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 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 through two-, three-, and four-dimensional spectra 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

Secondary Structures Can Be Characterized by Regular Patterns of NOEs K. Wüthrich (1986) NMR of Proteins and Nucleic Acids, Wiley Interscience

Angular Dependence of 3-bond J-couplings  Bax, et al. (1994) Measurement of Homo- and Heteronuclear J-couplings from Quantitative J Correlation, Methods Enzymol., 239,

Detection of Hydrogen Bonds h3 J NC’ -0.2 to -0.9 h2 J HC’ -0.6 to 1.3 h3 J HC  0.0 to 1.4 Ref: Grzesiek, et al. (2001) Methods Enzymol., 338,

Anisotropic Tumbling w.r.t. to B o Results in Residual Dipolar Couplings Magnitude of the dipole- dipole interaction is orientation dependent w.r.t. to the static magnetic field (B o ) Isotropic tumbling w.r.t. B o normally averages dipolar couplings to zero Small, but non-zero, magnetic susceptibility results in residual dipolar couplings that appear as apparent J-splittings

Induced Residual Alignment of Diamagnetic Proteins Lipid Bicelles LC (Tjandra & Bax, Science, 1997) Purified Bacteriophage Particles (Pf1) LC(Hansen et al, J. Am. Chem. Soc, 1998) Deformed Pores in Nondenaturing Polyacrylamide Gel (Sass et al, J. Biomol. NMR, 2000) dimyristoyl-phos- phatidylcholine (DMPC) dihexanoyl-phos- phatidylcholine (DHPC)

RDC for Proteins in Solution Correlate Very Well With Predictions from High-Resolution Crystal Structures

NMR Structure Determination 1.Start with a peptide chain of random starting conformation 2.Subject protein to a classical mechanical treatment (such as “simulated annealing”) that minimizes the total energy 3.Simulated annealing protocol is a common one used To minimize the energy. Protein is heated in the computer, which allows molecular motions to occur, and then is slowly cooled to minimize the energy (avoids local minima in the energy landscape)