NMR-based Structural Biology for Studying Biomolecular Interactions Karel Kubíček zobrazovani.png y:\Presentations\080513_KampusBohunice_Mornstein\composition_earth_seawater_human_body.png em_spectrum.jpg NMR sample spectrum 1pulse 11_HSQC_15N_zoom1 1)Structure 2) 2)Relaxation properties 3) 3)Interaction at atomic level resolution 4) 4)Analysis 5) 5)Image 200-500 ml of 100-1000 mM compound Method of choice Data to be analyzed Results NMR hardware 1)Magnet 2)Spectrometer 3)Control units IMG_1617.jpg Pc240013 Pc240009 NMR spectrometer Earth’s Magnetic Field ~ 50mT solenoid.gif Ampere’s law & solenoid Magnet - superconducting solenoids immersed into He bath - He-bath ~4 K further improved to ~2.1 K with J-T pump - field strength 25-28 Tesla - (Nb, Ta)3Sn superconductor of 0.81 mm with ~271 filaments buried in OFHC copper matrix - wirestrain n4a02f3 500px-20T_superconducting_magnet.png fig6 fig6 c-mag-4 Bore cca 55 mm He-refill N2-refill J. Emsley & R. Feeney, Progr.. NMR Spectroscopy 1995, 28, 1 ‘10 1945 60 1961 220 ‘65 1973 360 1979 500 1987 600 ‘92 750 1000 ‘97 2000 ‘68 decoupling decoupling TROSY FT and nD DNP in high fields 1GHz_Bruker.jpg Time [year] Quench an abnormal termination of magnet operation Occurs when part of the superconducting coil enters the normal (resistive) state. This can occur i)because the field inside the magnet is too large ii)the rate of change of field is too large (causing eddy currents and resultant heating in the copper support matrix) iii)or a combination of the two. iv)a defect in the magnet can cause a quench. v) v) MOVIE: https://www.youtube.com/watch?v=d-G3Kg-7n_M 6914430-0-large probe probe4 probe5 NMR Probe(head) LRC Spectrometer CBU Control board unit FGU Frequency gen. u. Shimms Temperature Unit AcquisitionControler Transmitter TMP3_1 TMP6_1 TMP4_1 NMR Spectrometer - Overview Signal - sl(t)=S sl(t) l TMP5_1r TMP5_2r TMP5_3r NMR radiofrequency pulse Pulzy: a)tvrdé – 7-30 ms@-3~+3dB b)selektivní – ms~s@>30db c)adiabatické earth-magfield.jpg magnetic field = 0 magnetic field > 0 For NMR, nuclear spin is needed!!! Spin analogy to a compass needle Electron Proton Neutron Atom = In the planetary model of the atom, an electron orbits a nucleus, forming a closed-current loop and producing a magnetic field with a north pole and a south pole. Molecule is hence a group of small magnetic fields and each atom within the molecule experiences different local magnetic field. A picture containing game Description automatically generated NMR - Refresh 1)nuclear spin ¹ 0 (1H, 13C, 15N, 31P) - number of neutrons and the number of protons both even Þ NO nuclear spin - number of neutrons plus the number of protons odd Þ half-integer spin (i.e. ½, 3/2, 5/2) - number of neutrons and the number of protons both odd Þ integer spin (i.e. 1, 2, 3) 2) n=g*B (1) - when placed in a magnetic field of strength B, a nuclei with a net spin can absorb a photon, of frequency n. The frequency n depends on the gyromagnetic ratio, g of the nuclei 3)from quantum mechanics we know that nucleus with spin I can have 2I +1 orientations Þ nuclei with a spin ½ can have two orientations in an external magnetic field– low / high energy N S N S N S E=h n (2) Nuclear Magnetic Resonance Refresh From (1) and (2): E=h g B N S N S N S N S CW vs. Fourier transform NMR Problem of NMR the magnitude of the energy changes in NMR spectroscopy small Þ sensitivity is a major limitation Solution I. increase sensitivity by recording many spectra, and then add them together; because noise is random, it adds as the square root of the number of spectra recorded. For example, if 100 spectra of a compound were recorded and summed, then the noise would increase by a factor of 10, but the signal would increase in magnitude by a factor of 100 Þ large increase in sensitivity. However, if this is done using a CW-NMR, the time needed to collect the spectra is very large (one scan takes 2 - 8 minutes). FID CW vs. Fourier transform NMR Solution II. FT-NMR Þ all frequencies in a spectrum are irradiated simultaneously with a radio frequency pulse. Following the pulse, the nuclei return to thermal equilibrium. A time domain emission signal is recorded by the instrument as the nuclei relax. A frequency domain spectrum is obtained by Fourier transformation. 1pulse FT time domain (FID – free induction decay) frequency domain RF pulse 90° 1dspec_1 etoh2_1 EtOH protein Each proton = 1 NMR signal 1dspec_1 Each (non-exchangeable) proton = 1 NMR signal We -NH -NH/-NH2/arom Ha Hb -CH3 H2O Each (non-exchangeable) proton = 1 NMR signal Size Relaxation FID NMR line(width) after FT slow (i.e. long t2 time) medium fast A close up of a glove Description automatically generated A picture containing ball, game Description automatically generated A yellow ball Description automatically generated A close up of a logo Description automatically generated Time Time Hz Hz Hz Size Relaxation FID NMR line(width) after FT slow (i.e. long t2 time) medium fast A close up of a logo Description automatically generated A close up of a logo Description automatically generated Time Time Hz Hz Hz e.g. Cholesterol Biomolecules 5-30 kDa Large molecules 50+ kDa NMR data processing window_function NMR data processing Window functions: 1) improvements od S/N ratio 2) increasing resolution cavanagh1 cavanagh2A Exp Lor.-Gauss Kaiser w. function NMR data processing – window functions – apodization y:\Presentations\080430_UFKL_NMRSeminar\plot_NMR.png cavanagh3 zero_filing NMR data processing – Zero Filling, Linear prediction Linear prediction Zero filling 240 points 64 pts LP to 128 pts LP to 240 pts |nmrPipe -fn POLY -time \ |nmrPipe -fn SP -off 0.33 -end 0.98 -pow 2 -c 1.0 \ |nmrPipe -fn ZF -size 2048 \ |nmrPipe -fn FT -auto \ |nmrPipe -fn PS -p0 -76.0 -p1 0.0 -di \ |nmrPipe -fn EXT -x1 11.0ppm -xn 6.0ppm -sw \ |nmrPipe -fn POLY -ord 3 -auto \ |nmrPipe -fn TP \ I)Solvent suppression II)Window function III)Zero-filling IV)FT V)Transpose (in case of multidimensional spectra) F2 NMR data processing - summary 11_HSQC_15N_zoom1 ft_only_zoom1 11_HSQC_15N ft_only NMR as a tool for study structure, dynamics and interactions of biomolecules 1)Structure determination of NAs and proteins 2)Protein – metal interaction 3)Protein – ligand interaction For most of the modern applications, enrichment by 13C, 15N and often 2H needed! 1) Isotope Ground state spin Natural abundance [%] Rel. Sensitivity 1H ½ ~100__ 1.00x10+0 13C ½ 1.10 1.59x10-2 15N ½ 0.37 1.04x10-3 19F ½ 100__ 8.30x10-1 31P ½ ~100__ 6.63x10-2 12C 0 98.90 - 16O 0 ~100__ - D:\karelk\Presentations\070314-UFKLSeminar\NMR-11.jpg r1,2 r1,2; r1,3; r2,3≤ 6 Å 1Å=1.10-10m NOE: NMR as a tool for study structure, dynamics and interactions of biomolecules 0) AA/NA sequence, resonance assignment, standard chemical shifts 1)Structure determination of proteins/NAs 2)NMR can provide detailed information about the structure at the atomic level resolution relying on the spatial proximity of two interacting protons – nuclear Overhauser enhancement (NOE) 3)Additional structural information can be obtained (residual dipolar couplings – RDCs, J-couplings, backbone chemical shifts - CSI) 4) http://www.fbreagents.com/basics_nmr/9proteins.htm Structure calculation Iterative procedure of structure determination by NMR N C Nrd1 CID PDB ID: 2LO6 Uncertainty of the final structure represented as a family of 10-20 structures with deviation among individual members indicated by RMSD (typically <1.5 Å2) Final structure 1H / ppm Studying interactions by NMR titration 1)Slow exch. regime (on the NMR timescale) – individual peaks for each of the studied states (e.g. free / complexed forms of a protein), peak intensity representing population of a given state 2)Intermediate exchange regime 3)Fast exchange regime – single peak whose chemical shift position is given by the molar ratio of the states present in solution Slow (KD<1 mM) Free Bound 100% 0% 50% 50% 0% 100% Intermediate (KD ~1-10 mM) Free Bound 100% 0% 50% 50% 0% 100% Fast exchange regime (KD>10 mM) Free Bound 100% 0% 50% 50% 0% 100% backbone.eps CID-15N-HSQC_JCh.png 1H-15N HSQC, cca 155 aa, well folded, 600MHz, 293K 15N-1H HSQC – Heteronuclear Single Quantum Coherence 1)1 peak ≅ 1 amino acid 2)good estimate of the protein folding status 3)no information about sequential assignment (which peak is which amino acid) 4)for sequential assignment third dimension needed (13C) 5)once assignment of the peaks known – HSQC is optimal tool for monitoring interactions by NMR through titrations (i.e. stepwise addition of small amounts of ligand to the nearly constant volume solution with the isotopically enriched molecule) CID-15N-HSQC_JCh.png Screen Shot 2013-10-09 at 9.53.04 AM.png 1H-15N HSQC, cca 155 aa, well folded, 600MHz, 293K 1H 1D, Cavanagh et al., 2007 backbone.eps CID-CTD_191108_tit0_bis.png Interaction of Nrd1-CID with C-terminal domain (CTD) NMR Titration - 15N enriched CID + unlabeled CTD-Ser5P in n-steps, n=6 in our case - peaks corresponding to the interacting residues of CID change their chemical shift (position in the spectrum) => interaction surface, binding constant, stoichiometry We studied the interaction between CTD and CID by monitoring the chemical shift changes of the CID residues – one peak represents one amino acid in this spectra - upon addition of the CTD. In this way, we indentified the interacting surface of the Nrd1-CID cid-ctd.trans_model.perturb.png Nrd1 CID interaction surface – CID residues experiencing the largest chemical shift variations upon the interaction with 5-phospho-Ser CTD shown in blue with side-chains in stick representation … that are located on three helices of the CID. cid-ctd.trans_model.mutants.2.png CTD-CID interaction with mutants studied by fluorescence anisotropy cid_barChart.png Additionally, still analyzing the chemical shift perturbations, we have selected the residues crucial for the interaction and have mutated them to observe a dramatic drop in the affinity as measured by fluorescence anisotropy. Interligand NOEs between CID and CTD – 900MHz, 150ms, 293K CID resonances Next, we determined the NMR structure of the complex between Nrd1-CID and Ser5P-CTD by measuring intermolecular contacts that are defined by these NOEs, where in one frequency domain, we acquired the resonances of the protein and in the second dimension the resonances of the ligand. Transferred-NOE •NOE = pbound.NOEbound + pfree.NOEfree • •tc,bound >> tc,free (and pL,free >> pL,bound) • • NOEbound > NOEfree The correlation time is roughly defined as the time taken for a molecule to rotate one radian or move a distance of the order of its own dimension. Usually it is assumed that the correlation time depend on temperature according to an Arrhenius type of function; Correlation time - The characteristic time between significant fluctuations in the local magnetic field experienced by a spin due to molecular motions. freeDisco080ms1 boundDisco080ms1 Transferred NOE Experiments tr-NOESY~600mM Discodermolide without and with ~12mM tubulin 800MHz, mixing time=80ms disco w/o tub disco:tub 50:1 8 7 6 ppm Screen Shot 2013-10-16 at 2.33.46 PM.png Screen Shot 2013-10-16 at 2.33.58 PM.png Screen Shot 2013-10-16 at 2.34.17 PM.png Screen Shot 2013-10-16 at 2.34.41 PM.png protein ligand2 ligand1 Magnetization to be transferred Transferred magnetization Note the weak “signal” They “compete” for same place but never “meet” interligand NOE Experiments . interligand NOE Experiments . inerligEpoA inerligTbs (50)Tbs + (1)Tub interligand NOE Experiments . inerligEpoA inerligEpoATbs inerligTbs (50)Tbs + (1)Tub (50)Tbs+(50)EpoA+(1)Tub interligand x-peaks, 100-450ms, 900MHz interligand NOE Experiments . inerligEpoA inerligEpoATbs inerligTbs (50)Tbs + (1)Tub (50)Tbs+(50)EpoA+(1)Tub interligand x-peaks, 100-450ms, 900MHz