Laser Light Scattering

- Basic ideas – what is it?

- The experiment – how do you do it?

- Some examples systems – why do it?

Double Slit Experiment

screen

Coherent beamExtra path length

+ +

= =

Light Scattering Experiment

Laser at fo

Scattered light

Scatterers in solution (Brownian motion)

ffo

Narrow line incident laserDoppler broadenedscattered light

f

0 is way off scale f ~ 1 part in 1010 - 1015

More Detailed Picturedetector

Inter-particle interference

time

Detected intensity

Iaverage

How can we analyze the fluctuations in intensity?

Data = g() = <I(t) I(t + )>t = intensity autocorrelation function

Intensity autocorrelation• g() = <I(t) I(t + )>t

For small

For larger

g()

c

What determines correlation time?• Scatterers are diffusing – undergoing Brownian

motion – with a mean square displacement given by <r2> = 6Dc (Einstein)

• The correlation time c is a measure of the time needed to diffuse a characteristic distance in solution – this distance is defined by the wavelength of light, the scattering angle and the optical properties of the solvent – ranges from 40 to 400 nm in typical systems

• Values ofc can range from 0.1 s (small proteins) to days (glasses, gels)

Diffusion• What can we learn from the correlation time?• Knowing the characteristic distance and

correlation time, we can find the diffusion coefficient D

• According to the Stokes-Einstein equation

where R is the radius of the equivalent sphere and is the viscosity of the solvent

• So, if is known we can find R (or if R is known we can find

6Bk TDR

Why Laser Light Scattering?1. Probes all motion

2. Non-perturbing

3. Fast

4. Study complex systems

5. Little sample needed

Problems: Dust and

best with monodisperse samples

Some Examples

Superhelical DNAwhere = Watson-Crick-Franklin double stranded DNA

pBR322 = small (3 million molecular weight) plasmid DNA

Laser light scattering measurements of D vs give a length L = 440 nm and a diameter d = 10 nm

DNA-drug interactions: intercalating agent PtTS produces a 26o unwinding of DNA/molecule of drug bound

Since D ~ 1/size, as more PtTS is added and DNA is “relaxed,” we expect a minimum in D

As drug is added DNA first unwinds to open circle and then overwinds with opposite handedness. At minimum in D the DNA is unwound.

This told us that there are 34 superhelical turns in native pBR

pBR is a major player in cloning – very important to characterize well

Antibody molecules

• Technique to make 2-dimensional crystals of proteins on an EM grid (with E. Uzgiris at GE R&D)

Change pH

60o120o

Conformational change with pH results in a 5% change in D – seen by LLS and modeled as a swinging hinge

Aggregating/Gelling SystemsStudied at Union College

• Proteins:– Actin – monomers to polymers and networks

Study monomer size/shape, polymerization kinetics, gel/network structures formed, interactions with other actin-binding proteins

Epithelial cell under fluorescent microscope

Actin = red, microtubules = green, nucleus = blue

Why?

Aggregating systems, con’t– BSA (bovine serum albumin) amyloid- insulin– Chaperones

• Polysaccharides:– Agarose– Carageenan

Focus on the onset of gelation –

what are the mechanisms causing gelation? how can we control them? what leads to the irreversibility of gelation?

what factors cause or promote aggregation?

what is the structure of the aggregates?

how can proteins be protected from aggregating?

Collaborators and $$• Nate Poulin ’14 & Christine Wong ‘13• Michael Varughese ’11 (med school)• Anna Gaudette ‘09• Bilal Mahmood ’08 & Shivani Pathak ’10 (both in med school)• Amy Serfis ‘06 & Emily Ulanski ’06 (UNC, Rutgers )• Shaun Kennedy (U Michigan, Ann Arbor in biophysics)• Bryan Lincoln (PhD from U Texas Austin, post-doc in Dublin)• Jeremy Goverman (medical school)• Shirlie Dowd (opthamology school)• Ryo Fujimori (U Washington grad school)• Tomas Simovic (Prague)• Ken Schick, Union College• J. Estes, L. Selden, Albany Med• Gigi San Biagio, Donatella Bulone, Italy

Thanks to NSF, Union College for $$