one-dimensional spectra provides 1. chemical shifts & relative intensities 2. j-couplings
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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
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