NMR parameters intensity chemical shift coupling constants 1D 1 H spectra of nucleic acids and proteins

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1 Lecture #2 M230 NMR parameters intensity chemical shift coupling constants Juli Feigon 1D 1 H spectra of nucleic acids and proteins

2 NMR Parameters A. Intensity (area) 1D NMR spectrum: integrated intensity of resonances is α # protons giving rise to signal. e.g. HN O CH 3 3 equiv. protons O N H 1 proton Relative intensity 3:1 B. Chemical Shift 1) Defines location of NMR line along rf axis v Determined by electronic environment around proton v Measured relative to a reference for aqueous solution, usually DSS (2,2-dimethyl-2-silapentane-5-sulfate). If defined in Hz, depends on magnitude of field; use absolute scale: PPM ν = frequency in Hz Gives + values of ppm relative to 0 for DSS

3 Macromolecules protons resonate ~ 0-16 ppm ( MHz 10,000 Hz) lowfield highfield (term from CW days) 16 ppm 0 ppm downfield less shielded higher frequency upfield defined as reference frequency (highly shielded) Use: (a) can classify types of protons by chemical shift e.g. aromatic, sugars, methyl (b) conformational changes will be manifested by chemical shift changes (due to changes in induced shift)

4 Factors that determine chemical shift: 2) Intrinsic chemical shift characteristic of a particular chemical group (due to electronic environment of covalent bonds) 3) Induced chemical shift shift from intrinsic position by the influence (thru space) of neighboring chemical centers Important types of induced shifts: a) H-bonding Causes large downfield shifts (up to ~5 ppm) e.g. N H N imino protons in base pairs b) ring current shifts Important effect of aromatic aa s and n.a. bases Give rise to chemical shift dispersion for ds na s and native proteins c) paramagnetic shifts (won t discuss in this course)

5 Recall: protons in benzene (aromatic) ring are intrinsically downfield shifted by electronic π cloud above and below ring Ring current field gives rise to ring current shifts induced in aromatic rings by external field B 0 applied perpendicular to ring [Electrons circulating around ring induce a magnetic moment that opposes the applied field at center of ring. Dipole field falls off as r -3 ] Extrinsic field from ring A opposes B 0 at Hb Hb is shielded and ring current shifted upfield Intrinsic field at attached proton Ha adds to B 0 proton is deshielded and appears at higher frequency higher ppm lower field

6 In general, protons in macromolecules located above or below an aromatic ring will be ring current shifted upfield. Especially important for stacked bases in nucleic acids (and for chemical shift dispersion of folded proteins). Maps of ring current shift calculations to predict size of induced shift Magnitude up to ~1.5 ppm For stacked base upfield vs H-bonding ~5 ppm downfield Ring current shielding values for adenine in a plane 3.4 Å away

7 C. Spin-spin coupling constants (J-coupling) 1) Splitting of proton resonances into multiplet structure due to weak interactions between protons on neighboring carbons. Qualitative explanation = OR slightly higher state A proton B Orientation of one proton has a small influence on orientation of electron. This gets communicated to 2nd nucleus due to Pauli exclusion principle for electrons. Info on spin orientation of A gets communicated to B via bonding electrons. J is field independent (since not dependent on B 0 ) expressed in Hz. Magnitude of interaction between A & B is given by coupling constant J AB

8 Spin-spin coupling J A B n J A B n Weakly coupled. We will usually assume this. Called AX spin system. Multiplet structure is recognizable for A Δν AB B even though intensities are distorted J J Deviation in intensities. Lines in center more intense, towards edges less intense. Can use 1st order analysis for Δν AB J A B J A B Strongly coupled. AB system. Convention: Use letters far apart in alphabet to denote weak coupling. # bonds # J HA H B spins Δν AB e.g. 3 J AB *Make sure you review rules for appearance of multiplets due to spin-spin splitting (weak coupling case) if you don t know them already.

9

10 2) Spin-spin coupling important for spectral identification (assignments) 3) Conformational analysis Karplus relation: J = A cos 2 φ + B cosφ + C, where A, B, C are empirical constants ϕ = 0 (cis) ϕ = 180 (trans) Size of coupling gives information on dihedral angle. If coupling constant can be measured, it can be used to determine dihedral angles. Example: sugar pucker (deoxyribose) 2 endo (B-DNA) S type J 1 2 ~ 10 Hz 3 endo (A-DNA) N type J 1 2 ~ Hz

11 Example: 3 J H NH α A very useful coupling constant for defining protein structure Measured coupling reveals that phi angle for residue is ~-120 o J = A cos 2 φ + B cosφ + C These coupling constants are determined for each residue as part of protein structure determination.

12 1D 1 H Spectra of Proteins and NA Nucleic Acids 1) Resonances Non-exchangeable Exchangeable (only seen in H 2 O) DNA Base AH8, GH8 AH2 aromatic TH6, CH6 CH5, TMe Deoxyribose - 1, 2, 2, 3, 4, 5, 5 iminos aminos RNA Same, except UH5, UH6 (like CH5, CH6) ribose, no 2 (2 only) 2) Spin systems (non-exchangeable) CH5-CH6, UH5-UH6 AX ~7 Hz TMe-TH6 A 3 X ~1.5 Hz deoxyribose XAMWTNP ribose H1 H5 (Assumes weak coupling; not true for H2, H2 & H5, H5 ) Conventions for identifying spin systems: AX weakly coupled AB strongly coupled A 3 X 3 chemically equivalent Closer in alphabet, more strongly coupled

13 ~1.5 Hz DNA deoxyribose TMe-TH6 ~1.5 Hz RNA ribose Minor groove: sugar, AH2, G amino 5 5 ~7 Hz Also, 31 P, and proton attached 13 C and 15 N And 13 C- 1 H, 15 N- 1 H, 31 P- 13 C, and 31 P- 1 H J coupling CH5-CH6 ~7 Hz UH5-UH6

14 3) 1D 1 H spectrum of a DNA dodecamer with N 6 A mod. A. Non-exchangeable 1) intensities Me 3x aromatics 2) chemical shifts à see regions on spectrum 3) coupling constants v CH5-CH6 v all sugar protons are coupled (more complicated) HDO H3 H4,H5 H5 m 6 A TMe C G C G A * A T T C G C G G C G C T T A * A G C G C * = m aromatic H1,H5 H2,H2 H8 H2 H6 * * **

15 3) cont 1D spectra of a DNA dodecamer with N 6 A mod. B. Exchangeable iminos aminos iminos A T G C amino aromatic C G C G A * A T T C G C G G C G C T T A * A G C G C * = m Strange appearance of spectrum has to do with the fact that the sample is in H 2 O (rather than D 2 O) M H vs mm DNA factor of 10 5 Dynamic range problem Need to suppress H 2 O signal

16 Proteins 1) Resonances R N C α H H O C Non-exchangeable: C α H R = aliphatic, aromatic, etc. Exchangeable: amide some aa side chains 2) Spin systems - non-exchangeable (more complicated than n.a.) see next page AX gly R= H A 3 X ala R= CH 3 AMX ser R= -CH 2 O H cys R= -CH 2 S H O asp R= -CH 2 C O H asn R= CH 2 C-N H 2 O = aromatics R= -CH 2 -ring (his, phe, tyr, trp) = exchangeable; not observed in D 2 O Example (more complicated) ε NH 3 CH 2 δ CH 2 γ CH 2 β CH 2 N C H Hα lysine O C Convention - written hi to low field A 2 (F 2 T 2 ) M P X γ δ ε α highest field β β similar chemical shifts lowest field Usually γ, δ, and ε are degenerate, but not always

17 Wüthrich p

18 3) 1D 1 H Protein spectrum in H 2 O -CH 3 Rnt1p dsrbd ~90 residues 800 MHz H 2 O -CH 2 - Backbone amide NH Plus sidechains resonances from: Trp, Phe, Tyr, His ring H Asn & Gln amides Arg NH Lys NH 2 CHα Later: 13 C chemical shifts imp. for aa assignment! 13 C and 15 N imp for 3D exp for sequential assignment

19 Wüthrich p.18

20 Example 1: Spectral dispersion greatly increases in folded protein Ubiquitin (76 aa) Unfolded. Bad spectral dispersion. Overlapped (degenerate) chemical shifts (i.e. all alanine methyl groups have similar chemical shift) Folded. Good spectral dispersion. Single peaks for atoms observed. Tertiary structure causes atoms to have unique magnetic environments. *Note the spectra also tell us about the purity of the sample. There are no major protonated inpurities in this sample. As the protein is ~1mM impurities in greater concentrations will show up as very large signals

21 Example 2: conformational changes are manifested by changes in chemical shift Z-DNA B-DNA Feigon et al. Science 230, (1985)

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