Biochemistry 530 NMR Theory and Practice. Gabriele Varani Department of Biochemistry and Department of Chemistry University of Washington

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1 Biochemistry 530 NMR Theory and Practice Gabriele Varani Department of Biochemistry and Department of Chemistry University of Washington

2 1D spectra contain structural information.. but is hard to extract: need multidimensional NMR 1D spectrum Dispersed amides: protein is folded Hα: protein contains β-sheet Downfield CH3: Protein is folded

3 Even 2D spectra can be (and indeed are) very crowded Realistic limit of homonuclear NMR: proteins of amino acids; spectra of larger proteins are too crowded

4 3D Heteronuclear NMR

5 Useful nuclei such as 15 N, 13 C are rare Isotope Spin Natural Magnetogyric ratio NMR frequency (I) abundance γ/10 7 rad T -1 s -1 MHz (2.3 T magnet) 1 H 1/ % H C 1/ N N 1/ O 5/ F 1/ Na 3/ P 1/ Cd 1/

6 Requirements for Heteronuclear NMR: isotope labeling Isotopically labelled proteins can be prepared straightforwardly in E. coli by growing cells in minimal media (e.g. M9) supplemented with appropriate nutrients ( 15 NH4Cl, 13 C-glucose) Metabolic pathways can be exploited and appropriate auxotrophic strains of E. coli can also be used for selective labelling: e.g. use acetate instead of glucose and obtain selective labeling of certain side chain CH 3 Isotopic labelling of protein expressed in eukaryotic cells is expensive but can be done (post-translational modifications can be studied but you need $$$$)

7 Nuclei such as 13 C, 15 N have low γ and therefore low s/n Isotope Spin Natural Magnetogyric ratio NMR frequency (I) abundance γ/10 7 rad T -1 s -1 MHz (2.3 T) 1 H 1/ % H C 1/ N N 1/ O 5/ F 1/ Na 3/ P 1/ Cd 1/

8 HH scalar couplings carry structural information but are small (1-10 Hz) 3 J HαN =5.9cos 2 φ-1.3cosφ J αβ =9.5cos 2 χ 1-1.6cosχ (Karplus, 1958)

9 Heteronuclear NMR exploits 1-bond scalar couplings 1-bond couplings are large ( Hz) compared to HH couplings (1-10 Hz) They are independent of conformation: no structural insight but wonderful for assignments

10 The basic building block of Heteronuclear NMR (INEPT) Polarization of 13 C and 15 N is low: start with 1 H polarization Use 1-bond scalar couplings to transfer magnetization from 1 H to the nucleus of interest Delay Δ must be set to 1/4J for optimal transfer: in the absence of relaxation, magnetization transfer is 100% efficient

11 The basic building block of Heteronuclear NMR (INEPT)

12 The basic building block of Heteronuclear (INEPT-1D)

13 The basic building block of 2D Heteronuclear NMR (HSQC) Polarization of 13 C and 15 N is low: start with 1 H, transfer to 1 H with INEPT (sensitivity increases by the ratios of γ, e.g. 10 for 15 N) Label magnetization with 15 N Larmor frequency in t1 and record 15 N evolution in the first dimension Go back to 1 H for detection with a reverse INEPT, i.e. from 15 N to 1 H and record 1 H evolution in the direct dimension (high s/n)

14 Section of 1 H- 15 N HSQC spectrum of protein-rna complex

15 The fundamental size limitation of NMR The delay τ to transfer magnetization must be τ << T 2 As protein size increases, T 2 becomes shorter and HSQC fails but Wuthrich and co. came up with TROSY that allows NMR to be performed at even 1 MDa (Nature 2002)

16 HSQC is the building block and foundation for very many heteronuclear NMR experiments Measurements of relaxation properties (motion) Spectral assignments 3D versions of NOESY and COSY spectra (structure)

17 The most important experiment for protein structure determination: 3D NOESY HSQC Measurements of 1 H- 1 H distances (as in 2D NOESY) Resolution is spread in a third dimension (usually 15 N but also 13 C; for nucleic acids mostly 13 C; for protein/nucleic acid complexes you can observe only the protein, only the RNA or only the contact from one to the other)

18 Magnetization transfer in 3D NOESY-HSQC

19 Schematic illustration of 3D NOESY HSQC A collection of many (e.g. 128) NOESY spectra each separated by the frequency of the attached 15 N nucleus

20 A NOESY plane from a 3D NOESY HSQC Only those amides whose 15 N resonates at ppm can be observed in this particular NOESY plane; other amides 1 H are observable at different planes

21 HSQC is a building block and foundation for very many heteronuclear NMR experiments Measurements of relaxation properties (motion) Spectral assignments 3D versions of NOESY and COSY spectra (structure)

22 Heteronuclear NMR exploits 1-bond scalar couplings 1-bond couplings are large ( Hz) compared to HH couplings (1-10 Hz) They are independent of conformation: no structural insight but great for assignments

23 Triple resonance experiments that rely on scalar couplings for magnetization transfer have made assignments trivial

24 A triple resonance experiment: HN(CO)CA

25 Different triple resonance experiments light up distinct nuclei HN(CO)CA (sequential) HNCA (intraresidue and sequential)

26 Different triple resonance experiments light up distinct nuclei H(CA)NH

27 Different triple resonance experiments light up distinct nuclei CBCA(CO)NH: sequential connections to the side chains

28 Protein and nucleic acid 3D structure generation from NMR Typical protocol for 3D structure generation from NMR data ( 1 H only)

29 Protein and nucleic acid 3D structure generation from NMR Typical protocol for 3D structure generation from NMR data (with 3D data)

30 How to judge the quality of an NMR structure?

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