Protein Function in Nanobioconjugates

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Protein Function in Nanobioconjugates

• CHEM 570
• March 27, 2008
• Jennifer A. Jamison
• Matthews Research Group

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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

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What are Nanobioconjugates?

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

Electrostatically
Covalently




S
S

S

-
-
S
S


-
S
-

S

S



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What are Nanobioconjugates?

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

Electrostatically
Covalently




S
S

S

-
-
S
S


-
S
-

S

S



5
What are Nanobioconjugates?

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

Electrostatically
Covalently




S
S

S

-
-
S
S


-
S
-

S

S



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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

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Applications

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

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Applications

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

These applications could change the way we
practice medicine!
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Example Application Breast Cancer Detection
Breast carcinoma cells, with green QDots With R.
Drezek (Rice University)
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Objective
To understand and manipulate protein-nanoparticle
interactions
Electrostatic
Non- Specific
What governs these types of interactions?
Targeted
Covalent
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Objective
To understand and manipulate protein-nanoparticle
interactions
Electrostatic
Non- Specific
What governs these types of interactions?
Targeted
Covalent
12
Objective
To understand and manipulate protein-nanoparticle
interactions
Electrostatic
Non- Specific
What governs these types of interactions?
Targeted
Covalent
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Quick Intro to Proteins.
Amino Acid
www.wikipedia.org
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Quick Intro to Proteins.
Amino Acid
Chains of amino acids make up proteins.
)8
8(
www.wikipedia.org
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Quick Intro to Proteins.
Amino Acid
Chains of amino acids make up proteins.
Amino Acid 2
Amino Acid 1
)8
8(
www.wikipedia.org
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Due to non-covalent interactions, proteins fold
into complex structures..
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Representations of 3D Protein Structures
Ribbon Linguini
Ball and Stick
Space-filling
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My Research
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My Model Nanobioconjugate System
15 nm
4.4 nm
Myoglobin
Immunoglobin G
Gold Nanocrystal
MW 1,000 kDa Isoelectric point 5.5
14 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
http//www.umass.edu/microbio/rasmol/igg_w.gif
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Synthesis of Aqueous Gold Nanoparticles

• 10-15 size distribution
• Particles gt 10 nm in diameter

• Solution of HAuCl4H2O

Dispersion of Gold Nanoparticles

AuCl2-

AuCl2-
AuCl2-
Au
AuCl2-
AuCl2-
AuCl2-
AuCl2-
AuCl2-

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Synthesis of Nanobioconjugates


or
Gold Nanoparticles
Unstable ?
Stable ?
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?
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Flocculation Assay
Add 1 sodium chloride to nanobioconjugates with
varying protein concentrations.
No protein / No NaCl
No protein NaCl
Protein NaCl
when the particles are completely covered by the
protein, they will not change colors
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Flocculation Assay
Increasing Protein Concentration
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Quantify Flocculation Assays
Ultra-Violet/Visible Spectroscopy
IgG (uM)
myoglobin (uM)
control
control
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We have determined stability and quantified the
number of proteins.

• Now, what about protein function?

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Gel Electrophoresis

• Separates proteins based on size

First, proteins are unfolded and charged.
Sodium Dodecyl Sulfate (SDS)
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Gel Electrophoresis
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Gel Electrophoresis
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Gel Electrophoresis
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Gel Electrophoresis
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Gel Electrophoresis
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Gel Electrophoresis
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Gel Electrophoresis
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Gel Electrophoresis
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Gel Electrophoresis
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Gel Electrophoresis
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Gel Electrophoresis
-

Apply a Voltage
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Gel Electrophoresis
-

Apply a Voltage
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Gel Electrophoresis
-
Large Proteins

Small Proteins
Apply a Voltage
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Electrophoresis Confirmation of IgG Orientation
IgG bands either missing or too light to detect!
myoglobin
IgG
IgG first
Myo first
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Nanobioconjugates for Epitope-Mapping
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Objective
To understand and manipulate protein-nanoparticle
interactions
Electrostatic
Non- Specific
What governs these types of interactions?
Targeted
Covalent
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Objective
To understand and manipulate protein-nanoparticle
interactions
Electrostatic
Non- Specific
What governs these types of interactions?
Targeted
Covalent
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Definitions
Antibody
Antigen
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Definitions
Paratope
Epitope
Antibody
Antigen
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Definitions
Paratope
Epitope
Antibody
Antigen
We actually dont know the epitope of Myoglobin!
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Epitope-Mapping

• Studying the interactions of antibodies with
  specific regions of protein antigens
• Very expensive and time-consuming!

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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

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Can we use nanobioconjugates to determine the
relative position of the epitope?
?
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Can we use nanobioconjugates to determine the
relative position of the epitope?
?
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Can we use nanobioconjugates to determine the
relative position of the epitope?
?
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Can we use nanobioconjugates to determine the
relative position of the epitope?
?
One mutants 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.
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Myoglobin Mutants
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Myoglobin Mutants
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Myoglobin Mutants

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

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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 .