Linear dichroism for the study of nucleci acids fibrous and membrane proteins
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Linear dichroism for the study of nucleci acids, fibrous and membrane proteins. Electrons  bonds  structure. UV/visible light: l ~ 180 nm – 800 nm, energy h n = hc/ l causes electrons to go to higher energy levels. l required depends on electron rearrangement needed.

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Electrons bonds structure
Electrons membrane proteins bonds  structure

UV/visible light:l ~ 180 nm – 800 nm, energy hn = hc/l

causes electrons to go

to higher energy levels.

l required depends on electron

rearrangement needed.

In solution: broad bands due to

diff. vibrational levels

in excited state &

molecules having slightly

different energy levels.

UV: –350 nm

Vis: 400 nm –

Excited electronic state

  • .

Ground electronic state

Ground vibn’l level

r


Proteins
Proteins membrane proteins

Far UV (250 – 180 nm) dominated by peptide group

Many buffers absorb here — so beware!

First n* 210 – 220 nm, weak (e~100 cm1dm3mol1)

First * 180 nm, stronger (e ~ 7000 cm1dm3mol1)

In -helix only has a component at 208 nm

?? ns* transition 175 nm

Side chains absorb in this region:

aromatics, Asp, Glu, Asn, Gln, Arg & His. Usually small.


Polarization of transitions
Polarization of transitions membrane proteins

Directionelectrons move during a transition

Oriented samples and polarised light:only light whose

electric field pushes the electrons along the polarisation

direction causes a transition

Nucleic acids: DNA bases: all transitions perpendicular

to helix axis


Transition polarisations
Transition polarisations membrane proteins

-strand

Poly proline II

-helix

tryptophan

tyrosine

adenosine


Linear dichroism
Linear dichroism membrane proteins

LD = A//- A^w.r.t. orientation axis

orientation methods:

stretched film or flow most common


Flow Aligned membrane proteinsLinear Dichroism

Sample

Plane Polarised

Light

Detector

LD = A// A

= absorbance // orientation direction  absorbance  to it


Quartz capillary membrane proteins~200 m

annular gap LD cell

with thermal control

50 m annular gap LD cell

with CaF2 optics,

demountable

components, inner

rotating cylinder


The problems
The Problems membrane proteins

  • Gene Detection.

  • SNP Detection.

  • Ligand binding.


PCR membrane proteins

Primer

Genomic DNA

Taq

Amplimer


Aligned ld
Aligned LD membrane proteins

Not Aligned

Aligned


Ld spectrum of dna
LD Spectrum of DNA membrane proteins


Inducing alignment
Inducing Alignment membrane proteins

Increasing

Alignment


Anthracene 9 carbonyl n spermine dna
Anthracene-9-carbonyl- membrane proteinsN’-spermine + DNA

  • Anthracene absorbance bands

  • broadened and red-shifted

  • Overlap with DNA, but

  • not at 280 nm


LD adjusted for change in S membrane proteins


Ld of dek woolfson et al s peptide fibres
LD of Dek Woolfson et al’s peptide fibres membrane proteins

280 nm

aromatic

222 nm, n-p*

20 mM tropomyosin

10 mM SAF-p1/02

100 mM matured


Protein membrane proteins

# amino

acids

% Secondary structure content

% Aromatic amino acids

a

b

o

Trp

Tyr

Phe

Ab1-42

42

-

90

10

-

2.3

7.1

Collagen type I

~3000

-

-

100

-

0.5

1.3

a1-antitrypsin

418

20

30

50

0.7

1.4

6.5

F-actin

337

17

18

65

1.1

4.2

3.2

Protein FibresTim Dafforn, Dave Halsall. Louise Serpell


F-actin membrane proteins


Collagen and f actin
Collagen and F-actin membrane proteins

Collagen

Cryo em

Collagen

Flow LD

206 nm //

Long axis

Collagen

CD

F-actin

CD

Aromatic

  • Flow LD

  • F-actin

  • 220/190

  • axis, 210

  • // axis


Membrane proteins
Membrane proteins membrane proteins

normal

the reduced LD, LDr = LD/isotropic absorbance, is:

where  is the angle the transition moment of interest makes with the normal to the cylinder surface (i.e. to the lipid long axis), S is the orientation factor that denotes the fraction of the liposome that is oriented as a cylinder perfectly parallel to the flow direction.


membrane proteins-helix

220 nm n-* polarised  helix axis

208 nm // helix axis

Helix // lipids at 208 nm LD<0, 220 nm >0

Gramicidin


Ld of nanotubes
LD of nanotubes membrane proteins

  • Single walled nanotubes (as received)

  • UV spectroscopy: ? p-plasmon band

Abs of SWNT

In SDS < CMC

LD of SWNT

+ anthracene

LD of anthracene

expanded

LD of SWNT


DNA binding modes membrane proteins

1. Intercalation between base pairs:

DNA lengthens and stiffens

2. In major groove:

more options for selectivity

3. In minor groove:

planar aromatics with AT rich DNA

4. External binding:

non specific


DNA binding & structure control by transition metal supramolecular helicates

Alison Rodger and Mike Hannon

Isabelle Meistermann, Karen Sanders, Chris Isaac, Jemma Peberdy, Laura Childs, Syma Khalid, Mark Rodger

University of Warwick

Virtudes Moreno, Barcelona (AFM)

Einar Sletten, Norway (NMR)


An inexpensive approach supramolecular helicates

Simple imines can be used for supramolecular assembly

e.g. FeCl2

  • Yield 87%

  • Isolate by filtration

  • Cost per gramme: 8p (0.8FFr;

  • 0.13 US$; 0.23DM; 230ItLi; 50GrD)

X-ray structure

M.J. Hannon, C.L. Painting, A. Jackson, J. Hamblin, and W. Errington, Chem. Commun., 1997, 1807.


Probing binding by absorbance supramolecular helicates

  • Only very small changes observed in the MLCT bands at ~ 580 nm.

  • Larger changes in ligand based band at ~ 330nm.

  • Changes confirm binding.

  • Small changes suggest binding does not cause major perturbations in the helical structure.

Absorbance spectra of ct-DNA with 20 mM [Fe2L3]Cl4


Stretched film LD of the di-iron helicate supramolecular helicates

  • MLCT bands at ~580 nm are radially (xy) polarised.

  • The ligand band at the ~320 nm contains both radial (xy) and long axis (z) contributions

  • Aim to understand flow LD …

LD of spectra of [Fe2L3]Cl4 in a stretched polyvinylalcohol film


Kinking the DNA supramolecular helicates

The flow LD experiment not only reveals a binding angle of 60°(±10 °) but also reveals changes in the DNA region.

The decrease in the magnitude of the LD signal in the DNA region with respect to free DNA means a decrease in DNA orientation.

This indicates that the [Fe2L3]Cl4 helicate is kinking the DNA.

LD spectra of free and bound ct-DNA


AFM studies with linear DNA supramolecular helicates

with complex

no complex

mm

mm

And more

more

mm

mm


NMR structure supramolecular helicates


free DNA supramolecular helicates

DNA:M = 10:3

DNA:P = 10:3

DNA:rac = 10:3

AFM images of a fragment of 200 pb linear DNA

incubated at 37ºC for 5 h with the iron cylinders.


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