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

More Sophisticated Techniques Are Required for Proteins and Other Macromolecules 1 H (ppm) 13 C (ppm) Thr Asp His Ala Thr Asp His Ala CC CO’CO’     CC C  C 

Two-Dimensional Data Sets Greatly Increase Spectral Resolution Procedure For Recording a 2D Spectrum Minimally Involves 1)Exciting the first nucleus 2)Recording the frequencies of the first nucleus 3) Utilizing some type of physical interaction to transfer NMR signal to second nucleus 4) Recording the frequencies of the second nucleus 1) Excite C 2) Record C frequencies 3) Transfer C to H 4) Record H frequencies Acquisition Time 2h Ile in D 2 O

2D Spectra Provide Enhanced Resolution 1 H (ppm) 13 C (ppm)   

Increasing the Information Content Through Additional Interactions Excite C Record C frequencies Transfer C to H Record H frequencies 1 H (ppm) 13 C (ppm)    Transfer H to H NOTE:  connectivities for blue and green would also be observed (not shown so as to avoid complexity in the diagram)

1 H- 15 N 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 C O ) Chemical Shifts Intraresidue Correlation (HNCA) Excite C  Record C  frequencies Transfer to intraresidue N Record N frequencies Transfer to HN Record H frequencies Interresidue Correlation (HN(CO)CA) Excite C  Record C  frequencies Transfer to intraresidue C O Transfer to interresidue N Record N frequencies Transfer to interresidue HN Record H frequencies

Triple-resonance Data Intraresidue Data (Both C  & C  ) Interresidue Data (Both C  & C  ) i+1i

Protein Chemical Shifts Indicate Secondary Structures with High Accuracy Assign Chemical Shifts (Referencing Relative to DSS) Compare Chemical Shifts to those in random coil peptides  -helix  -sheet CC CC CC HH positivenegative nonepositive 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/r IS 6 )f(  c ) r IS = 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 1 H- 1 H NOESY spectrum once the 1 H resonance assignments are complete NOESY Procedure: 1.Excite First Proton 2.Record Proton Frequencies 3.Transfer to Any proton 5 Å or less by NOE 4. Record Proton Frequencies

NOE Analysis - Practical Aspects Protein of 150 residues typically has about 30 possible NOEs per residue; unambiguous identification of these can be difficult with 2D NOE methods alone NOE spectra can be simplified and extended into more than two dimensions by employing isotope-editing Procedure: Excite nitrogen Record nitrogen frequencies Transfer to attached proton (J-coupling) Record proton frequencies Transfer 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, recover interresidue NOEs per AA