Transcript Introduction to Biomolecular NMR

Introduction to Biomolecular NMR
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Nuclear Magnetic Resonance
Spectroscopy
• Certain isotopes (1H, 13C, 15N, 31P )
have intrinsic magnetic moment
n
m
• Precess like tops in magnetic field B0
Bo
w = g Bo
• In a 600 MHz spectrometer
– protons precess at 600 MHz
– 15N nuclei precess at ~60 MHz
– 13C nuclei precess at ~125 MHz
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Creating coherence
z
Mo
x
B1
•
Unless the spins are aligned (coherent),
their nett effect will be zero
•
B0 field aligns spins M0
•
B1 field rotates M0 into x-y plane
•
M0 rotates at speed n in x-y plane
•
Coils in x-y plane record fluctuating
magnetic field
•
B1 field must rotate about z-axis at
precession frequency n
Bo
y
i
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1D NMR experiment
z
z
Mo
90y
x
x
pulse
y
y
Mxy
Free Induction Decay
(FID)
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Free Induction Decay
FT
M

M(t) = cos(w t) exp(- t/T)
t
w
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Fourier transform spectroscopy
• System resonates at many
different frequencies (c.f.
church bell)
t
• Excite all frequencies
simultaneously using a ‘hard’
pulse
• Frequency analyse (Fourier
transform) to yield
component frequencies
w
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Two major causes of decay of signal
• Spin-lattice relaxation (T1 decay)
z
M
x
– loss of energy by spins leads to return of M
to z axis
M(t) = M(0) e-t / T1
y
– happens with time constant T1
• Loss of coherence due to dephasing (T2
decay)
z
M(t) = M(0) e-t / T2
x
y
M
• T1 >> T2
• T2 inversely related to homogeneity of B0
• No energy is lost during dephasing 
signal may be refocused
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NMR of proteins
• A sample of protein contains many protons
–
–
–
–
–
HN proton attached to N on backbone
Ha proton attached to Ca on backbone
Hb proton attached to Cb on backbone (typically 2)
Hg proton attached to Cg on backbone
Protons in H2O molecules (concentration 110 M as compared to ~1mM for protein)
• Different protons precess at different frequencies, depending on their
chemical environment
w = - g (1-s) B
0
s depends on the chemical shielding; e.g. how exposed the nucleus is to the
solvent or how close it is to a heavy atom such as C or N
– protons in water correspond to s=0 (no chemical shielding)
– protons in the protein may have s>0 (to the right of the water peak) or s<0 (to
the left)
–
• Define a B0-independent scale:
– known as ppm’s
 = wH2O - w ) / ( 106 wH2O ) = s / 106
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1D NMR spectrum of a protein
• In terms of ppm scale, peaks appear
at same place irrespective of the
strength of B0
 larger proteins have more
overlapping peaks
• But line width is independent of B0
– roughly  T2-1
– increases with size of protein
 less overlap at higher field
• Also strength of signal increases
with B0
• Conclusion: going to higher field
increases sensitivity and resolution
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Interactions between nuclei (couplings)
• Coupled springs
– transfer of energy back and forth
• Scalar coupling
–
–
–
–
mediated through overlap of electronic orbitals
“through bond” coupling
useful for assigning particular peaks to particular protons
determine covalent structure of the protein molecule
• Dipolar coupling
– results from interaction of dipolar fields of nuclei
– “through space” coupling
– useful for determining non-covalent structure (folded shape) of molecule
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Simplest 2D experiment
Correlation spectrOScopY experiment
90y
90x
t2
COSY pulse sequence
t1
•
•
•
•
Pair of coupled nuclei s1 and s2
Record whole series of 1D experiments, each with a different value of t1
Second 90 pulse transfers magnetization from s1 to s2
Data acquired during t2 tells us the precession frequency (w2) of s2
S(t2) = cos(w2t2)
• During t1 magnetization is on s1 and therefore precesses at frequency w1
– initial magnitude at beginning of t2 depends on t1 and w1
S(t1,t2) = cos(w1t1) cos(w2t2)
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The amplitiude of the 1D spectrum acquired during t2 varies sinusoidally with a different
frequency as a function of the interval t1, indicating that during t1 the magnetization is on a
spin with the corresponding frequency
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2D NMR spectrum
Fourier transform in both
t1and t2 gives S(w1, w2),
which when plotted as
contour function gives a peak
at coordinates w1 and w2
w1
w2
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2D COSY spectrum
• Magnetization which stays on
same nucleus during t1and t2 has
the same frequency in both
dimensions
 along the diagonal
• Magnetisation which jumps from a
nucleus with frequency w1 during
t1 to one with frequency w2 during
t2 is represented by a cross-peak at
cooordinates (w1,w2)
• The furthest that magnetisation is
able to jump is the distance of 3
bonds; i.e
– HN - Ha
– Ha - Hb
– Hb - Hg
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COSY spectrum of a small molecule
• COSY spectrum
directly confirms
covalent structure of
molecules
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TOCSY
TOtal Correlation SpectroscopY
• TOCSY is an ‘relayed’ extension of
COSY
– uses scalar coupling
• Cross-peaks appear between all spins
which can be connected by relaying
• Magnetisation still can’t be transferred
across peptide bond (3-bond limit still
applies)
 amino acids still form isolated spin
systems
• Useful for recognising particular amino
acids
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Heteronuclear NMR
Peptide bond
• 3-bond limit means that crosspeaks are never observed between
protons in different amino acids;
i.e. there is no magnetization
transfer across the peptide bond
• Magnetization can be transferred if
the intervening nuclei are
magnetic; i.e. 13C and 15N.
• This is achieved by producing the
protein recombinantly in bacteria
grown with 15N-ammonium
chloride and 13C-glucose as the
sole nitrogen and carbon sources
respectively
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3D experiments
wCa
wN
wHN
• The previous experiments can be
extended to two indirect
dimensions, t1 and t2
• The real time interval during
which all the FID’s are recorded is
called t3, or the direct dimension.
• S is a function of t1, t2, and t3; to
get the spectrum it must be
Fourier transformed inall three
time dimensions.
• If the magnetization is on a
nucleus with frequency w1 in t1,
w2 in t2 and w3 in t3, the spectrum
will have a ‘peak’ centred at
coordinates (w1, w2, w3)
• In 3D a peak is more like a ball
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Heteronuclear assignment experiments
• 3D HNCA experiment
– protein must be isotopically
enriched with 1H, 13C and 15N
• Peaks represented as balls in 3D
space at coordinates corresponding
to:
–
–
–
1H
shift of an amide proton (HN)
15N shift of attached N
13C shift of attached C
a
• At same 1H and 15N values,
another peak corresponding to 13C
shift of Ca of preceding residue
 makes it possible to walk along
sequence to assign entire backbone
residue i-1
residue i
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
2




1
3
wC


4
5

wN
wHN
Assignment of all HN, N and Ca resonances of a pentapeptide in a HNCA spectrum by
‘walking’ along the backbone. In each case the black sphere represents the in-residue
Ca , the grey sphere the Ca of the preceding residue
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NOE effect provides structural
information
• Nuclear Overhauser Effect
produces coupling between
protons which are close in
space (though not necessarily
covalently bonded)
• NOE cross-peaks  R-6
 only observed for R < 5 Å
• NOESY is 2D experiment in
which cross peak intensities are
proportional to NOE between
corresponding protons
NOESY spectrum of lysozyme
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Basic method for protein structure
determination by NMR
• ASSIGN all peaks using COSY-type spectra
• Identify all cross peaks between assigned diagonal peaks on
NOESY spectra
• Convert NOESY cross-peaks to distance constraints between
corresponding protons
• Find 3D structure which optimally satisfies distance constraints
as well as protein stereochemistry
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Structure determination
• Molecular modelling with energy function
Etotal = Ecovalent geometry + ENOE restraints
• Use optimisation algorithm to find molecular structure with
lowest value of Etotal which still satisfies all NMR-derived
distance constraints
• Generate family of structures
• Resolution generally not as good as X-ray, but may be better
reflection of molecules in-vivo
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