Download I. Physical Biochemistry Protein Interactions Ultraviolet(UV

Department of Chemistry and Biochemistry
University of Lethbridge
Biochemistry 4200
I. Physical Biochemistry
Protein Interactions
Ultraviolet(UV)-Spectroscopy
UV light is electromagnetic radiation with a wavelength between 10 – 400 nm.
Wavelengths between 250 – 310 nm (near UV) are used to study proteins
as they are primarily absorbed by aromatic amino acids (Phe (260 nm),
Tyr (278 nm), Trp (292 nm)) within proteins.
UV absorption by homogeneous protein samples depends on the number,
type, conformation, and microenvironment of all its aromatic amino acids.
Binding of a ligand to a protein can alter the conformation (and/or
microenvironment) of one or more aromatic amino acids in the protein
and give rise to an altered UV-absorption spectra.
What is measured at 190-220 nm?
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Ultraviolet(UV)-Spectroscopy
Typical Experiment:
Required solutions, etc.:
1. dilute solution of protein in buffer
2. a stock solution of concentrated ligand in the same buffer
3. stock solution of buffer
4. quartz cuvettes (Why?)
How to continue? What do we need to meaure?
Ultraviolet(UV)-Spectroscopy
1) Blank
Place buffer in cuvette, measure absorbance at each wavelength
between 250 – 310 nm. This is the absorption due to the buffer
solution. Set value at each point to baseline.
2) Protein Spectra
Place protein solution in cuvette and measure the absorbance at
each wavelength. This is the absorbance due to the protein in your
sample.
3) Ligand Spectra
Place ligand solution in cuvette and measure the absorbance at
each wavelength. This is the absorbance due to the ligand (in the
absence of protein). Typically, small ligands have little or no UV
absorbance in this region of the spectrum.
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Ultraviolet(UV)-Spectroscopy
4) Protein-Ligand complex Spectra
Place mixture of Protein-ligand complex in cuvette, measure
absorbance at each wavelength. This is the combined absorbance
of the protein and ligand in your sample.
5) Difference Spectra
Mathematically, each point of the protein spectra and each point of
the ligand spectra are subtracted from the equivalent point of the
protein-ligand spectra. The result is the UV absorbance associated
with the conformational changes in the protein due to ligand target
binding.
protein
protein
+ligand
Ultraviolet(UV)-Spectroscopy
Applications:
Characterization of proteins (e.g. spectroscopic fingerprint)
Determination of concentration (Aλ = ελ / c)
Protein interactions
Limitations:
Ligand binding not always results UV changes
No information on the nature of interaction
Sample purity
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CD Spectroscopy
Circular dichroism (CD) refers to the difference in absorption between
Right- and left-handed circular polarized light by a chiral sample.
∆ A = (AL – AR) / A
CD Spectroscopy
Circular dichroism (CD) refers to the difference in absorption between
Right- and left-handed circular polarized light by a chiral sample.
∆ A = (AL – AR) / A
Circular dichroism (∆ A) is typically converted to
2
-1
-1
mean molar ellipticity per residue ( Θmr : deg cm mol residue )
Circular polarized radiation is the result of two plane polarized
beams that are normal to one another.
The resulting rotational component is either clockwise (right-handed)
or counter-clockwise (left-handed).
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CD Spectroscopy
Right-handed circular polarized
Plane polarized light
pertendicular to plane
+ phase shifted within plane
Left-handed circular polarized
Plane polarized light
pertendicular to plane
+ phase shifted within plane
CD Spectroscopy
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CD Spectroscopy
CD spectra are run in the far-UV region of about 190 – 250 nm where
Peptide bonds absorb.
The technique is particularly sensitive to secondary structures.
CD can be used to estimate the secondary structural content of a protein.
In general, the CD signal at 215 nm indicates the sheet content and
The signal at 208 nm and 222 nm are used to calculated the helical
content.
CD Spectroscopy
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CD Spectroscopy
Example:
2
-1
-1
An all helix polypetide has an ellipticity of -38000 deg cm mol res at 222 nm
An all random polypetode has an ellipticity of -12000 deg cm2 mol-1 res-1 at 222 nm
An unknown protein has an ellipticity of -23000 deg cm2 mol-1 res-1 at 222 nm
CD Spectroscopy
(-23000 - -38000) / (-12000 - -38000) = 0.58
1.00 – 0.58 = 0.42 → 42 % helix
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CD Spectroscopy
Notes on the accuracy of the method (compared to known structures):
helix
sheet
turn
other
95-100%
< 75%
< 25%
< 90 %
Surface Plasmon Resonance
Plasmon resonance refers to the loss of a small fraction of the
total energy of incident radiation at specific angles of incidence.
Polarized light striking a metal surface transfers
energy to the delocalized electrons of the metal, reducing the
intensity of the reflected light.
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Surface Plasmon Resonance
For thin metal film, the primary determinant of the intensity of the
reflected light is the refractive index of the material coating it.
The thin metal film (Au) is coated with immobilized sample. Solutions
containing ligand are then passed across the immobilized sample.
If binding occurs, the refractive index of the coated metal film is changed
and both the intensity and angle of reflection are changed.
Surface Plasmon Resonance
Radiation used is typically in the near infra-red at 780 nm
The plasmon resonance signal is proportional to the molecular
mass of the ligand.
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Surface Plasmon Resonance
Allows to determine kinetic parameters: kon and koff
Typical response curve:
What is the problem here ?
Analytical Ultracentrifugation
Analytical ultracentrifugation utilizes
Extremely high centrifugal fields
e.g. 500,000 x g
Experiments yield molecular mass
information. Allows to asses oligomeric
state of proteins in solution.
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Analytical Ultracentrifugation
Theory of the technique is straightforward
1- downward force in centrifugal field:,
force
down
= m (1 – νp ρs) ω2 r
νp = specivic volume
ρs = density of solvent
ω = rotaion (radians)
2 – force opposing sedimentation (frictiona)
f = friction coefficient
v = velocity
forceopp = f v
3 – In the case of a sedimenting macromolecule
f v < m (1 – νp ρs) ω2 r
Analytical Ultracentrifugation
3 – In the case of a sedimenting macromolecule
rearranged
f v < m (1 – ν ρ ) ω2 r
p s
v
ω2 r
m
<
f
v
<
ω2 r
S
(1 – νp ρs)
Sedimentation coefficient
Substitution of particle mass by molar mass (M) and Avogadro’s number:
S
<
M
Nf
(1 – νp ρs)
Measuring the rate of sedimentation allows to calculate S and than M
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Analytical Ultracentrifugation
Experimentally, the rate of
sedimentation can be monitored
by UV-Absorption.
Fluorescence Resonance Energy Transfer
Fluorescence resonance energy transfer (FRET)
is a distance-dependent interaction between the electronic excited states of
two dye molecules in which excitation is transferred from a donor molecule
to an acceptor molecule without emission of a photon.
E
Distance, Å
The efficiency of FRET is dependent on the distance between the two dyes.
FRET is an important technique for investigating a variety of biological
phenomena that produce changes in molecular proximity.
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Fluorescence Resonance Energy Transfer
Primary conditions for FRET
Donor and acceptor molecules must be in close proximity (typically 10–100 Å).
The absorption spectrum of the acceptor must overlap the
fluorescence emission spectrum of the donor.
Donor and acceptor transition dipole orientations must be approximately
parallel.
Fluorescence Resonance Energy Transfer
Förster Radius
The distance at which energy transfer is 50% efficient
is defined by the Förster radius (R0).
The magnitude of Ro is dependent on the spectral properties
of the donor and acceptor dyes.
R0 = 44Å
E
Distance, Å
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Fluorescence Resonance Energy Transfer
Protein Dynamics:
Arrangement of the Kirromycin Stalled EF-Tu on the
Ribosome


Donor
P
A
30Å
EF-Tukirr
30Å
60Å
60Å
P
A
Fluorescence
50S
30S
Acceptor
1.1
0.9
0.7
+ Phe-tRNAPhe Qsy8
0.5
0
2
4
6
Time, s
8
10
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