NOESY Spectra
Transcript of NOESY Spectra
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Large Molecules
NOESY Spectra
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Large Molecules
NOESY Spectra
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Small Molecules
NOESY Spectra
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Small Molecules
NOESY Spectra
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Hb
Ha
Hc
C
Hc
Ha
Hb
δ (f2)
δ (f1)
NOESY (NOE SpectroscopY)
(+)
(-) Small molecule (+) Large molecule
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-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
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Transverse cross-relaxation
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Transverse cross-relaxation
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Spin locking
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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.
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Transverse Solomon equations
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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
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ROESY (Rotating framE SpectroscopY)
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ROESY (Rotating framE SpectroscopY)
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ROESY (Rotating framE SpectroscopY)
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ROESY (Rotating framE SpectroscopY)
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ROESY (Rotating framE SpectroscopY)
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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
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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.
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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
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Relaxation and molecular size
78910 ppm78910 ppm78910 ppm78910 ppm 78910 ppm78910 ppm78910 ppm78910 ppm
6-residue peptide 180-residue protein
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Free M13
M13 bound to CaM
M. Ikura and A. Bax, J. Am. Chem. Soc. 114,2433 (1992)
Relaxation and molecular size
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Transferred NOEs and bound conformations
IF SF
SB IB
NOE σF
σB
Exchange k
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NOE Kinetics and correlation time
IF SF NOE
σF IB SB NOE σB
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Transferred NOEs and bound conformations
NOE int.
time
Mixing time
L
L Measured TrNOE Intensity
NOE of free L
τ
LE
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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
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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
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Transferred NOEs and bound conformations
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D81
H57
S139
A157C159
R161
P6P5P4
P3
P2
P1
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Basic aaAcidic aaHydrophobic aa
R161
D81
H57
C159
R155
F154 P6P5
P4P3P2
P1