linear dichroism for the study of nucleci acids fibrous and membrane proteins
Download
Skip this Video
Download Presentation
Linear dichroism for the study of nucleci acids, fibrous and membrane proteins

Loading in 2 Seconds...

play fullscreen
1 / 32

Linear dichroism for the study of nucleci acids, fibrous and membrane proteins - PowerPoint PPT Presentation


  • 103 Views
  • Uploaded on

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.

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 ' Linear dichroism for the study of nucleci acids, fibrous and membrane proteins' - keaton-knox


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
electrons bonds structure
Electrons  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

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

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

-strand

Poly proline II

-helix

tryptophan

tyrosine

adenosine

linear dichroism
Linear dichroism

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

orientation methods:

stretched film or flow most common

slide7

Flow AlignedLinear Dichroism

Sample

Plane Polarised

Light

Detector

LD = A// A

= absorbance // orientation direction  absorbance  to it

slide9

Quartz capillary ~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
  • Gene Detection.
  • SNP Detection.
  • Ligand binding.
slide11
PCR

Primer

Genomic DNA

Taq

Amplimer

aligned ld
Aligned LD

Not Aligned

Aligned

inducing alignment
Inducing Alignment

Increasing

Alignment

anthracene 9 carbonyl n spermine dna
Anthracene-9-carbonyl-N’-spermine + DNA
  • Anthracene absorbance bands
  • broadened and red-shifted
  • Overlap with DNA, but
  • not at 280 nm
ld of dek woolfson et al s peptide fibres
LD of Dek Woolfson et al’s peptide fibres

280 nm

aromatic

222 nm, n-p*

20 mM tropomyosin

10 mM SAF-p1/02

100 mM matured

slide18

Protein

# 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

collagen and f actin
Collagen and F-actin

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

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.

slide22

-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
  • 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

slide24

DNA binding modes

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

slide25

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)

slide26

An inexpensive approach

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.

slide27

Probing binding by absorbance

  • 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

slide28

Stretched film LD of the di-iron helicate

  • 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

slide29

Kinking the DNA

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

slide30

AFM studies with linear DNA

with complex

no complex

mm

mm

And more

more

mm

mm

slide32

free DNA

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.

ad