Post on 15-Feb-2022
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Large Molecules
NOESY Spectra
Large Molecules
NOESY Spectra
Small Molecules
NOESY Spectra
Small Molecules
NOESY Spectra
Hb
Ha
Hc
C
Hc
Ha
Hb
δ (f2)
δ (f1)
NOESY (NOE SpectroscopY)
(+)
(-) Small molecule (+) Large molecule
-1
-0.5
0
0.5
0.01 0.1 1 10 100
0.1 1 10 100
0.4
-0.5
-1.0
ωτc
Small molecules
Large molecules
Extreme narrowing limit
NOESY (NOE SpectroscopY)
Higher temperature Lower temperature
Lower viscosity Higher viscosity
Lower field Higher field
Changing τc
Changing ω
Transverse cross-relaxation
Transverse cross-relaxation
Spin locking
Spin-locking – Theory
z
x’
y’
z
x’
y’
90
BSL (x) BSL
• BSL is a fluctuating magnetic field • It is static in the rotating frame. This is why these experiments are commonly called rotating-frame experiments.
Transverse Solomon equations
90 90 90
t1 tm
NOESY t2
ROESY (Rotating framE SpectroscopY)
90
t1 tm
ROESY t2
-Izcos(ΩIt1) -Szcos(ΩIt1) B0
-Ixcos(ΩIt1) -Sxcos(ΩIt1) BSL
ROESY (Rotating framE SpectroscopY)
ROESY (Rotating framE SpectroscopY)
ROESY (Rotating framE SpectroscopY)
ROESY (Rotating framE SpectroscopY)
ROESY (Rotating framE SpectroscopY)
Hb
Ha
Hc
C
Hb
Ha
Hc
δ (f2)
δ (f1)
(+)
(-) Small molecule ~0 “intermediate” molecules
(+) Large molecule (-) Every molecule using ROESY
NOESY vs ROESY
NOESY vs ROESY
Comparison of diagonal-peak and cross-peak amplitudes for NOESY and ROESY for a set of rotational correlation times. All simulations are for the case of two protons separated by 2 Å @ 500 MHz.
Transferred NOEs and bound conformations
• The information about the bound conformation is transferred to the free molecule by chemical exchange between bound and free species
• In a typical TrNOE experiment a low concentration of protein (≤0.1mM) and 10 to 30 molar equivalents of ligand are used
Relaxation and molecular size
78910 ppm78910 ppm78910 ppm78910 ppm 78910 ppm78910 ppm78910 ppm78910 ppm
6-residue peptide 180-residue protein
Free M13
M13 bound to CaM
M. Ikura and A. Bax, J. Am. Chem. Soc. 114,2433 (1992)
Relaxation and molecular size
Transferred NOEs and bound conformations
IF SF
SB IB
NOE σF
σB
Exchange k
NOE Kinetics and correlation time
IF SF NOE
σF IB SB NOE σB
Transferred NOEs and bound conformations
NOE int.
time
Mixing time
L
L Measured TrNOE Intensity
NOE of free L
τ
LE
Applicability limits of TrNOE
• The rate of exchange between free and bound states needs to be sufficient for an appreciable magnetization flux to occur between them.
• In this case:
<σ> = xFσF + (1-xF)σB
Applicability limits of TrNOE • <σ> should be dominated by the bound
term: xFσF <<(1-xF)σB
The extent to which this inequality is fulfilled depends on:
• the relative tumbling rates and distances in the free and bound states
• the equilibrium constant Kd
Transferred NOEs and bound conformations
D81
H57
S139
A157C159
R161
P6P5P4
P3
P2
P1
Basic aaAcidic aaHydrophobic aa
R161
D81
H57
C159
R155
F154 P6P5
P4P3P2
P1