One-dimensional Spectra Provides 1. Chemical shifts & Relative Intensities 2. J-couplings.

One-dimensional Spectra Provides

1. Chemical shifts & Relative Intensities

2. J-couplings

More Sophisticated Techniques Are Required for

Proteins and Other Macromolecules

1H (ppm) 13C (ppm)

Thr

Asp

His

Ala

Thr

Asp

His

Ala

C CO ’ C CC

Two-Dimensional Data Sets Greatly Increase

Spectral Resolution

Procedure For Recording a 2DSpectrum Minimally Involves

1) Exciting the first nucleus2) Recording the frequenciesof the first nucleus3) Utilizing some type of physical interactionto transfer NMR signal to second nucleus

4) Recording the frequencies of the second nucleus

1) Excite C2) Record C frequencies

3) Transfer C to H

4) Record H frequencies

Acquisition Time 2hIle in D2O

2D Spectra Provide Enhanced Resolution

1H (ppm)

13C

(pp

m)

Increasing the Information Content Through Additional Interactions

Excite C

Record C frequencies

Transfer C to H

Record H frequencies

1H (ppm)

13C

(pp

m)

Transfer H to H

NOTE: connectivities for blue and green would also be observed (not shown so as to avoid complexity in the diagram)

1H-15N Correlation Spectrum of a 26 kDa Protein

3D Triple-Resonance Methods for Sequential Resonance Assignment of Proteins

Strategy: Correlate Chemical Shifts of Sequentially Related Amides to the Same C (or C or CO) Chemical Shifts

Intraresidue Correlation (HNCA)

Excite CRecord C frequenciesTransfer to intraresidue NRecord N frequenciesTransfer to HNRecord H frequencies

Interresidue Correlation (HN(CO)CA)

Excite CRecord C frequenciesTransfer to intraresidue CO

Transfer to interresidue NRecord N frequenciesTransfer to interresidue HNRecord H frequencies

Triple-resonance Data

Intraresidue Data(Both C & C)

Interresidue Data(Both C & C)

i+1i

Protein Chemical Shifts IndicateSecondary Structures with High Accuracy

Assign Chemical Shifts (Referencing Relative to DSS)

Compare Chemical Shifts to those in random coil peptides

-helix -sheet

CCC

H

positive negative

none positive

positive negative

negative positive

Wishart, et al., Biochemistry, 31, 1647 (1992)

Wishart, et al., J. Biomol. NMR, 4, 171 (1994)

Identification of Close Interproton Distances

Protons separated in space by about 5 Å or less will influence the relaxation properties of one another (via dipole-dipole interactions): Known as the Nuclear Overhauser Effect, or NOE

Importantly, note that this effect is in general distinct from the interaction between nuclei via J-couplings; J-couplings are mediated by electron orbital overlap between chemically bonded nuclei and are thus are only observed between nuclei separated by about 4 chemical bonds, or less

NOEs instead can be observed in theory between any two possible protons within a molecule separated by 5 Å or less (irregardless of the number of chemical bonds by which the atoms are separated)

NOE (1/rIS6)f(c) rIS = internuclear distance

f(c) = statistical quantity which describes the timescale with which a molecule reorients in solution

NOEs in Structure Determination

NOEs can be identified from a 2-D 1H-1H NOESY spectrum once the 1H resonance assignments are complete

NOESY Procedure:

1. Excite First Proton2. Record Proton Frequencies3. Transfer to Any proton 5 Å or less by NOE4. Record Proton Frequencies

NOE Analysis - Practical Aspects

Protein of 150 residues typically has about 30 possible NOEsper residue; unambiguous identification of these can be difficultwith 2D NOE methods alone

NOE spectra can be simplified and extended into more than twodimensions by employing isotope-editing

Procedure:

Excite nitrogenRecord nitrogen frequenciesTransfer to attached proton (J-coupling)Record proton frequenciesTransfer to any proton 5 Å or less (NOE)Record Proton Frequencies

Isotope Editing Enhances Spectral Resolution

Typically 3D 15N-edited NOESY 3D 13C-edited NOESY

4D 13C-edited, 13C-edited 4D 15N-edited, 13C-edited

Typically, recover10 - 15 interresidueNOEs per AA