Chemical exchange

Literature: P. J. Hore, Nuclear Magnetic Resonance, Oxford, 1995

Chemical exchange is manifested in NMR spectra

Slow exchange: separate signals for each conformer

Fast exchange: signals are averaged

Chemical exchange

The chemical exchange leads to broadened lines unless it

is very slow or very fast. The exchange rate can be

derived from the line broadening.

Coalescence: k = ( )/

• Fast exchange requires that k > .

(The averaging is more efficient if the lifetime of the states, 1/k, is short.)

• If is large, the two lines won’t average so easily.

Fast exchange

Intermediate exchange

More complicated lineshapes which can be expressed as sums of two phase-

and frequency-shifted Lorentzians

Reeves & Shaw, Can. J. Chem. 48, 3641 (1970)

The NMR time-scale

• The NMR time-scale is defined by the

difference in frequency (Hz) between the exchanging sites, .

• The notation slow, fast and intermediate

exchange refers to that time-scale.

• The NMR time-scale depends on the

spectrometer frequency and the nucleus

considered (1H, 13C etc).

Unsymmetrical two-site exchange

The ratio of the rate constants

is the equilibrium constant:

kA/kB = K

• Fast exchange: the frequency

of the signal is at the population-

weighted average of the individual

signals.

• Slow exchange: the signal of

the less populated state

broadens first.

Tyrosine ring flips in BPTI

• BPTI was the first protein

studied in depth by NMR.

• The 180o ring flip of Tyr35 is slow on

the NMR time scale at 4 oC and fast

at 72 oC.

• The signals of protons 3 and 5

collapse first because their chemical

shift difference is smaller than that of

protons 2 and 6.

MVTFHTNHGD IVIKTFDDKA PETVKNFLDY CREGFYNNTI FHRVINGFMI

QGGGFEPGMK QKATKEPIKN EANNGLKNTR GTLAMARTQA PHSATAQFFI

NVVDNDFLNF SGESLQGWGY CVFAEVVDGM DVVDKIKGVA TGRSGMHQDV

PKEDVIIESV TVSE

NN

O

CCC

O

CC

HH H H

Amino-acid selective labelling of proteins

E. coli prolyl cis-trans isomerase (PpiB)

• The 15N-HSQC spectrum correlates the chemical

shifts of directly bonded 15N and 1H.

• 15N-labelled proteins are made in E. coli using

15N-labelled media.

• The 15N-HSQC spectrum can be simplified by

using a medium of 19 unlabelled amino acids and one 15N-labelled amino acid only the

labelled residues are observed.

NN

O

CCC

O

CC

HH H H

MVTFHTNHGD IVIKTFDDKA PETVKNFLDY CREGFYNNTI FHRVINGFMI

QGGGFEPGMK QKATKEPIKN EANNGLKNTR GTLAMARTQA PHSATAQFFI

NVVDNDFLNF SGESLQGWGY CVFAEVVDGM DVVDKIKGVA TGRSGMHQDV

PKEDVIIESV TVSE

15N-HSQC spectrum of 15N-arginine labelled PpiB

The 15N-HSQC cross-peak of Arg87 can be assigned by double-selective labelling,

using 15N-Arg and 13C-Ala. Only the 15N of Arg87 will show scalar coupling to 13C.

An HNCO experiment transfers magnetizationvia the pathway H N C.

15N-Arg labelled PpiB

15N-Arg/13C-Ala double-labelled PpiB

Only the cross-peak of Arg87 is visible.

Addition of a ligand (signals from the ligand are circled).

Only the cross-peak of Arg87 shifts.

Therefore, the ligand binds near Arg87.

HSQC

HNCO

HSQC

+ ligand

Arg87

FEBS Lett. 524, 159 (2002)

Titration of an RNA-binding protein with RNA

Change in chemical shifts with increasing

RNA concentration,

(a) showing 1:1 binding

(b) allowing determination of the binding constant

Superimposition of 15N-HSQC spectra

in the presence of increasing amounts

of RNA

• Binding affinity can be measured for 10 μM < Kd < 1 M.

• Identifies binding site.

• Uniquely suited for weak binding.

• Used very often in drug development.

Ligand binding by NMR

• DHFR undergoes a catalytic cycle with 2 substrates.

• In each state, DHFR has a somewhat different

conformation and is in equilibrium with minor

conformational species.

• The rates of conformational changes in the protein

govern the rates of the individual steps in the cycle.

Science 313, 1638 (2006)