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

H2N CH C

CH2

O

C

NH2

O

N CH C

CH2

O

N

NH

N CH C

CH2

O

CH2

C

OH

O

N CH C

CH2

OH

O

OH

H H H

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

C C NN

C

O HH

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)

N CH C

R2

O

CH C

R1

O

N

H H 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 -sheetCCCO

H

positive negativenone positive

positive negativenegative positive

Wishart, et al., Biochemistry, 31, 1647 (1992)Wishart, et al., J. Biomol. NMR, 4, 171 (1994)

Identification of Close Interproton DistancesProtons 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/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 throughtwo-, three-, and four-dimensional spectra 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

N

HH3C

H2C O

NH2H

H

NH3+

COO-

NOE Analysis - Practical AspectsProtein 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

N

HH3C

H2C O

NH2H

H

NH3+

COO-

Isotope Editing Enhances Spectral Resolution

N

HH3C

H2C O

NH2H

H

NH3+

COO-

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

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

HN

NCO(i-1)

CO(i)

HR

HHN

Bax, et al. (1994) Measurement of Homo- and Heteronuclear J-couplings from Quantitative J Correlation, Methods Enzymol., 239, 79-105

Detection of Hydrogen Bonds

C

CON H

C

CON H

C

CON H

h3JNC’ -0.2 to -0.9

h2JHC’ -0.6 to 1.3

h3JHC 0.0 to 1.4

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

Anisotropic Tumbling w.r.t. to BAnisotropic Tumbling w.r.t. to Boo

Results in Residual Dipolar CouplingsResults 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

ISθ = 90° I

S

θ = 54.7°θ = 0°I

SBdd ∝ −1 Bdd ∝ +2Bdd = 0

€

Bdd

= Dmax

AB

IAz

IBz

Bz ( 3 cos

2

θ − 1 )

Induced Residual Alignment of Diamagnetic Induced Residual Alignment of Diamagnetic ProteinsProteins

• 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)

O

12

O

O

12

O

P

O

-O O

N(CH3)+

O

5

O

O

5

O

P

O

-O O

N(CH3)+

RDC for Proteins in Solution Correlate Very Well WithPredictions 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 usedTo 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)

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ENOE = f r(r − R)

r R

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ETot = ENOE + E J−coupling + EH−bond + ERDC + ...