 W8 W28 K54 Y33 F34 I56 W28

20 Chemical shift changes:fast exchange: GYF binding to spliceosomal SmB Wt(rot)/Wt_SmB(grün) G32 T21 F34 D36 S30 Y33 M25 W8 F20 V29 I56 K54 W28 W8 Ne W28 Ne

21 Binding of the single-site mutant Y33A(rot)/Y33A_SmB(grün) G32 T21 F34 D36 S30 Y33 M25 W8 F20 V29 I56 K54 W28 W8 Ne W28 Ne

22 (Non-)Binding of the double mutant Y33AW8R(rot)/Y33AW8R_SmB(grün)

23 Slow exchange: Linewidth analysis can be used to map binding sites Walters et al., PNAS 1999, 96, k off values of 3.2 s 1 (A) or 25.6 s 1 (B) for chemical shift differences of 0 Hz (black) and 500 Hz (red). R 1, R 2, and a are 23 Hz, 250 Hz, and 0.8, respectively

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25 STD-NMR can be used to observe binding in complex mixtures J. Am. Chem. Soc., 2005, 127 (3), pp

26 Cross saturation: detection of 15 NH groups of a deuterated acceptor protein Takahashi et al., Nat. Struct. Biol.(2000) Variant: Selective protonation of an otherwise deuterated donor protein Igarashi et al., JACS 2008

27 Selective protonation of amino acids with different efficiencies Arginine Feasible for: Ala, Arg, Cys, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Trp, and Tyr Asparate

28 Identification of donor residues close in space to affected acceptor amides

29 Identification of donor and acceptor interfaces

30 ..to be continued in later sessions

31 Paramagnetic relaxation enhancement Iwahara & Clore, Nature, 2006

32 At higher salt concentrations, T 2 relaxation data cannot be explained by the specific complex Model system: Homeodomain of human HOXD9 in complex with a 24-base-pair DNA duplex Incorporation of conjugated deoxy(d)t-edta-mn 2+ into the DNA {Γ obs 2 (i) Γ calc 2 (i) Q=( Γ obs 2 ) (i) 2

33 Low-affine interactions likely contribute to formation of the specific complex 15 N- 1 H-correlation spectra indicate that the structure of the specific complex does not change significantly when going from 20 mm to 160 mm salt! Structural representations of the measured intermolecular PRE profiles. At 20mM NaCl, the data is compatible with the structure of the complex bound to the specific site. At 160 mm NaCl the observed PRE are interpreted as footprints of minor species that are in rapid exchange with the specific complex.