Post on 13-Jan-2016
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 .