Biochemistry 530 NMR Theory and Practice

Gabriele Varani Department of Biochemistry and

Department of Chemistry University of Washington

1D spectra contain structural information .. but is hard to extract: need multidimensional NMR

1D spectrum

Dispersed amides: protein is folded

Hα: protein contains β-sheet

Downfield CH3: Protein is folded

1D vs 2D homonuclear NMR Spectroscopy

Basic structure of 1D experiment

Basic structure of 2D experiment (e.g. NOESY)

Jeener and Ernst, 1972

2D homonuclear NMR Spectroscopy

Basic structure

Two basic experiments: NOESY and COSY

How to generate a 2D experiment: NOESY

1. Record numerous 1D experiments by systematically incrementing time t1 from 0 to tmax; the experiment generates a matrix of points S(t1,t2) which encodes the frequency information with respect to both t1 and t2

Typical number of repetitions (t1 increments) for homonuclear NMR: 256-512

How to process the data to obtain a 2D spectrum

2. Execute the FT for each experiment (each t1 value) with respect to time t2 to generate an interferogram S(t1, ω2) 3. Execute the FT with respect to t1 (for each ω2 value) to generate the final 2D spectrum S(ω1,ω2)

1D vs 2D NMR spectra of a protein

1D spectrum

amides Hα Side chain CH2

Side chain CH3

NH2

1D vs 2D NMR spectra of a protein

2D projection representation

2D contour representation

“cross-peaks” in multidimensional NMR carry structural information

2D contour representation: peaks outside diagonal are called cross-peaks

Diagonal is closely related to 1D spectrum

Nuclear Overhauser Effect SpectroscopY (NOESY)

The NOESY experiments detects interactions between spins that are close in space and dipolar coupled: the magnetization transfer mechanism is essentially the same as FRET in optical spectroscopy (fluorescence)

1. Excite with 90o pulse

2. During t1, spins are labeled with their Larmor frequency

3. The second 90o pulse exchanges rotates magnetization to -z

5. The final 90o pulse makes the signal observable and signal is acquired in t2

Nuclear Overhause Effect SpectroscopY (NOESY)

4. Magnetization is exchanged between spin I and S during the mixing time

Cross-peaks appear between spins which are close in space (<5-6 A) (assignments and structure)

When magnetization is exchanged, spin I signal contains information on spin S and viceversa (cross-peaks)

Nuclear Overhause Effect SpectroscopY (NOESY)

The mixing coefficients, αIS = αSI are proportional to the NOE between these two nuclei

The NOE is related to the distance r between the two spins and the correlation time τc (the time for reorientation of the IS vector in the molecule): structure and motion

Nuclear Overhause Effect SpectroscopY (NOESY)

NOE ∝ r IS-6f (τc) τm

A simple example: a small immunogenic peptide

A simple example: a small immunogenic peptide

COSY and NOESY connectivities in the polypeptide unit

COSY (broken lines) and NOESY (continuous lines)

Protein chemical structure dictates ‘local’ NOE interactions

Sequential and medium range interactions in polypeptides

Protein secondary structure determines local NOE interactions

Sequential and medium range NOE interactions in regular turns

Protein 3D structure determines local NOE interactions

Sequential and medium range NOE interactions in an α-helix

Protein 3D structure determines NOE interactions

Medium and long range NOE interactions in a parallel and anti-parallel β-sheets

Patterns of NOE interactions define protein secondary structure

Observable NOE interactions (<5 A) in regular protein secondary structures

NOE interactions, scalar couplings (and chemical shifts) can be combined to define protein secondary structure

NOE interactions (<4.5 A) and scalar coupling patterns in regular protein secondary structures

A simple example: a small immunogenic peptide

A simple example: a small immunogenic peptide

COherence transfer SpectroscopY (COSY)

Cross-section of cross-peak between HN and Hα proton allows measurement of scalar couplings for residue C57

COherence transfer SpectroscopY (COSY)

The COSY experiments detects interactions (correlation) between spins that are scalar coupled: beware, it can only be understood through quantum mechanics

COherence transfer SpectroscopY (COSY)

1. Excite with 90o pulse

2. During t1, spins are labeled with their Larmor frequency

3. During t1, if spins are scalar coupled, the signal encodes this information as well

4. The second 90o pulse exchanges magnetization between spins: spin I now has memory of spin S and viceversa

5. Signal is acquired in t2

COherence transfer SpectroscopY (COSY)

Cross-peaks appear between spins which are scalar coupled (assignments)

± ±

±

±The cross-peak fine structure contains information on scalar coupling (structure)

When magnetization is exchanged, spin I signal contains information on spin S and viceversa (cross-peaks)

Structural information: scalar couplings directly gives you the torsion angles that define protein or n.a. structure

(Karplus, 1958)

3JHαN=5.9cos2φ-1.3cosφ +2.2

3Jαβ=9.5cos2χ1-1.6cosχ1+1.8

Cross-peaks in COSY experiments occur only between residues that are scalar coupled; in turns, these couplings can be measured in COSY experiments

COSY and NOESY connectivities in the polypeptide unit

COSY (broken lines) and NOESY (continuous lines)

Amino acid chemical structure

Different pattern of scalar couplings

Amino acid identification from scalar coupling patterns

Different pattern of scalar couplings allows amino acid type identification in correlated spectra (COSY, 2QF-COSY, TOCSY)

This is the first step towards complete spectral assignments of a protein spectrum (at least before heteronuclear NMR)

Amino acid chemical structure leads to distinct shift for each residue: random coil chemical shift values

Residue NH αH βH Others Gly 8.39 3.97

Ala 8.25 4.35 1.39

Val 8.44 4.18 2.13 γCH3 0.97, 0.94

Ile 8.19 4.23 1.90 γCH2 1.48, 1.19 γCH3 0.95 δCH3 0.89

Leu 8.42 4.38 1.65,1.65 γH 1.64 γCH3 0.94, 0.90

Prob 4.44 2.28,2.02 γCH2 2.03, 2.03 δCH2 3.68, 3.65

NOE interactions, scalar couplings (and chemical shifts) can be combined to define protein secondary structure

NOE interactions (<4.5 A) and scalar coupling patterns in regular protein secondary structures