3d triple resonance methods for sequential resonance assignment of proteins n.
Skip this Video
Download Presentation
3D Triple-Resonance Methods for Sequential Resonance Assignment of Proteins

Loading in 2 Seconds...

play fullscreen
1 / 15

3D Triple-Resonance Methods for Sequential Resonance Assignment of Proteins - PowerPoint PPT Presentation

  • Uploaded on

3D Triple-Resonance Methods for Sequential Resonance Assignment of Proteins. Strategy: Correlate Chemical Shifts of Sequentially Related Amides to the Same C a (or C b or C O ) Chemical Shifts. Intraresidue Correlation (HNCA). Interresidue Correlation (HN(CO)CA. Excite C a. Excite C a.

I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
Download Presentation

PowerPoint Slideshow about '3D Triple-Resonance Methods for Sequential Resonance Assignment of Proteins' - frayne

Download Now An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.

- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
3d triple resonance methods for sequential resonance assignment of proteins
3D Triple-Resonance Methods for Sequential Resonance Assignment of Proteins

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

Intraresidue Correlation (HNCA)

Interresidue Correlation (HN(CO)CA

Excite Ca

Excite Ca

Record Ca frequencies

Record Ca frequencies

Transfer to intraresidue N

Transfer to intraresidue CO

Record N frequencies

Transfer to interresidue N

Transfer to HN

Record N frequencies

Record H frequencies

Transfer to interresidue HN

Record H frequencies

triple resonance data
Triple-resonance Data



Intraresidue Data

(Both Ca & Cb)

Interresidue Data

(Both Ca & Cb)

protein chemical shifts indicate secondary structures with high accuracy
Protein Chemical Shifts IndicateSecondary Structures with High Accuracy

Assign Chemical Shifts (Referencing Relative to DSS)

Compare Chemical Shifts to those in random coil peptides















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

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

identification of close interproton distances
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)

rIS = internuclear distance

f(tc) = statistical quantity which describes the

timescale with which a molecule reorients in


NOE µ (1/rIS6)f(tc)

noes in structure determination
NOEs in Structure Determination

NOEs can be identified through

two-, three-, and four-dimensional

spectra once the 1H resonance

assignments are complete

NOESY Procedure:

Excite First Proton

Record Proton Frequencies

Transfer to Any proton 5 Å or

less by NOE

4. Record Proton Frequencies

noe analysis practical aspects
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


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
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

10 - 15 interresidue

NOEs per AA

secondary structures can be characterized by regular patterns of noes
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
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, 79-105

detection of hydrogen bonds
Detection of Hydrogen Bonds

h3JNC’ -0.2 to -0.9

h2JHC’ -0.6 to 1.3

h3JHCa 0.0 to 1.4

Ref: Grzesiek, et al. (2001) Methods Enzymol., 338, 111-133

anisotropic tumbling w r t to b o results in residual dipolar couplings
Anisotropic Tumbling w.r.t. to BoResults in Residual Dipolar Couplings
  • Magnitude of the dipole-dipole interaction is orientation dependent w.r.t. to the static magnetic field (Bo)
  • Isotropic tumbling w.r.t. Bo 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
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)


phatidylcholine (DHPC)


phatidylcholine (DMPC)


RDC for Proteins in Solution Correlate Very Well With

Predictions from High-Resolution Crystal Structures


NMR Structure Determination

Start with a peptide chain of random starting conformation

Subject protein to a classical mechanical treatment (such as “simulated annealing”) that minimizes the total energy

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)