NMR-based Structural Biology for Studying Biomolecular Interactions Karel Kubíček NMR sample spectrum 1) Structure 2) Relaxation properties 3) Interaction at atomic level resolution 4) Analysis 5) Image 200-500 µl of 100-1000 µM compound Method of choice Data to be analyzed Results NMR hardware 1)Magnet 2)Spectrometer 3)Control units NMR spectrometer Earth’s Magnetic Field ~ 50µT 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 Bore cca 55 mm He-refill N2-refill J. Emsley & R. Feeney, Progr.. NMR Spectroscopy 1995, 28, 1 ‘101945 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 SpectrometerFrequency[Hz] 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. MOVIE: https://www.youtube.com/watch?v=d-G3Kg-7n_M NMR Probe(head) Spectrometer CBU Control board unit FGU Frequency gen. u. Shimms Temperature Unit AcquisitionCon troler Transmitter NMR Spectrometer - Overview Signal - sl(t)=S sl(t) l NMR radiofrequency pulse Pulzy: a) tvrdé – 7-30 µs@-3~+3dB b) selektivní – ms~s@>30db c) adiabatické 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 closedcurrent 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. 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 Energy Magnetic Field N S N S Energy Magnetic Field Abs.E. Magnetic Field change field change frequency 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). 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. FT time domain (FID – free induction decay) frequency domain RF pulse 90° EtOH protein Each proton = 1 NMR signal 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 Time Inducedvoltage Time Inducedvoltage Hz Hz Hz Size Relaxation FID NMR line(width) after FT slow (i.e. long t2 time) medium fast Time Inducedvoltage Time Inducedvoltage Hz Hz Hz e.g. Cholesterol Biomolecules 5-30 kDa Large molecules 50+ kDa NMR data processing NMR data processing Window functions: 1) improvements od S/N ratio 2) increasing resolution Exp Lor.-Gauss Kaiser w. function NMR data processing – window functions – apodization 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 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! 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__ - 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) http://www.fbreagents.com/basics_nmr/9proteins.htm Structure calculation Iterative procedure of structure determination by NMR N CNrd1 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 15N/ppm 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 µM) Free Bound 100% 0% 50% 50% 0% 100% Intermediate (KD ~1-10 µM) Free Bound 100% 0% 50% 50% 0% 100% Fast exchange regime (KD>10 µM) Free Bound 100% 0% 50% 50% 0% 100% 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) 1H-15N HSQC, cca 155 aa, well folded, 600MHz, 293K 1H 1D, Cavanagh et al., 2007 water 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 Nrd1 CID interaction surface – CID residues experiencing the largest chemical shift variations upon the interaction with 5-phospho-Ser CTD shown in blue with sidechains in stick representation CTD-CID interaction with mutants studied by fluorescence anisotropy Interligand NOEs between CID and CTD – 900MHz, 150ms, 293K CID resonances CTDresonances Transferred-NOE NOE = pbound .NOEbound + pfree .NOEfree tc,bound >> tc,free (and pL,free >> pL,bound) NOEbound > NOEfree 0 20 40 60 80 -20 -40 -60 -80 -100 0.1 1 10 100 ηmax ωτ NOE Transferred NOE Experiments tr-NOESY~600µM Discodermolide without and with ~12µM tubulin 800MHz, mixing time=80ms disco w/o tub disco:tub 50:1 8 7 6 ppm 876ppm 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 . (50)Tbs + (1)Tub (50)EpoA+(1)Tub interligand NOE Experiments . (50)Tbs + (1)Tub (50)Tbs+(50)EpoA+(1)Tub (50)EpoA+(1)Tub interligand x-peaks, 100-450ms, 900MHz interligand NOE Experiments . (50)Tbs + (1)Tub (50)Tbs+(50)EpoA+(1)Tub (50)EpoA+(1)Tub interligand x-peaks, 100-450ms, 900MHz