Protein Function in Nanobioconjugates CHEM 570 March 27, 2008 Jennifer A. Jamison Matthews Research...
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![Page 1: Protein Function in Nanobioconjugates CHEM 570 March 27, 2008 Jennifer A. Jamison Matthews Research Group.](https://reader035.fdocuments.in/reader035/viewer/2022070410/56649ec75503460f94bd3fa9/html5/thumbnails/1.jpg)
Protein Function in Nanobioconjugates
CHEM 570March 27, 2008
Jennifer A. JamisonMatthews 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
SS
SS
S S
S
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What are Nanobioconjugates?
• A biomolecule (protein, DNA, RNA, etc.) attached to a nanomaterial
Electrostatically Covalently
-
-
-
--
--
- ---
-- -
--
++
++
++
+
++ + +
+ +
S
SS
SS
S S
S
![Page 5: Protein Function in Nanobioconjugates CHEM 570 March 27, 2008 Jennifer A. Jamison Matthews Research Group.](https://reader035.fdocuments.in/reader035/viewer/2022070410/56649ec75503460f94bd3fa9/html5/thumbnails/5.jpg)
What are Nanobioconjugates?
• A biomolecule (protein, DNA, RNA, etc.) attached to a nanomaterial
Electrostatically Covalently
-
-
-
--
--
- ---
-- -
--
++
++
++
+
++ + +
+ +
S
SS
SS
S S
S
![Page 6: Protein Function in Nanobioconjugates CHEM 570 March 27, 2008 Jennifer A. Jamison Matthews Research Group.](https://reader035.fdocuments.in/reader035/viewer/2022070410/56649ec75503460f94bd3fa9/html5/thumbnails/6.jpg)
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
Figure 2: Cellular Imaging (w/Drezek, Rice)Figure 2: Cellular Imaging (w/Drezek, Rice)Breast carcinoma cells, with green QDotsWith R. Drezek (Rice University)
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ObjectiveTo understand and manipulate protein-nanoparticle interactions
Non-Specific
Targeted
Electrostatic
Covalent
What governs these types of interactions?
![Page 11: Protein Function in Nanobioconjugates CHEM 570 March 27, 2008 Jennifer A. Jamison Matthews Research Group.](https://reader035.fdocuments.in/reader035/viewer/2022070410/56649ec75503460f94bd3fa9/html5/thumbnails/11.jpg)
ObjectiveTo understand and manipulate protein-nanoparticle interactions
Non-Specific
Targeted
Electrostatic
Covalent
What governs these types of interactions?
![Page 12: Protein Function in Nanobioconjugates CHEM 570 March 27, 2008 Jennifer A. Jamison Matthews Research Group.](https://reader035.fdocuments.in/reader035/viewer/2022070410/56649ec75503460f94bd3fa9/html5/thumbnails/12.jpg)
ObjectiveTo understand and manipulate protein-nanoparticle interactions
Non-Specific
Targeted
Electrostatic
Covalent
What governs these types of interactions?
![Page 13: Protein Function in Nanobioconjugates CHEM 570 March 27, 2008 Jennifer A. Jamison Matthews Research Group.](https://reader035.fdocuments.in/reader035/viewer/2022070410/56649ec75503460f94bd3fa9/html5/thumbnails/13.jpg)
Quick Intro to Proteins….
Amino Acid
www.wikipedia.org
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Quick Intro to Proteins….
Chains of amino acids make up proteins….
…)∞∞(…
Amino Acid
www.wikipedia.org
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Quick Intro to Proteins….
Chains of amino acids make up proteins….
…)∞∞(…
Amino Acid #2
Amino Acid #1
Amino Acid
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
Space-filling Ribbon“Linguini”
Ball and Stick
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My Research
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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
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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-
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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?
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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
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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
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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)
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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…
Sodium Dodecyl Sulfate (SDS)
First, proteins are unfolded and charged….
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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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+-
Apply a Voltage
Gel Electrophoresis
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+-
Apply a Voltage
Gel Electrophoresis
![Page 39: Protein Function in Nanobioconjugates CHEM 570 March 27, 2008 Jennifer A. Jamison Matthews Research Group.](https://reader035.fdocuments.in/reader035/viewer/2022070410/56649ec75503460f94bd3fa9/html5/thumbnails/39.jpg)
+-
Apply a Voltage
Large Proteins
Small Proteins
Gel Electrophoresis
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Electrophoresis: Confirmation of IgG OrientationIg
G fi
rst
Myo
firs
t
myo
glob
in
IgG
IgG bands either missing or too light to detect!
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Nanobioconjugates for Epitope-Mapping
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ObjectiveTo understand and manipulate protein-nanoparticle interactions
Non-Specific
Targeted
Electrostatic
Covalent
What governs these types of interactions?
![Page 43: Protein Function in Nanobioconjugates CHEM 570 March 27, 2008 Jennifer A. Jamison Matthews Research Group.](https://reader035.fdocuments.in/reader035/viewer/2022070410/56649ec75503460f94bd3fa9/html5/thumbnails/43.jpg)
ObjectiveTo understand and manipulate protein-nanoparticle interactions
Non-Specific
Targeted
Electrostatic
Covalent
What governs these types of interactions?
![Page 44: Protein Function in Nanobioconjugates CHEM 570 March 27, 2008 Jennifer A. Jamison Matthews Research Group.](https://reader035.fdocuments.in/reader035/viewer/2022070410/56649ec75503460f94bd3fa9/html5/thumbnails/44.jpg)
Definitions
Antibody Antigen
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Definitions
Antibody
Paratope
Antigen
Epitope
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Definitions
Antibody
Paratope
Antigen
Epitope
We actually don’t 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?
?
![Page 50: Protein Function in Nanobioconjugates CHEM 570 March 27, 2008 Jennifer A. Jamison Matthews Research Group.](https://reader035.fdocuments.in/reader035/viewer/2022070410/56649ec75503460f94bd3fa9/html5/thumbnails/50.jpg)
Can we use nanobioconjugates to determine the relative position of the epitope?
?
![Page 51: Protein Function in Nanobioconjugates CHEM 570 March 27, 2008 Jennifer A. Jamison Matthews Research Group.](https://reader035.fdocuments.in/reader035/viewer/2022070410/56649ec75503460f94bd3fa9/html5/thumbnails/51.jpg)
Can we use nanobioconjugates to determine the relative position of the epitope?
?
![Page 52: Protein Function in Nanobioconjugates CHEM 570 March 27, 2008 Jennifer A. Jamison Matthews Research Group.](https://reader035.fdocuments.in/reader035/viewer/2022070410/56649ec75503460f94bd3fa9/html5/thumbnails/52.jpg)
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.
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Myoglobin Mutants
4 myoglobin mutants:
K63C, H81C, E105C, and G121C
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Myoglobin Mutants
K63C
H81
C
E105
C
G12
1C
WT
WT
alon
e
IgG
alo
ne
4 myoglobin mutants:
K63C, H81C, E105C, and G121C
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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?)
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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 .