Protein Function in Nanobioconjugates

CHEM 570March 27, 2008

Jennifer A. JamisonMatthews Research Group

Outline• Nanobioconjugates

– Definition– Motivation: Why do we care?

• My Research– Objective– Introduction to Proteins– Model Sytem– Overview of Gel Electrophoresis– Stability and Stoichiometry

• Application: Epitope-Mapping

• Conclusions

What are Nanobioconjugates?

• A biomolecule (protein, DNA, RNA, etc.) attached to a nanomaterial

Electrostatically Covalently

-

-

-

--

--

- ---

-- -

--

++

++

++

+

++ + +

+ +

S

SS

SS

S S

S

What are Nanobioconjugates?

• A biomolecule (protein, DNA, RNA, etc.) attached to a nanomaterial

Electrostatically Covalently

-

-

-

--

--

- ---

-- -

--

++

++

++

+

++ + +

+ +

S

SS

SS

S S

S

What are Nanobioconjugates?

• A biomolecule (protein, DNA, RNA, etc.) attached to a nanomaterial

Electrostatically Covalently

-

-

-

--

--

- ---

-- -

--

++

++

++

+

++ + +

+ +

S

SS

SS

S S

S

Why Study Nanobioconjugates?

• Hybrid materials = Hybrid properties

• Exploit nano properties in the bio applications– Fluorescence, magnetism, etc.

• Use bio properties to affect nano synthesis– Self-assembly, nano-patterning

Applications

• Drug Delivery• Electron Microscopy Tags• Sensing/Detection• MRI Contrast Agents• Consumer Products• Etc.

Applications

• Drug Delivery• Electron Microscopy Tags• Sensing/Detection• MRI Contrast Agents• Consumer Products• Etc.

These applications could change the way we practice medicine!

Example Application: Breast Cancer Detection

Figure 2: Cellular Imaging (w/Drezek, Rice)Figure 2: Cellular Imaging (w/Drezek, Rice)Breast carcinoma cells, with green QDotsWith R. Drezek (Rice University)

ObjectiveTo understand and manipulate protein-nanoparticle interactions

Non-Specific

Targeted

Electrostatic

Covalent

What governs these types of interactions?

ObjectiveTo understand and manipulate protein-nanoparticle interactions

Non-Specific

Targeted

Electrostatic

Covalent

What governs these types of interactions?

ObjectiveTo understand and manipulate protein-nanoparticle interactions

Non-Specific

Targeted

Electrostatic

Covalent

What governs these types of interactions?

Quick Intro to Proteins….

Amino Acid

www.wikipedia.org

Quick Intro to Proteins….

Chains of amino acids make up proteins….

…)∞∞(…

Amino Acid

www.wikipedia.org

Quick Intro to Proteins….

Chains of amino acids make up proteins….

…)∞∞(…

Amino Acid #2

Amino Acid #1

Amino Acid

www.wikipedia.org

Due to non-covalent interactions, proteins fold into

complex structures…..

Representations of 3D Protein Structures

Space-filling Ribbon“Linguini”

Ball and Stick

My Research

My Model Nanobioconjugate System

MW = 17 kDa

Isoelectric point ≈ 7.0

http://www.umass.edu/microbio/rasmol/igg_w.gif

**15 nm*4.4 nm

*Kent, M.S.; Yim, H.; Sasaki, D.Y.; Langmuir 2004,20, 2819-2829**Hainfeld, J.F.; Powell, R.D. Journal of Histology & Cytochemistry 2000, Vol. 48(4): 471-480

MW ≈ 153 kDa

Isoelectric point ≈ 6.1-8.5

14 nmMW ≈ 1,000 kDa

Isoelectric point ≈ 5.5

Myoglobin

Immunoglobin G

Gold Nanocrystal

Synthesis of Aqueous Gold Nanoparticles

Solution of HAuCl4•H2O

Trisodium Citrate

Dispersion of Gold Nanoparticles

10-15% size distributionParticles > 10 nm in

diameter

++++++

+++

Au

AuCl2-

AuCl2-

AuCl2-

AuCl2-

AuCl2-

AuCl2-

AuCl2-

AuCl2-

Synthesis of Nanobioconjugates

Gold Nanoparticles

+

Unstable Stable

=or

But, how do we know if the proteins are actually attached?

How do we determine the number of proteins on the surface of the nanoparticles?

Flocculation Assay

Add 1% sodium chloride to nanobioconjugates with varying protein concentrations….

…when the particles are completely covered by the protein, they will not change colors

No protein / No NaCl No protein + NaCl Protein + NaCl

Flocculation Assay

0.00 0.02 0.10 0.20 0.38 0.57 0.95 1.40 1.90

[protein]

No ProteinNo NaCl

Increasing Protein Concentration

Quantify Flocculation Assays

300 400 500 600 700 800

Ab

sorb

ance

(a.

u.)

Wavelength (nm)

0 uM 0.4 uM 1.6 uM 2.4 uM 3.2 uM 4 uM 0 uM + no NaCl

300 400 500 600 700 800

Ab

sorb

ance

(a.

u.)

Wavelength (nm)

0 uM .1 uM .3 uM .5 uM .7 uM .9 uM 1 uM 0 uM + no NaCl

Ultra-Violet/Visible Spectroscopy

0 10.1 0.3 0.5 0.7 0.9 0 1.6 43.22.40.4control control

[IgG] (uM) [myoglobin] (uM)

We have determined stability and quantified the number of proteins.

Now, what about protein function?

Gel Electrophoresis

• Separates proteins based on size…

Sodium Dodecyl Sulfate (SDS)

First, proteins are unfolded and charged….

Gel Electrophoresis

Gel Electrophoresis

Gel Electrophoresis

Gel Electrophoresis

Gel Electrophoresis

Gel Electrophoresis

Gel Electrophoresis

Gel Electrophoresis

Gel Electrophoresis

Gel Electrophoresis

+-

Apply a Voltage

Gel Electrophoresis

+-

Apply a Voltage

Gel Electrophoresis

+-

Apply a Voltage

Large Proteins

Small Proteins

Gel Electrophoresis

Electrophoresis: Confirmation of IgG OrientationIg

G fi

rst

Myo

firs

t

myo

glob

in

IgG

IgG bands either missing or too light to detect!

Nanobioconjugates for Epitope-Mapping

ObjectiveTo understand and manipulate protein-nanoparticle interactions

Non-Specific

Targeted

Electrostatic

Covalent

What governs these types of interactions?

ObjectiveTo understand and manipulate protein-nanoparticle interactions

Non-Specific

Targeted

Electrostatic

Covalent

What governs these types of interactions?

Definitions

Antibody Antigen

Definitions

Antibody

Paratope

Antigen

Epitope

Definitions

Antibody

Paratope

Antigen

Epitope

We actually don’t know the epitope of Myoglobin!

Epitope-Mapping

• Studying the interactions of antibodies with specific regions of protein antigens

• Very expensive and time-consuming!

Epitope-Mapping

• Studying the interactions of antibodies with specific regions of protein antigens

• Very expensive and time-consuming!

• Why should anyone care?– Development of new vaccines & diagnostics

Can we use nanobioconjugates to determine the relative position of the epitope?

?

Can we use nanobioconjugates to determine the relative position of the epitope?

?

Can we use nanobioconjugates to determine the relative position of the epitope?

?

Can we use nanobioconjugates to determine the relative position of the epitope?

?

One mutant’s interaction with the gold nanoparticles should occlude the epitope region and either prevent IgG from binding or decrease its binding affinity in comparision to the other mutants.

Myoglobin Mutants

4 myoglobin mutants:

K63C, H81C, E105C, and G121C

Myoglobin Mutants

K63C

H81

C

E105

C

G12

1C

WT

WT

alon

e

IgG

alo

ne

4 myoglobin mutants:

K63C, H81C, E105C, and G121C

Myoglobin Mutants

K63C

H81

C

E105

C

G12

1C

WT

WT

alon

e

IgG

alo

ne

4 myoglobin mutants:

K63C, H81C, E105C, and G121C

• K63C mutant does not interact as well with IgG (near epitope?)

Summary & Conclusions

• Nanobioconjugates– Nanomaterial + Biomolecule– Have both nano and bio properties– Stability can be quantified– Stoichiometry can be evaluated

• Epitope-Mapping– Nanobioconjugates provide an easier and cheaper

way of determining epitopes .