Biochemistry 530 NMR Theory and Practice David Baker Autumn Quarter 2014 Slides Courtesy of Gabriele Varani
Recommended NMR Textbooks Derome, A. E. (1987) Modern NMR Techniques for Chemistry Research, Pergamon Press Wüthrich, K. (1986) NMR of Proteins and Nucleic Acids, John Wiley and Sons Roberts, G. C. K. (1993) NMR of Macromolecules: A Practical Approach, Oxford Univ. Press Cavanagh, J., et al. (1996) Protein NMR Spectroscopy, Principles and Practice, Academic Press Evans, J. N. S. (1999) Biomolecular NMR Spectroscopy, Oxford Univ. Press
Useful websites http://www.ch.ic.ac.uk/local/organic/nmr.html NMR Spectroscopy. Principles and Application. Six second year lectures given at Imperial College, U.K. http://uic.unl.edu/nmr_theory.html http://www.shu.ac.uk/schools/sci/chem/tutorials/molspec/nmr1.htm Theoretical principles of NMR Courtesy of Sheffield Hallam University, U.K. http://www.nature.com/nsb/wilma/v4n10.875828203.html links to various NMR and structural biology web sites simulation and analysis software; NMR research groups, etc.
Origin of the NMR signal Nuclear subatomic particles have spin 1. If the number of neutrons and protons are both even the nucleus has 0 spin i.e. 12 C (6 neutrons + 6 protons = 12) has I = Ø spin 2. If the number of neutrons plus protons is odd the nucleus has a half-integer spin (1/2, 3/2, 5/2) i.e. 13 C (7 neutrons + 6 protons = 13) has I = 1/2 spin 3. If the number of neutrons and protons are both odd then the nucleus has an integer spin (1, 2, 3) i.e. 14 N (7 neutrons + 7 protons = 14) has I = 1 spin; spin 1 nuclei are quadrupolar (relax fast) For high resolution applications, we use spin ½ nuclei ( 1 H, 13 C, 15 N, 31 P in biology);
Nuclear spins and the energy levels field in a magnetic A nucleus of spin I has 2I + 1 possible orientations (a nucleus with spin 1/2 has 2 possible orientations) Each level is given a magnetic quantum number m For a spin ½ nucleus like 1 H, the two possible values +1/2 and -1/2 of m are In the absence of an external magnetic field, these orientations have equal energy; if a magnetic field is applied, the energy levels are split
Nuclear spins and the energy levels field in a magnetic The energy of a particular level is given by: m=1/2 E = γ h m B o Energy m=- 1/2 where: γ is the the gyromagnetic ratio, a nuclear property (a measure of the polarizability of the nucleus) h is Planck's constant divided by 2π (h = h/2π ) B o is the strength of the magnetic field The difference in energy between levels (the transition energy) ΔE =(1/2-(-1/2)) γ hb o =γ hb o =hω o If the magnetic field is increased, so is ΔE (as ΔE increases, so does sensitivity)
Nuclear precession in a magnetic field: classical description semi- $ # The nucleus possesses a magnetic moment M proportional to its spin I # In a magnetic field, the axis of rotation will precess about the magnetic field B o : dm/dt = -γ M x B o The frequency of precession (ω o Larmor frequency) is identical to the transition frequency (ω o = -γb o ) The precession may be clockwise or anticlockwise depending on the sign of the gyromagnetic ratio (+γ or -γ)
NMR properties of nuclei of common use in biology Isotope Spin Abundance Magnetogyric ratio NMR frequency (I) γ/10 7 rad T -1 s -1 MHz (2.3 T magnet) Deriva2on for 1 H: (26.7519*10 7 rad/t/s)*2.3 T/(2π rad)=97.9 MHz 1 H 2 H 13 C 14 N 15 N 17 O 19 F 23 Na 31 P 113 Cd 1/2 1 1/2 1 1/2 5/2 1/2 3/2 1/2 1/2 99.985 % 0.015 1.108 99.63 0.37 0.037 100 100 100 12.26 26.7519 4.1066 6.7283 1.9338-2.712-3.6279 25.181 7.08013 10.841-5.9550 100.000000 15.351 25.145 7.228 10.136783 13.561 94.094003 26.466 40.480737 22.193173
Origin of a macroscopic (observable) NMR signal # $ $# # Out of a large collection of moments, a surplus have their z component aligned with the applied field, so the sample becomes magnetized in the direction of the main field B o The parallel orientation is of lower energy than the antiparallel At equilibrium, spins will be distributed according to Boltzmann distribution between the two energy states A net magnetization parallel to the applied magnetic field arises because of the small population difference between states
S/N in NMR is poor because close According to Boltzmann distribution: energy levels are so n 1 Energy n 2 If the system is exposed to a frequency: then the energy absorbed is proportional to the difference if (as is the case for optical spectroscopy), then be in their ground state configuration essentially all the molecules will
S/N in NMR is poor because close energy levels are so n 1 Energy n 2 If instead (as is the case for NMR spectroscopy), then the net absorption of energy will be very small because the rate of upward transitions is equal to the rate of downward transitions For this reason, we use magnets of increasing strength to separate energy level more and increase the sensitivity of the experiment
The chemical shift scale The frequency of absorption of the NMR signal depends on the external field as we have seen ν 0 = γ B 0 & Let us now introduce a quantity that describes the fact that different nuclei in the sample experience slightly different magnetic fields because of chemical structure and conformation ν = (1-σ) γ B 0 Finally, let us introduce a scale that is field-independent, so that we can compare directly data recorded on different spectrometers: δ=(ν ν o )/ν 0 x10 6 We use a standard sample (e.g. DSS) to reference all of our spectra, so that we can report the resonance frequency for our proton in a universal, field-independent manner
Amino acid chemical structure leads to distinct shift for each residue: random coil chemical shift values Residue! NH! H! H! Others! Met! 8.42! 4.52! 2.15,2.01! CH2 2.64, 2.64 CH3 2.13! Cys! 8.31! 4.69! 3.28,2.96! Trp! 8.09! 4.70! 3.32,3.19! 2H 7.24 4H 7.65 5H 7.17 6H 7.24 7H 7.50 NH 10.22! Phe! 8.23! 4.66! 3.22,2.99! 2,6H 7.30 3,5H 7.39 4H 7.34! Tyr! 8.18! 4.60! 3.13,2.92! 2,6H 7.15 3,5H 6.86! His! 8.41! 4.63! 3.26,3.20! 2H 8.12 4H 7.14!
1D NMR spectrum of a protein the chemical shift scale 1D spectrum amides NH 2 H α& Side chain CH 2 Side chain CH 3
How are experiments recorded: the Radiofrequency field (RF)! #,!%&'())*+(,- $ $# %&!'()*+ # Modern NMR use radiofrequency pulses to generate observable signals and manipulate the spin state of the system under study: this is the basis of 2D/3D NMR but also of all FT NMR The alternating voltage applied across the ends of the coil in the NMR probe induces an alternating magnetic field in the sample The geometry of the coil is arranged so that this field is perpendicular to the applied field (in the xy plane) This oscillating B 1 field is substantially smaller than the external field B o (thousands of times smaller), and close to the Larmor frequency
FT-NMR spectroscopy relies on weak RF pulses to excite signal! $ $ $# # # # %&!'()*+!,-. /0 $% # 1. Static (z-axis) magnetization M o is not observable as NMR signal $ # %&!'()*+!,&& $# $. /0 # 12++!'2+3+**456 152!748+9!7 2. Turn RF on: M o driven around x axis 3. Turn RF off after a p/2 pulse (control pulse power or length)! $% $ 4. M o precesses in the xy plane at its Larmor frequency!*46! 57 $# 2+3+4:+2!,-!35*! 57 # 57 2+3+4:+2!354) 5; 748+!<*+3= 5. The receiver records the radio signal generated by the precessing magnetic moment M x,y
The NMR Signal and Spectrum The emission signals are oscillatory and physically damped (damped harmonic oscillations) This signal is called the Free Induction Decay or FID The actual spectrum is recovered from the FID via Fourier transformation, which transforms the time interferogram into a frequency spectrum Without FT NMR, it would take the square of the time to obtain an equivalent signal/noise ration
Example: FID and a 1D spectrum FID Fourier Transform 1D spectrum
The effect of relaxation! # $%&'!()'*+,- %3345%4$' No relaxation: infinitely sharp lines #.!/0!12 6 '67(#$8-9 + # $%&'!()'*+,-! /89.!(/8-9 + #.!/0!12 Relaxation has two components: inhomogeneity (line width, T 2 ) and return to thermal equilibrium (T 1 )
1D spectra contain structural information.. but is hard to extract: need multidimensional NMR 1D spectrum Dispersed amides: protein is folded Hα: protein contains β- sheet Downfield CH3: Protein is folded
1D vs 2D homonuclear NMR Spectroscopy *+!,-. #$%#%&'() & /$&$0&'() Basic structure of 1D experiment 12+! 6+!,-. & * 7 Basic structure of 2D experiment (e.g. NOESY) #$%#%&'() $3(45&'() 7'8')9 /$&$0&'() & 6 Jeener and Ernst, 1972
2D homonuclear NMR Spectroscopy! 01!234 & / 5 #$%#%&'() $*(+,&'() 5'6')7 -$&$.&'() & 0 Basic structure 89 89 89 2;><= & / 5?@1 89 89 Two basic experiments: NOESY and COSY :;<= & /
cross-peaks in multidimensional NMR carry structural information Diagonal is closely related to 1D spectrum 2D contour representation: peaks outside diagonal are called cross-peaks
1D Nuclear spectra contain Overhauser structural Effect information SpectroscopY.. but (NOESY) is hard to extract: need multidimensional NMR!!! 01!234 Biochemistry 530 NMR Theory and Practice & / #$%#%&'() $*(+,&'() 5'6')7 -$&$.&'() Gabriele Varani 5'6')7 %.K,'#$ = : B 1 D C Department of Biochemistry and Ha: protein contains b-sheet Department of Chemistry.()<'-$#!%!&G(!<')!<8<&$5H!@!I!AJ >?6 >?6 >?6 & / 5 Dispersed The NOESY amides: experiments detects interactions between protein spins is that folded are close in space and dipolar Downfield coupled: CH 3 : the magnetization transfer mechanism Protein is essentially is folded the same as FRET in optical spectroscopy (fluorescence) & 0 5 University of Washington & 0 1D spectrum
Nuclear Overhause Effect SpectroscopY (NOESY) 1D spectra contain structural information.. but is hard to extract: need multidimensional NMR Biochemistry 530,/0-.>*)!?!&@/!-+.0!-#-&*5A!7!B!8C pulse NMR Theory and Practice 1 % $ 9 $ &!-.0! 7!& ' $&!-.0! 8!& ' % $% #!,/-! $#!,/-! 7!& ' 8!& ' 23 23 Dispersed amides: protein is folded ; $ $ $ # $# 4 $ Gabriele 5 5..06 Varani 1. Excite with 90 o $%!,/-! Department of 8!& ' %!,/-! 7!& ' Biochemistry $ $ and : Ha: protein contains b-sheet 23 Department of Chemistry $ $%!,/-! 8!& ' %!,/-! 7! & ' <5..06= # # $ University of Washington # & '! ()** +)*,*--./0 #!' 77!,/-! 7!& '! (! 87!,/- 8!& ' ) $#!' 88!,/-! 8! & '! (!! 78!,/- 7!& ' ) # # 2. During t1, spins are labeled with their Larmor frequency 3. The second 90 o pulse exchanges rotates magnetization 1D spectrum to -z 4. Magnetization is exchanged between spin I and S during the mixing time Downfield CH 3 : 5. The final 90 Protein is folded o pulse makes the signal observable and signal is acquired in t2
!! 1D spectra Nuclear contain Overhause structural Effect SpectroscopY information.. but (NOESY) is hard to extract: need multidimensional NMR Biochemistry 530. / 4 #$ %&''!(&')'**+,- #$ 0)12+&' NMR Theory and. 3 Practice #$ 4+$+-5 6#! 77!),* 7!. /! 8!$#! 99!),* 9!. /! 8!#! 97!),* 9!. /!8!$#! 79!),* 7!. / :.6!),*! 7!. 3!8!),*! 9!. 3!: 7 @ 3 Dispersed amides: protein is folded 9 9 97 9 9 9 > 7? Gabriele Varani <+05,-0=!('0;* Department of Biochemistry and Ha: protein contains b-sheet Department of Chemistry 99 9 > 9? 7 )&,**('0;* 7 77 7 > 7? 79 7 > 9? @ University 7 of / Washington 7 9 When magnetization is exchanged, spin I signal contains information on spin S and viceversa (crosspeaks) 1D spectrum Cross-peaks appear between spins which are close in space (<5-6 A) (assignments and Downfield structure) CH 3 : Protein is folded
!! Nuclear Overhause Effect SpectroscopY (NOESY) 1D spectra contain structural information.. but is hard to extract: need multidimensional NMR Biochemistry 530. / 4 #$ %&''!(&')'**+,- #$ 0)12+&' NMR Theory and. 3 Practice #$ 4+$+-5 6#! 77!),* 7!. /! 8!$#! 99!),* 9!. /! 8!#! 97!),* 9!. /!8!$#! 79!),* 7!. / :.6!),*! 7!. 3!8!),*! 9!. 3!: 7 @ 3 Dispersed amides: protein is folded 9 9 97 9 9 9 > 7? Gabriele Varani <+05,-0=!('0;* Department of Biochemistry and and motion Ha: protein contains b-sheet Department of Chemistry 99 9 > 9? 7 )&,**('0;* 7 77 7 > 7? 79 7 > 9? @ University 7 of / Washington 7 9 The mixing coefficients, a IS = a SI are proportional to the NOE between these two nuclei The NOE is related to the distance r between the two spins and the correlation 1D spectrum time t c (the time for reorientation of the IS vector in the molecule): structure Downfield CH 3 : Protein is folded NOE r IS -6 f (t c ) m
1D spectra A simple contain example: structural a small information immunogenic.. but peptide is hard to extract: need multidimensional NMR Biochemistry 530 NMR Theory and Practice Dispersed amides: protein is folded Gabriele Varani Department of Biochemistry and Ha: protein contains b-sheet Department of Chemistry Downfield CH 3 : Protein is folded University of Washington 1D spectrum
1D Patterns spectra of contain NOE interactions structural information define protein.. but secondary is hard to extract: need structure multidimensional NMR Biochemistry 530 NMR Theory and Practice Gabriele Varani Department of Biochemistry and Ha: protein contains b-sheet Department of Chemistry Dispersed amides: protein is folded Downfield CH 3 : Observable NOE interactions Protein (<5 is A) folded in regular protein secondary structures University of Washington 1D spectrum
1D spectra COherence contain transfer structural SpectroscopY information.. (COSY) but is hard to extract: need multidimensional NMR! 65!HIJ! Biochemistry 530 NMR Theory and Practice 34 34 Gabriele Varani, -, 6 7189$:; Department of Biochemistry = <. 5 and Ha: protein contains b-sheet Department of Chemistry 12%#$>;:!7!,?2!#@$%!#0#,;AB!'!C!*D, - A @:;@7:7,$2% ;F2G9,$2% A$$%K >;,;1,$2% Dispersed The COSY amides: experiments detects interactions protein (correlation) is folded between spins that are Downfield scalar CH 3 : coupled: beware, it can only be understood Protein is folded through quantum mechanics University of Washington, 6 1D spectrum
1D spectra COherence contain transfer structural SpectroscopY information (COSY).. but is hard to extract: need multidimensional NMR 12%#$>;:!7!,?2!#@$%!#0#,;AB!'!C!*D = / #& & Biochemistry 530 NMR Theory and Practice 34 E:;;!@:;1;##$2%. Dispersed $!#$%!& *!!(! ) + amides: '* +, - protein is folded / #%!12#!& '!!(! ) '* +, - %!12#!& *!!_! ) + '* +, - #$!#$%!& '!!(! ) + 0 '* +, - $!#$%!& + *!!(! ) '* +, - # + Gabriele 34 Varani / 5 Department of Biochemistry, 6 7189$:; exchanges 0 and + Ha: protein contains b-sheet Department of Chemistry #$!#$%!& '!!(! ) '* +, - # < / #% % University of Washington 1. Excite with 90 o pulse, - 2. During t1, spins are labeled with their 0 Larmor frequency 3. During t1, if spins are scalar coupled, the signal encodes this 1D spectrum information as well 4. The second 90 o pulse magnetization between spins: spin I now has Downfield memory CH 3 of : spin S and Protein viceversa is folded 5. Signal is acquired in t2
1D spectra COherence contain transfer structural SpectroscopY information (COSY).. but is hard to extract: need multidimensional NMR! 7 Biochemistry 530. NMR / Theory 0)12+&' and Practice #$ %&''!(&')'**+,- #$ #!$/435*+-!6 7!!8! 9 7: ;!. /!8!!*+-!6 7!!<! 9 7: ;!. / =!!!!!$/435*+-!6 7!!8! 9 7: ;!. 3!8!!*+-!6 7!!<! 9 7: ;!. 3 = %#!$/435*+-6 :!!8! 9 7: ;!. /!8!!*+-!6 :!!<! 9 7: ;!. / =!!!!!$/435*+-!6 :!8! 9 7: ;!. 3!8!!*+-!6 :!!<! 9 7: ;!. 3 = Dispersed amides: protein is folded : B 3 : 97: 97: :!D!9 7:43 7!D!9 7:43 : C 7 ; : : C : ;. 3?+0@,-0A!('0>* Gabriele Varani Cross-peaks appear 1D spectrum between spins which are scalar coupled (assignments) Department of Biochemistry and Ha: protein contains b-sheet Department of Chemistry )&,**('0>* 7 7 C 7 ; 7 C : ; 7!D!9 7:43 :!D!9 7:43 When magnetization is exchanged, spin I signal contains information on spin S and viceversa (crosspeaks) The cross-peak fine Downfield structure CH contains 3 : Protein information is folded on scalar coupling (structure) University 7 B / of Washington
1D Structural spectra contain information: structural scalar information couplings.. directly but is hard gives to you the torsion extract: angles need that multidimensional define protein or NMR n.a. structure! Biochemistry 530 NMR Theory and Practice # % $ # % % # ' Dispersed amides: protein is folded! # $ %&'()*+, -.'!)*+ /0,',!! Gabriele Varani (Karplus, 1958) Department % of Biochemistry % Cross-peaks and in COSY Ha: protein experiments contains b-sheet occur only Department of Chemistry # $ # # &! %('&)*+, -.'1)*+ 0.'2!! between residues that are Downfield CH 3 : scalar coupled; in turns, these Protein is folded couplings can be measured in COSY experiments University of Washington 1D spectrum
1D COSY spectra and contain NOESY connectivities structural information the polypeptide.. but is hard unit to extract: need multidimensional NMR Biochemistry 530 NMR Theory and Practice Gabriele Varani Department of Biochemistry and Ha: protein contains b-sheet Department of Chemistry Dispersed amides: protein is folded Downfield CH 3 : COSY (broken lines) and NOESY Protein (continuous is folded lines) University of Washington 1D spectrum
Amino acid identification from scalar coupling patterns Different pattern of scalar couplings allows amino acid type identification in correlated spectra (COSY, 2QF-COSY, TOCSY) This is the first step towards complete spectral assignments of a protein spectrum (at least before heteronuclear NMR)
NOE interactions, scalar couplings (and chemical shifts) can be combined to define protein secondary structure NOE interactions (<4.5 A) and scalar coupling patterns in regular protein secondary structures
Even 2D spectra can be (and indeed are) very crowded Realistic limit of homonuclear NMR: proteins of 100-120 amino acids; spectra of larger proteins are too crowded
3D Heteronuclear NMR -/0123!#!$$%&'(!+*6# % 4#%#5%&'(,/0123 %- %,!#!$$%&'( #)'*+%&'( 4#%#5%&'(./0123 %- %,!#!$$%&'( #)'*+%&'( #)'*+%&'( %. 4#%#5%&'(
Useful nuclei such as 15 N, 13 C are rare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
How are experiments recorded: the Radiofrequency field Requirements for Heteronuclear (RF) NMR: isotope labeling! $ Modern NMR use radiofrequency pulses to # $# Isotopically labelled proteins can generate be prepared observable straightforwardly in E. coli by growing signals cells and in manipulate minimal the media (e.g. M9) supplemented with spin appropriate state of the nutrients system ( 15 NH4Cl, 13 C-glucose) # under study: this is the,!%&'())*+(,- basis of 2D/3D NMR but Metabolic %&!'()*+ pathways can be exploited also of and all appropriate FT NMR auxotrophic strains of E. coli can also be used for selective The alternating labelling: voltage e.g. use applied acetate across instead the of ends glucose of the and coil obtain the NMR probe selective induces labeling an alternating of certain side magnetic chain field CH 3 in the sample The geometry Isotopic of labelling the coil of protein is arranged expressed so in that eukaryotic this field cells is is perpendicular expensive to the but applied can be done field (in (post-translational the xy plane) modifications can be studied but you need $$$$) This oscillating B 1 field is substantially smaller than the external field B o (thousands of times smaller), and close to the Larmor frequency
Heteronuclear NMR exploits 1-bond scalar couplings 1-bond couplings are large (20-150 Hz) compared to HH couplings (1-10 Hz) They are independent of conformation: no structural insight but wonderful for assignments
The Heteronuclear basic building NMR block exploits of Heteronuclear 1-bond scalar NMR couplings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olarization of 1-bond couplings 13 C and are 15 N is low: start with large (20-150 Hz) compared 1 H polarization 1=60<65!=4<6ID;0C6Q to HH couplings!e!'fgh (1-10 78 Hz) Use 1-bond 9'($&>7 %&$'# B566!C5616DD4@< scalar couplings to transfer magnetization from ()!!&!*'+!%!&+'$, 9'($&>8 1 ()!!&!*'+!-!&+'$, H to the nucleus of interest They are independent of conformation: no structural!e!'fgh 78 insight B566!C5616DD4@< but wonderful for assignments Delay must be '+ set to 1/4J for optimal transfer: in the 94<IC;0D6!8%> absence of relaxation, magnetization transfer is 100% efficient
The Heteronuclear basic building NMR block exploits of of Heteronuclear 1-bond scalar NMR (INEPT-1D) couplings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olarization of 1-bond couplings 13 + C ): (!= and 6 are 15 N is low: start with large (20-150 Hz) compared 1 H polarization 1=60<65!=4<6ID;0C6Q to HH F # # couplings!e!'fgh (1-10 78 Hz) G 9'($&>7 <0HI2-.!34!J Use 1-bond %&$'# B566!C5616DD4@< scalar couplings %&'*() to transfer 3-!C!0K<44.L magnetization from ()!!&!*'+!%!&+'$, %&'(: 9'($&>8 1 ()!!&!*'+!-!&+'$, H to the nucleus of interest They are independent * of conformation: * no structural!e!'fgh &'(%#! 78 &'#%$! )(!*+,-./*01 insight B566!C5616DD4@< )%$!*+,-./*01 but 2*3+1,-#*,-4+5 wonderful for assignments Delay must be '+ 2*3+1,-#*,-4+5 set to 1/4J for optimal transfer: in the 94<IC;0D6!8%> absence of relaxation, magnetization transfer is 100% efficient
The Heteronuclear basic building NMR block exploits of 2D Heteronuclear 1-bond scalar NMR couplings (HSQC) Polarization of 13 C and 15 N is low: start with 1 H, transfer to 1 H with 1-bond INEPT (sensitivity couplings are increases large (20-150 by the Hz) ratios compared of, e.g. to 10 HH for N) couplings (1-10 Hz) Label magnetization with 15 N Larmor frequency in t1 and record 15 N evolution They are independent the first dimension of conformation: no structural insight but wonderful for assignments 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)
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)
HSQC The is most the important building block experiment and foundation for protein for structure very many determination: heteronuclear NMR 3D NOESY experiments HSQC Measurements of relaxation 1 H- 1 H distances properties (as in (motion) 2D NOESY) Spectral Resolution assignments is spread in a third dimension (usually 15 N but also 13 C; for nucleic acids mostly 13 C; for protein/nucleic acid complexes 3D versions you of can NOESY observe and COSY only the spectra protein, (structure) only the RNA or only the contact from one to the other)
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