Lecture #2 M230 Juli Feigon NMR parameters intensity...

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

Transcript of Lecture #2 M230 Juli Feigon NMR parameters intensity...

Lecture #2 M230 Juli FeigonNMR parameters

intensitychemical shiftcoupling constants

1D 1H spectra of nucleic acids and proteins

NMR Parameters

1D NMR spectrum: integrated intensity of resonances is α # protons giving rise to signal.

CH3

O

HN

O NH

e.g.3 equiv. protons

1 protonRelative intensity 3:1

A. Intensity (area)

B. Chemical Shift

1) Defines location of NMR line along rf axisv Determined by electronic environment around protonv 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 HzGives + values of ppmrelative to 0 for DSS

Macromoleculesprotons resonate ~ 0-16 ppm (20 ppm @ 500 MHz ⇒ 10,000 Hz)

16 ppm 0 ppmupfielddownfield

less shieldedhigher frequency

defined asreference frequency(highly shielded)

lowfield highfield

(term from CW days)

Use: (a) can classify types of protons by chemical shifte.g. aromatic, sugars, methyl

(b) conformational changes will be manifested by chemical shift changes(due to changes in induced shift)

Important types of induced shifts:a) H-bonding

Causes large downfield shifts (up to ~5 ppm)

b) ring current shiftsImportant effect of aromatic aa’s and n.a. bases

Give rise to chemical shift dispersion for ds na’s and native proteins

N · · · · H—N imino protons in base pairse.g.

Factors that determine chemical shift:

2) Intrinsic chemical shiftcharacteristic of a particular chemical group(due to electronic environment of covalent bonds)

3) Induced chemical shiftshift from intrinsic position by the influence (thru space) of neighboringchemical centers

c) paramagnetic shifts (won’t discuss in this course)

Recall: protons in benzene (aromatic) ring are intrinsically downfield shifted byelectronic π cloud above and below ring

Ring current field gives rise to ring current shifts inducedin aromatic rings by external field B0 applied perpendicularto ring

[Electrons circulating around ring induce a magneticmoment that opposes the applied field at center of ring.Dipole field falls off as r-3]

Intrinsic field at attached protonHa adds to B0 ⇒ proton isdeshielded and appears at higherfrequency ≡ higher ppm ≡ lower field

Extrinsic field fromring A opposes B0 atHb ⇒ Hb is shieldedand ring currentshifted upfield

Ring currentshielding values foradenine in a plane3.4 Å away

Maps of ring current shiftcalculations to predictsize of induced shift

Magnitude up to ~1.5 ppmFor stacked base → upfieldvs H-bonding → ~5 ppm downfield

In general, protons in macromolecules located above or below an aromatic ringwill be ring current shifted upfield. Especially important for stacked basesin nucleic acids (and for chemical shift dispersion of folded proteins).

A B

proton

C. Spin-spin coupling constants (J-coupling)1) Splitting of proton resonances into multiplet structure due to

weak interactions between protons on neighboring carbons.

Qualitativeexplanation Orientation of one proton has

a small influence on orientationof electron. This getscommunicated to 2nd nucleusdue to Pauli exclusion principlefor electrons.

OR slightlyhigher state

Info on spin orientation of A gets communicatedto B via bonding electrons.

J is field independent (since not dependent on B0) ∴ expressed in Hz.

=

Magnitude of interaction between A & B is given by coupling constant JAB

Spin-spin coupling

Weakly coupled.We will usually assume this.Called AX spin system.

Deviation in intensities.Lines in center more intense, towards edges less intense.

Strongly coupled.AB system.

Multiplet structureis recognizablefor

even though intensitiesare “distorted”

Can use 1st orderanalysis for

Convention: Use lettersfar apart in alphabet todenote weak coupling.

*Make sure you review rules for appearance of multiplets due to spin-spinsplitting (weak coupling case) if you don’t know them already.

BΔνABA

JABn

JABn

ΔνAB

JJ

ΔνAB

JA

B

JA

B

#JHAHB← spins# bonds →

e.g. 3JAB

2) Spin-spin coupling important for spectral identification (assignments)3) Conformational analysis

Karplus relation:

⇒ Size of coupling givesinformation on dihedral angle.∴ If coupling constant can bemeasured, it can be used todetermine dihedral angles.

Example: sugar pucker (deoxyribose)

ϕ = 0° (cis)ϕ = 180° (trans)

2’endo (B-DNA)S typeJ1’2’ ~ 10 Hz

3’endo (A-DNA)N typeJ1’2’ ~ 1.5-3.3 Hz

J = A cos2φ + B cosφ + C, where A, B, C are empirical constants

Example: 3JHNHα

A very useful coupling constant fordefining protein structure

Measured couplingreveals that phi angle for residue is ~-120o

These coupling constants are determined for each residue as part of protein structure determination.

J = A cos2φ + B cosφ + C

Nucleic Acids1) Resonances

Non-exchangeable Exchangeable (only seen in H2O)

DNA Base AH8, GH8AH2TH6, CH6CH5, TMe

Deoxyribose - 1’, 2’, 2’’, 3’, 4’, 5’, 5’’

RNA Same, except UH5, UH6 (like CH5, CH6)ribose, no 2’’ (2’ only)

iminosaminos

2) Spin systems (non-exchangeable)CH5-CH6, UH5-UH6 AX ~7 HzTMe-TH6 A3X ~1.5 Hzdeoxyribose XAMWTNPribose

Conventions for identifying spin systems:AX weakly coupledAB strongly coupledA3X 3 chemically equivalent

Closer in alphabet,more strongly coupled

(Assumes weak coupling; not true for H2’, H2’’ & H5’, H5’’)H5’’H1’

aromatic

1D 1H Spectra of Proteins and NA

CH5-CH6 ~7 HzUH5-UH6

~7 Hz

~1.5 Hz

Minor groove:sugar, AH2, G amino

TMe-TH6 ~1.5 Hz1’ 3’ — 4’

2’

2’’

— 5’

5’’

5’

5’’1’ —2’ — 3’ — 4’

DNAdeoxyribose

RNAribose

Also, 31P, and proton attached 13C and 15NAnd 13C-1H, 15N-1H, 31P-13C, and 31P-1H J coupling

3) 1D 1H spectrum of a DNA dodecamer with N6A mod.A. Non-exchangeable

1) intensities Me 3x aromatics2) chemical shifts

à see regions on spectrum3) coupling constants

v CH5-CH6v all sugar protons are coupled (more complicated)

1 2 3 4 5 6 7 8 9 10 11 12 C G C G A A T T C G C GG C G C T T A A G C G C12 11 10 9 8 7 6 5 4 3 2 1

** * = m6

m6AHDO

aromaticH1’,H5

H3’

H4’,H5’H5”

H2’,H2”

TMe

H8H2

H6

* * **

3) cont 1D spectra of a DNA dodecamer with N6A mod.B. Exchangeable

iminosaminos

Strange appearance of spectrum has to do with the fact that the sample is in H2O (rather than D2O) -- 110 M H vs mM DNA ⇒ factor of 105

⇒ Dynamic range problemNeed to suppress H2O signal

iminos

A•T G•C aminoaromatic

1 2 3 4 5 6 7 8 9 10 11 12 C G C G A A T T C G C GG C G C T T A A G C G C12 11 10 9 8 7 6 5 4 3 2 1

** * = m6

Proteins1) Resonances

Non-exchangeable:CαHR = aliphatic, aromatic, etc.

Exchangeable:amidesome aa side chains

2) Spin systems - non-exchangeable (more complicated than n.a.) → see next page

AX gly R= HA3X ala R= CH3AMX ser R= -CH2O H

cys R= -CH2S H

asp R= -CH2C

asn R= CH2C-N H2

aromatics R= -CH2-ring(his, phe, tyr, trp)

OO H

=

O

= exchangeable;not observed in D2O

N C C

R O

Example(more complicated)

lysine

Convention -written hi to low field

A2 (F2 T2) M P XNH3

CH2

CH2

CH2

CH2

ε

δγ

βN C

O

H Hα

highestfield

lowestfield

β βδ ε

similar chemical shifts

Usuallyγ, δ, and εare degenerate,but not always

H

C

γ α

Wüthrich p. 15-16

3) 1D 1H Protein spectrum in H2O

Rnt1p dsRBD~90 residues800 MHz

Backbone amide NHPlus sidechains resonances from:Trp, Phe, Tyr, His ring HAsn & Gln amidesArg NHLys NH2

H2O

-CH2-

-CH3

CHα

Later: 13C chemical shifts imp. for aa assignment!13C and 15N imp for 3D exp for sequential assignment

Wüthrich p.18

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

Example 1: Spectral dispersion greatly increases in folded protein

Feigon et al. Science 230, 82-84 (1985)

Z-DNA

B-DNA

Example 2: conformational changes are manifested by changes in chemical shift