Protein spectroscopy and dynamics

1
Protein spectroscopy and dynamics

• Vibrational spectroscopy
• Time-resolved spectroscopy
• Hemoglobin
• Myoglobin
• Enzymes
• Protein Folding

2
Dynamics in Proteins

• Dynamics consist of
• Protein relaxation in response to
  - ligand/substrate binding
  - electron transfer
• Protein folding.
  - cyclic compared to
  b-sheet peptides
  - unfolded - molten globule -
  folded
• Time-resolved vibrational spectroscopy is a tool
  for investigation of structural changes.

3
Vibrational Spectroscopy

• Quantum theory
• Normal modes
• Infrared absorption
• Raman scattering

4
Classical approach harmonic approximation
Differences with QM approach The solution is
oscillatory. Any energy is possible.
Q
5
Quantum theory of vibration
Harmonic approximation
Energy is quantized
v is the quantum number Allowed transitions v
v 1, v v - 1
Q
6
The bonding electronic state gives rise to a
potential energy surface for the nuclear motion
Harmonic approximation
7
There is a potential energy surface that
corresponds to each electronic state of the
molecule
The shift in the nuclear displacement arises
from the fact that the bond length increases in
the s state compared to the s state. We will
show that the overlap of the vibra- -tional wave
functions is key to understanding the shape of
absorption bands.
8
There are 3N-6 vibrational degrees of freedom in
a molecule with N atoms
Three degrees of freedom are required for
translation. Three degrees of freedom are
required for rotation. For example, in H2O there
are 9 total degrees of freedom and 3 vibrational
degrees of freedom. In C6H6 there are 36 degrees
of freedom and 30 vibrational degrees of
freedom. Exception In linear molecules there are
only 2 rotational degrees of freedom and
therefore the number of vibrations is 3N - 5.
9
The vibrational degrees of freedom can be
expressed as normal modes.
All normal modes have the same form for the
harmonic oscillator wavefunction and differ only
in the force constant k and mass m. The total
wavefunction is a product of normal modes. The
total nuclear wavefunction for water is
c1c2c3. The normal mode wavefunctions of water
correspond to the symmetric stretch, bend, and
asymmetric stretch. These are linear combinations
of the stretching and bending internal
coordinates of H2O.
10
Normal modes of water
In water vapor n1 n3, but symmetries are
different, G1 ¹ G3. (G is the
symmetry) However, the third overtone of 1 has
the same symmetry as the combination band G1 G1
G1 G1 G3 G3 . Strong anharmonic coupling leads
to strong overtones at 11,032 and 10,613 cm-1.
11
Comparison of harmonic and anharmonic potentials
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Frequency shift due to molecular interactions
Hydrogen bonding lowers O-H force constant and
H-O-H bending force constant.
vapor liquid n1 3825 3657 n2 1654
1595 n3 3935 3756
The intermolecular hydrogen bonding stretching
mode is difficult to observe.
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VibrationalTransitions
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Transition dipoles
In order for infrared light to be absorbed the
polarization must be aligned with the
direction of the transition moment. For a
vibrational mode this is determined by the
directional change in the dipole moment. This is
shown below for the bending mode of H2O.
15
Transition dipoles
The change in ground state dipole moment during
vibration interacts with light. The first term
is static and does not contribute to the
transition. Calling the vibrational
wave- functions ci the transition moment is
16
Dipole derivatives
The vibrational wavefunctions ci are
Gaussians, thus the transition moment for
transition from vibrational state 0 to
vibrational state 1 is The transition
dipole moment is proportional to the dipole
derivative. This is true for any normal mode of
vibration (i.e. harmonic).
17
Absorption of infrared radiation leads to
vibrational transitions
v 0
18
Absorption of infrared radiation leads to
vibrational transitions
v 1
v 0
19
The selection rule for vibrational transitions is
Dv 1
v 2
v 1
v 0
20
Analysis of isotope effects
Vibrational spectra are analyzed within
the harmonic approximation.
Reduced mass
Classical harmonic oscillator equation
21
Raman spectroscopy
Goal Study vibrational frequencies of the heme
and the axial ligands in order to obtain
information on the coupling of protein motion
and electrostatics with the heme iron
22
Resonance Raman spectrum is obtained by a laser
light scattering experiment
Detector
Lens
Spectrograph
Sample
Laser
Inelastic light scattering produces a frequency
shift. There is exchange of energy between the
vibrations of the molecule and the incident
photon.
23
Resonance Raman is a two photon process
Incident photon from a laser. Scattered
photon has an energy shift. The difference
is because the molecule is left in an
excited vibrational state.
hn
24
The iron in heme is the binding site for oxygen
and peroxide
Heme is iron protoporphyrin IX. Functional
aspects in Mb
O O
25
The iron in heme is the binding site for oxygen
and peroxide
Heme is iron protoporphyrin IX. Functional
aspects in Mb 1. Discrimination against CO
binding.
O C
26
The iron in heme is the binding site for oxygen
and peroxide
Heme is iron protoporphyrin IX. Functional
aspects in Mb 1. Discrimination against CO
binding. 2. O2 is the physiologically relevant
ligand, but it can oxidize iron (autooxidation).
3
27
Porphine orbitals
eg
eg
a2u
a1u
28
The four orbital model is used to represent the
highest occupied and lowest unoccupied MOs of
porphyrins
The two highest occupied orbitals (a1u,a2u) are
nearly equal in energy. The eg orbitals are
equal in energy. Transitions occur from a1u eg
and a2u eg.
M1
29
The transitions from ground state p orbitalsa1u
and a2u to excited state p orbitals egcan mix
by configuration interaction
Two electronic transitions are observed. One is
very strong (B or Soret) and the other is weak
(Q). The transition moments are MB M1 M2 MQ
M1 - M2 0
M1
M2
30
Absorption spectra for MbCO and deoxy Mb
Soret Band
Q Band
31
Resonance Raman spectrum for excitation of heme
Soret band
Soret Band B Band Excitation Laser
Q Band

Raman spectrum
32
Soret (B) band Resonance Raman spectra of MbCO
and Deoxy Mb
n8
33
B band Resonance Raman spectra of MbCO and Deoxy
Mb
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Hemoglobin

• Time scale for the R-T switch
• The trigger mechanism

35
The cooperative R - T switch
Hemoglobin is composed of two a and two b
subunits whose structure s resemble myoglobin.
Eaton et al. Nature Struct. Biol. 1999, 6, 351
36
The frequency of the iron-histidine vibration
shows strain in T state
The comparison of photolyzed HbCO in the R state
and the equilibrium T state. HbCO at 10 ns
Fe-His 230 cm-1 Deoxy Hb Fe-His 216 cm
-1 The lower frequency indicates weaker bonding
interaction and coupling to bending modes.
lexc 435 nm Fe-His
HbCO 10 ns R-state
Deoxy Hb T-state
37
The heme iron center moves out of the heme plane
and the porphyrin macrocycle domes upon
deligation of CO
CO is photolyzed
Fe displacement
Planar Heme
Domed Heme
38
The ligation of CO changes the spin state of the
heme iron
S 0
S 2
Low spin Fe(II)
High spin Fe(II)
39
The motion of the F-helix tugs on the proximal
histidine and introduces strain
The frequency lowering in the T state arises from
weaker Fe-His ligation and from anharmonic
coupling introduced by the bent conformation of
the proximal histidine.
40
Time-resolved resonance Raman can follow the R -
T structure change
Time evolution
HbCO 10 ns 100 ns 400 ns 1 ms 8 ms 15
ms 40 ms 60 ms 120 ms Deoxy Hb
Strain is introduced in stages as
intersubunit contacts are made. Based on the
x-ray data it was proposed that the iron
displacement from the heme plane is a trigger for
the conformational changes.
200 210 220 230 240 Raman Shift
(cm-1)
Scott and Friedman JACS 1984, 106, 5877
41
Ultrafast resonance Raman spectroscopy shows that
heme doming occurs in 1 ps
Equilibrium HbCO Difference spectra obtained by
subtraction of the red spectrum from spectra
obtained at the time delays shown.
The evidence suggests that heme iron displacement
is an ultrafast process that is independent of
viscosity.
Franzen and Martin Nature Structural Biology
1994, 1, 230
42
Dehaloperoxidase The First Enzymatically
Active Globin
NC State University
43
DHP oxidizes tribromophenol
DHP DBQ H2O
DHP TBP H2O2
44
Many Peroxidases belong to the Cytochrome c
Peroxidase family
PDB 1A2F Cytochrome c Peroxidase (CCP) Class
All a proteins Superfamily Heme
peroxidases Family CCP-like
Goodin and McCree Scripps Institute
PDB 2ATJ Horseradish Peroxidase (HRP) Class All
a proteins Superfamily Heme
peroxidases Family CCP-like
Hendrickson et al. Biochemistry (1998) 37, 8054
45
Dehaloperoxidase is a peroxidase that belongs to
the globin family
PDB 1A6G Myoglobin (Mb) Class All a
proteins Superfamily
Globin-like Family Globins
Vojetchovsky, Berendzen, Schlichting
PDB 1EW6 Dehaloperoxidase (DHP) Class All a
proteins Superfamily
Globin-like Family Globins
Lebioda et al. J.Biol.Chem. 275 18712 (2000)
46
Amphitrite ornata
1 cm
DHP is the coelomic hemoglobin
47
Comparison of DHP and Mb Structures
Mb DHP
Superimpose hemes
48
Overlay of active sites
Mb DHP
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Functional questions
Where is the pull?
Mb DHP
Where is the push?
50
Dehaloperoxidase looks like Mb, but
dehalogenates halophenols
Franzen et al., JACS (1998), 120, 4658-4661
51
Mechanism for phenol oxidation by HRP
2nd Electron transfer to Compound II
Electron transfer to Compound I
H

HRP Horseradish peroxidase Heme, Histidines,
Arginine, Calciums
52
X-ray structure of a substrate analog in the
binding site of DHP
4-iodophenol in internal site Unprecedented in
globins
Lebioda et al., J.Biol.Chem. (2000) 275, 18712
53
Globins have ferrous iron and bind O2
Heme is iron protoporphyrin IX. Functional
aspects in Mb 1. Discrimination against CO
binding. 2. O2 is the physiologically relevant
ligand, but it can oxidize iron (autooxidation).
O O
2
54
Peroxidases have ferric iron and bind H2O2
OH / HO

• Heme is iron protoporphyrin IX.
• Functional aspects in HRP
• Activation involves formation
• of compounds I and II.
• 2. Edge electron transfer to
• substrate.

3
55
Original ansatz DHP oxidation state must change
How can a protein be both and globin and a
peroxidase? The functional environment must
change in response to a stimulus. What is the
trigger for the function switch? Substrate
binding must be the key.
2 / 3 ?
56
Globins and Peroxidases diverged 1.8 billion
years ago Implicit meaning Ancestral protein
was both a hemoglobin and a peroxidase Terrebelli
d polychaetes do not figure in the
scheme. Convergent evolution? Divergent
evolution?
Hardison, J. Exp. Biol. 1998, 102, 1099
57
Fe-histidine stretching mode of deoxy
dehaloperoxidase
Fe-His mode
The frequency of the Fe-His mode is intermediate
between that of myoglobin (HHMb) and horseradish
peroxidase (HRP).
Franzen et al., JACS (1998), 120, 4658-4661
58
The ligation at the sixth position changes the
spin state of the heme iron
S 1/2
S 5/2
High spin Fe(III)
Low spin Fe(III)
59
Symmetric and non-totally symmetric modesA1g
B1g
n4
n10
Electron density Core size

Marker Modes
60
high spin
DHP core size marker mode study

n4
n10
n2
n3
DHP is intermediate. It has more low Spin
character Than Mb, but less than HRP
Belyea, Franzen et al. Biochemistry, 2006
61
low spin
DHP core size marker mode study

n4
n10
n2
n3
DHP is intermediate. It has more low Spin
character Than Mb, but less than HRP
Belyea, Franzen et al. Biochemistry, 2006
62
Core size marker comparison

n10
DHP core size in ferric form looks more like HRP
than Mb.
Belyea, Franzen et al. Biochemistry 2006
63
Model for proximal hydrogen bonding
Peroxidase Catalytic Triad Asp-His-Fe
Fe
His
Asp
This is the push In peroxidase mechanism
Franzen JACS 2001,123, 12578
64
Model for proximal hydrogen bonding
Dehaloperoxidase Catalytic Triad CO-His-Fe?
Fe
His
Backbone CO
Hydrogen bond strength is intermediate between Mb
and HRP/CcP.
Franzen JACS 2001,123, 12578