protein spectroscopy and dynamics l.
Download
Skip this Video
Loading SlideShow in 5 Seconds..
Protein spectroscopy and dynamics PowerPoint Presentation
Download Presentation
Protein spectroscopy and dynamics

Loading in 2 Seconds...

play fullscreen
1 / 64

Protein spectroscopy and dynamics - PowerPoint PPT Presentation


  • 99 Views
  • Uploaded on

Protein spectroscopy and dynamics. Vibrational spectroscopy Time-resolved spectroscopy Hemoglobin Myoglobin Enzymes Protein Folding. Dynamics in Proteins. Dynamics consist of:

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

PowerPoint Slideshow about 'Protein spectroscopy and dynamics' - makya


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
protein spectroscopy and dynamics

Protein spectroscopy and dynamics

Vibrational spectroscopy

Time-resolved spectroscopy

Hemoglobin

Myoglobin

Enzymes

Protein Folding

dynamics in proteins
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.
vibrational spectroscopy

Vibrational Spectroscopy

Quantum theory

Normal modes

Infrared absorption

Raman scattering

classical approach harmonic approximation
Classical approach: harmonic approximation

Differences with QM approach:

The solution is oscillatory.

Any energy is possible.

Q

quantum theory of vibration
Quantum theory of vibration

Harmonic approximation

Energy is quantized

v is the quantum number

Allowed transitions

v’ ® v + 1, v’ ® v - 1

Q

the bonding electronic state gives rise to a potential energy surface for the nuclear motion
The bonding electronic state gives rise to a potential energy surface for the nuclear motion

Harmonic approximation

there is a potential energy surface that corresponds to each electronic state of the molecule
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.

there are 3n 6 vibrational degrees of freedom in a molecule with n atoms
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.

the vibrational degrees of freedom can be expressed as normal modes
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.

normal modes of water
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 G3G3 .

Strong anharmonic coupling

leads to strong overtones

at 11,032 and 10,613 cm-1.

frequency shift due to molecular interactions
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.

transition dipoles
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.

transition dipoles15
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:

dipole derivatives
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).

slide20

Analysis of isotope effects

Vibrational spectra are analyzed within the

harmonic approximation.

Reduced mass

Classical harmonic oscillator equation

raman spectroscopy
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

resonance raman spectrum is obtained by a laser light scattering experiment
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.

resonance raman is a two photon process
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

the iron in heme is the binding site for oxygen and peroxide
The iron in heme is the binding site for oxygen and peroxide

Heme is iron protoporphyrin IX.

Functional aspects in Mb

O

|||

O

the iron in heme is the binding site for oxygen and peroxide25
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

the iron in heme is the binding site for oxygen and peroxide26
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+

slide28
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

slide29
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

resonance raman spectrum for excitation of heme soret band
Resonance Raman spectrum for excitation of heme Soret band

Soret Band

B Band Excitation Laser

Q Band

Raman spectrum

hemoglobin

Hemoglobin

Time scale for the R-T switch

The trigger mechanism

the cooperative r t switch
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

the frequency of the iron histidine vibration shows strain in t state
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.

Hb*CO 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

Hb*CO

10 ns

R-state

Deoxy Hb

T-state

slide37
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

the ligation of co changes the spin state of the heme iron
The ligation of CO changes the spin state of the heme iron

S = 0

S = 2

Low spin Fe(II)

High spin Fe(II)

the motion of the f helix tugs on the proximal histidine and introduces strain
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.

time resolved resonance raman can follow the r t structure change
Time-resolved resonance Raman can follow the R - T structure change

Time evolution

Hb*CO

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

ultrafast resonance raman spectroscopy shows that heme doming occurs in 1 ps
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

slide42

Dehaloperoxidase:

The First Enzymatically

Active Globin

NC State University

dhp oxidizes tribromophenol
DHP oxidizes tribromophenol

DHP + DBQ + H2O

DHP + TBP + H2O2

slide44

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

slide45

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)

slide46

Amphitrite ornata

~1 cm

DHP is the coelomic hemoglobin

slide49

Functional questions

Where is the pull?

Mb

DHP

Where is the push?

dehaloperoxidase looks like mb but dehalogenates halophenols
Dehaloperoxidase looks like Mb, but dehalogenates halophenols

Franzen et al.,

JACS (1998), 120, 4658-4661

mechanism for phenol oxidation by hrp
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

slide52

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

globins have ferrous iron and bind o 2
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+

peroxidases have ferric iron and bind h 2 o 2
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+

original ansatz dhp oxidation state must change
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+ ?

slide56

Globins and Peroxidases

diverged 1.8 billion

years ago

Implicit meaning:

Ancestral protein was

both a hemoglobin and

a peroxidase

Terrebellid polychaetes

do not figure in the

scheme.

Convergent evolution?

Divergent evolution?

Hardison, J. Exp. Biol. 1998, 102, 1099

fe histidine stretching mode of deoxy dehaloperoxidase
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

the ligation at the sixth position changes the spin state of the heme iron
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)

symmetric and non totally symmetric modes a 1g b 1g
Symmetric and non-totally symmetric modesA1g B1g

n4

n10

Electron density Core size

Marker Modes

slide60

high spin

DHP core size

marker mode

study

n4

n3

n2

n10

DHP is intermediate.

It has more low

Spin character

Than Mb, but less

than HRP

Belyea, Franzen et al.

Biochemistry, 2006

slide61

low spin

DHP core size

marker mode

study

n4

n3

n2

n10

DHP is intermediate.

It has more low

Spin character

Than Mb, but less

than HRP

Belyea, Franzen et al.

Biochemistry, 2006

slide62

Core size marker comparison

n10

DHP core size in ferric form looks more like HRP than Mb.

Belyea, Franzen et al. Biochemistry 2006

model for proximal hydrogen bonding
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

model for proximal hydrogen bonding64
Model for proximal hydrogen bonding

Dehaloperoxidase Catalytic Triad

C=O-His-Fe?

Fe

His

Backbone C=O

Hydrogen bond strength

is intermediate between

Mb and HRP/CcP.

Franzen JACS 2001,123, 12578