Transcript Protein Function in Nanobioconjugates

Protein Function in
Nanobioconjugates
CHEM 570
March 27, 2008
Jennifer A. Jamison
Matthews 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
S
S
S
S
S
S
S
What are Nanobioconjugates?
• A biomolecule (protein, DNA, RNA, etc.) attached to
a nanomaterial
Electrostatically
+ + +
+ - - - +
+ + - +
-+
- - +
+ + +
Covalently
S
S
S
S
S
S
S
S
What are Nanobioconjugates?
• A biomolecule (protein, DNA, RNA, etc.) attached to
a nanomaterial
Electrostatically
+ + +
+ - - - +
+ + - +
-+
- - +
+ + +
Covalently
S
S
S
S
S
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
Breast
cells, with
greenRice)
QDots
Figurecarcinoma
2: Cellular Imaging
(w/Drezek,
With R. Drezek (Rice University)
Objective
To understand and manipulate protein-nanoparticle interactions
NonSpecific
Electrostatic
What governs
these types of
interactions?
Targeted
Covalent
Objective
To understand and manipulate protein-nanoparticle interactions
NonSpecific
Electrostatic
What governs
these types of
interactions?
Targeted
Covalent
Objective
To understand and manipulate protein-nanoparticle interactions
NonSpecific
Electrostatic
What governs
these types of
interactions?
Targeted
Covalent
Quick Intro to Proteins….
Amino Acid
www.wikipedia.org
Quick Intro to Proteins….
Amino Acid
Chains of amino acids make up
proteins….
∞(…
…)∞
www.wikipedia.org
Quick Intro to Proteins….
Amino Acid
Chains of amino acids make up
proteins….
Amino Acid #2
Amino Acid #1
∞(…
…)∞
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
*4.4 nm
**15 nm
Myoglobin
MW = 17 kDa
Isoelectric point ≈ 7.0
Immunoglobin G
MW ≈ 153 kDa
Gold Nanocrystal
14 nm
http://www.umass.edu/microbio/rasmol/igg_w.gif
Isoelectric point ≈ 6.1-8.5
MW ≈ 1,000 kDa
Isoelectric point ≈ 5.5
*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
Synthesis of Aqueous Gold Nanoparticles
10-15% size distribution
Particles > 10 nm in
diameter
Trisodium Citrate
Solution of HAuCl4•H2O
Dispersion of Gold Nanoparticles
+++
+++
AuCl2-
AuCl2AuCl2-
Au
AuCl2+++
AuCl2-
AuCl2AuCl2AuCl2-
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?
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
Flocculation Assay
0.00 0.02
0.10
0.20
0.38
0.57
[protein]
Increasing Protein Concentration
0.95
1.40
1.90 No Protein
No NaCl
Quantify Flocculation Assays
Ultra-Violet/Visible Spectroscopy
300
400
500
600
700
0 uM
0.4 uM
1.6 uM
2.4 uM
3.2 uM
4 uM
0 uM + no NaCl
Absorbance (a.u.)
Absorbance (a.u.)
0 uM
.1 uM
.3 uM
.5 uM
.7 uM
.9 uM
1 uM
0 uM + no NaCl
300
800
400
500
[IgG] (uM)
0.1
0.3
0.5
0.7
700
800
Wavelength (nm)
Wavelength (nm)
0
600
[myoglobin] (uM)
0.9
1 control
0
0.4
1.6
2.4
3.2
4 control
We have determined stability and
quantified the number of proteins.
Now, what about protein function?
Gel Electrophoresis
• Separates proteins based on size…
First, proteins are unfolded and charged….
Sodium Dodecyl Sulfate (SDS)
Gel Electrophoresis
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
+
Large Proteins
Small Proteins
Apply a Voltage
Electrophoresis: Confirmation of IgG Orientation
IgG
myoglobin
Myo first
IgG first
IgG bands either missing or too light to detect!
Nanobioconjugates for EpitopeMapping
Objective
To understand and manipulate protein-nanoparticle interactions
NonSpecific
Electrostatic
What governs
these types of
interactions?
Targeted
Covalent
Objective
To understand and manipulate protein-nanoparticle interactions
NonSpecific
Electrostatic
What governs
these types of
interactions?
Targeted
Covalent
Definitions
Antibody
Antigen
Definitions
Paratope
Antibody
Epitope
Antigen
Definitions
Paratope
Antibody
Epitope
Antigen
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
4 myoglobin mutants:
IgG alone
WT alone
WT
G121C
E105C
H81C
K63C
K63C, H81C, E105C, and G121C
Myoglobin Mutants
4 myoglobin mutants:
• K63C mutant does not interact as well with IgG (near epitope?)
IgG alone
WT alone
WT
G121C
E105C
H81C
K63C
K63C, H81C, E105C, and G121C
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 .