Electromagnetic

radiation

– techniques

for measuring

biomolecules

Absorbtion

spectroscopyUV-spectroscopy

Cirkular

dichroism (CD) spectroscopyIR-spectroscopy

FluorescenceNMR

Optical

activity

•

Originates

from the presence

of assymetric

carbon

atoms

and their

effects

on nearby

chromophores•

Is used

as a fundamental criterion

for

traces

of life

on, for example, meteorites•

Detection

of chirality

The dualism of light

Electric field

component

Magnetic

field

component

The oscillatory

electric

component

of light

induces

electronic

transitions

between

orbitals. The probability

for transition

is given by

ba

= ∫*a

b

dτ

:the transition

dipole

moment

This

vectorlike

entity

ba

oscillates

with the light

frequency

and has a specific

orientation

dependent

on the molecular

geometry.

Linear

polarized

light: The orientation

of the electric

field

component

is constant

but

the size

is modulated: this is used

to investigate

the orientation

of the transition

dipole

moment in single

molecules

Circular

polarized

light: The size

of the vector

is constant

but

its

orientation

is modulated. The absorbtion

of this type

of light

is sensitive to larger

structural

properties, dependent

on interactions

between

various

transition

dipole

moments.

Circular

dichroism is observed

by a difference

in absorbtion

between

right-

and

left

polarized

light.

The method

is based

on

difference

spectra between

right-

and

left

polarized

light, thus

resulting

in

both

positive and negative maxima.

Basic units

in circular

dichroism:

Molar differential extinction

coefficient:

= (L

-R

) = (AL

-AR

) / (c

l)unit: l mol-1

cm-1

Mean

Residue

Ellipticity[

] = 100

obs

/ (c

l)unit: degrees

cm2

dmol-1

These units can be converted into each other (more on the CD lab)

[

] =

* 3300

Theory

To be able

to explain

optical

activity

we

have

to take

the magnetic

properties

of light

into

account. Elektric

component

Magnetic

component

Dab

= |

∫*a

b

dτ

| 2

= 9.180 x 10-3

∫

/

d

In UV/VIS we

define

the dipole

strength

for a light- induced

electronic

transition

as

- size, not direction!

How

can

we

measure

the strength

of a light- induced

electronic

transition?

Dab

= |

<b

|| a

> | 2

= 9.180 x 10-3

∫

/

d

…and in another

way

of writing

(Dirac-notation):

The rotational

strength

is obtained

by integrating

the CD- band over the wavelength

region for transition

(the absorbtion

band) where

the electronic

transition

from state

0 to state

a occurs:

Roa

= (3hc/83N0

) ∫

(()/) d

The rotation strength, which

has vectorlike

properties,

can be calculated

from quantum

mechanical

principles

and

knowledge

about

the wave

functions

in the ground-

and excited

states

in an unsymmetrical

molecule

(Rosenfeld,1928).

Pure electronic

absorbtion

Pure magnetic

absorbtion

Optical activity!

To avoid

that Roa

becomes

0, both

the electric

and the magnetic

transition

dipole

moments must be

0.

The Einstein coefficients, which

determine

the absorbtion strength, are proportional to the electric

transition

dipole

moment.

B0a

| <0

|| a

> |2

In order to display optic

rotation, a molecule

must therefore be able

to absorb

light.

The shape

of the equation

requires

certain

parallellity between

magnetic

och electric

transition

dipole

moment in order that Roa

does

not become

0.

Vector

product: A

B = Ax

Bx

+ Ay

By

+ Az

Bz

One of the terms must be

0 !

The electric

and magnetic

transition

dipole

moments must have

at least

one

vector

component

0 in a

common

direction

to result

in optical

activity.

•

To disturb

the ortogonality

between

magnetic and electric

transition

dipole

moments,

a

certain

assymetry

in the molecule

is required.

•

Since

electric

transition

dipole

moments are symmetric, unsymmetric

interactions

with

other

transition

dipoles

is required.

•

Interactions

between

transition

dipoles

gives information on structure!

•

What

type

of information can

we

expect?

What

can

be measured

is related

to what

absorbs

where:

Peptide

bond

absorbance

gives information on structure...

... while

aromatic

side

chain

absorbance

gives information regarding

the packing

of the protein core.

Sensitivity

varies

by wavelength

Variants of the HIV-protein rp24, Ehrhard

et al., Biochemistry

1996

More

than

100-fold less sensitivity

in Near-UV:

calls

for higher

concentrations, larger

cuvette

path

lengths

Practical advice ;-)

NOTE #1:

Only

try to measure

CD in wavelength

areas with UV absorbtion!

NOTE #2:

CD is a difference

method: make sure sufficient

light

is available

after sample

passage!

NOTE #3:

Avoid

buffer

components

with high intrinsic

absorbance!

Secondary

structure

affects

spectra

in the far-UV

Estimating

structure

in novel

proteins / mutants:

Divide

the spectrum

into

structure

contributionsA()obs

= A()

+ A()

+ A()randomor

= ()

*c

*l + ()

* c

*l + ()random

*crandom

*l

What

is

() for the various

structures? -

single

reference

spectra-

Database

of reference

spectra-

Neural network

Variants include-

Different mathematical

methods

to optimize

data fit-

Different selections

of reference

spectra

Applying

CD to biomolecules

•

Estimate

structure

content•

Protein interactions

•

Ligand

bindning•

Folding/unfolding

•

Enantiomers, racemization•

Analysis for patenting etc

•

Experimental concerns

How

can

binding

events be analyzed

by CD?

Exempel 1: protein-DNA-bindning

But

if

two

proteins bind each

other?

Example

2: titin

and actinin

Identifying

and characterizing

protein-protein interactions

Campbell & Lumb, Biochemistry 2002

Which

part of the protein Jun binds KIX? aa

47-89 eller aa

1-57?

Binding

of small molecules

Chiu

et al., Biochemistry

2001

Detection

of change

within

the protein complex

holo

apo

We

can

analyze

the CD-change

at a certain

wave length

as we

titrate

a ligand

into

the protein.

By fitting of appropriate

binding

equations

to the data, the affinity

can

be determined.

Choice of wavelength

for analysis

Entire

spectrafrom 260 nm

as far down

as possible

(~185 nm)

gives best accuracy

but

takes

a while

to record

(5-30 minutes

per spectrum)

Single

wavelengthsmeasure

at one

wavelength, change

conditions

(temp,

lingand

concentration

etc) and measure

again: around 10-60 s per measurement

+ time for change

of conditions

Kinetics, folding-

and binding

analysisChoose

wavelength

where

major change

occurs!

(so you must record

entire

spectra

first)

Folding-unfolding

: stability

Mutations in a small calcium-

binding

protein, Calbindin

D9k

25 °

C, no urea 90 °

C, no urea

25 °C, 9M urea

Stability

of mutants – temperature, urea, GuHCl

Thermal

stability

• Why

does

stability

vary

with protein concentration?

•

Which

information regarding

the measurement

is lacking

in the figure

legend above?

Fig. 4. The change in fraction folded ofGCN4 as a function of temperature and protein concentration. The concentrations range from 1 M

with the lowest TM

to 20 M

with the highest TM

.

Does the protein have structure???

Grx4 lacks enzymatic

activity

in a cellular

HED-assay, detected

by absorbance

of NADPH at 340 nm...

(usual

values

>100)

-30

-20

-10

0

10

20

30

190 200 210 220 230 240 250 260

Wavelength (nm)

Ellip

ticity

(kde

g*cm

2 *dm

ol-1

)

181614121086420

pET15b

Grx4 CGFS

Grx4 SGFS

Grx4 CGFC

Grx4 CPYC

Specific activity (NADPH U/mg)

Tran

sfor

man

t

-20

-18

-16

-14

-12

-10

-8

-6

-4

-2

030 40 50 60 70 80 90 100

Temperature (oC)

Ellip

ticity

@22

2 nm

(kde

g*cm

2 *dm

ol-1

)

… but

CD shows that Grx4 has a mixed alfa-beta

fold

and that it is thermodynamically

stable, similar

to Grx1.

Grx4

Grx1

Grx4

Grx1

Bindning analysis

using

CD: everything

contributes

–

seek

largest

change!

CD-effekt:

Diazepam

binding

to Human Serum Albumin

Difference

spectrum

of diazepam

with and without

protein

The protein gives little

signal in near-UV...

Titration

with increasing

amount

of ’host’

Summary•

Polarized

light

•

Optical

activity•

Circular

dichroism

•

Secondary

structure

analysis•

Folding/unfolding

•

Protein interactions•

Ligand

bindning

•

Enantiomers, racemization•

Experimental concerns