This is my final presentation after finishing my Amgen Scholar Research at MIT.

1. Effect of Protein Charge Distribution on Protein-Polymer
Interactions in Solution
Brandon Clark†, Dr. Sieun Kim‡, Dr. Bradley D. Olsen*‡
†Department of Chemical Engineering, Worcester Polytechnic Institute
‡Department of Chemical Engineering, Massachusetts Institute of Technology
Background Our Research
Research Plan
1. Build neutral proteins with changing patch distributions
2. Comparing intermolecular interactions between proteins
3. Conjugate proteins with cationic, anionic and neutral polymers
4. Investigate protein-polymer conjugation to explore the induced charging
effects
1. Next Area of Study: Protein Charge Distribution (“Patchiness”)
3. Higher Protein Charge Hinders Self Assembly Due to
Repulsive Protein-Protein Interactions2
Small Angle X-Ray
Scattering shows
higher order in more
neutral Proteins
2. Goal: Model Protein Charge Distribution’s Effect on
Interactions with Polyelectrolytes and Neutral Polymers
𝐿2
𝑁
=
𝑃𝑎𝑡𝑐ℎ 𝑆𝑖𝑧𝑒
# 𝑜𝑓 𝑃𝑎𝑡𝑐ℎ𝑒𝑠
Neutral
Protein
No Patch High Patch
Adaptive Poisson-Boltzmann Solve
(APBS) displays electrostatic
potential surfaces for various GFP
proteins
1. Encapsulation via coacervation of
biomacromolecules in polyelectrolyte
complex micelles can improve drug
delivery and nanoreactor synthesis1
2. Higher Protein Charge Creates Aggregates and Coacervates
in Solution with Polyelectrolytes1
Turbidity and DLS: higher turbidity and
hydrodynamic radius as net charge increases
Gray: no coacervation
White: coacervation
Charge modified proteins

2. 2. AIBN: radical source
RAFT Polymerization
Step 1:
Step 2:
3. Chain Transfer Agent3
Maleimide-CTACTA
Methods: Polymerization
Polymerization for BioconjugationPolymerization for Separated Solutions
M1
(Neutral)
M2
(Cation)
M3
(Anion)
1. Monomers
Reactants
Polyelectrolyte Formation
Quaternized PDMAEMA: strong polycation PMAA: weak polyanionOriginal PDMAEMA: weak polycation
Most likely too strong to compare with PMAA due to natural
positive charge out of solution

3. Methods: Protein Synthesis
Bacterial Transformation
Lysed Cell
Ncol
Green Fluorescent
Protein
Hind3
Bioconjugation: Maleimide Thiol “Click” Reaction
Bioconjugate Solutions
1. IPTG
2. Cell Lysis
Ni-NTA column
Ni-NTA
6xHis-tag
On Proteins
Protein Purification
Bound
Protein
Eluting protein
using Imidazol
Filter
Solutions of separated
protein and polymer
Advantages of this click reaction
1.Site specific
2.No catalyst
3.Water soluble
4.Quick reaction

4. Bioconjugation with PNIPAMPolymerization
3. GPC Shows Polymerization of Monomers for Separated
Solutions
Results
1. NMR for Chain Transfer Agents
1. SDS Gel and LC/MS confirms Protein Yield and Bioconjugation
Maleimide-Functionalized PNIPAM MW: 48 kDa
Bioconjugates
Pure Proteins
(-20) (0) J HP NP HPR
2. Proteins Retain Glow before and after Bioconjugation
300 400 500 600 700 800
0
20
40
60
80
100
523
485
Intensity
m/z
507
[M+H]
[M+Na]
[M+K]
200 300 400 500 600 700 800
0
20
40
60
80
100
m/z
[M+Na]
247
1-Propanol Maleimide Anhydride
Mw = 224 Da
Maleimide Functionalized Benzo-CTA
Mw = 484 Da
2. LC-MS Mass Spectra for RAFT CTA
Expected molar masses were
seen using LC-MS.
This indicates presence of
desired compounds to use in
RAFT reactions.
LC/MS Molar Mass: 76kDa
Protein MW: 28 kDa
0 5 10 15 20 25 30
-0.00004
-0.00002
0.00000
0.00002
0.00004
0.00006
0.00008
0.00010
0.00012
Retention time (min)
5 10 15 20 25 30
-0.00004
-0.00002
0.00000
0.00002
0.00004
Retention time (min)
0 5 10 15 20 25 30
-0.00002
0.00000
0.00002
0.00004
0.00006
0.00008
0.00010
0.00012
0.00014
Retention time (min)
• PEG
• MW: 33 kDa
• PDI: 1.01
• PMAA
• MW: 54 kDa?
• PDI: 1.15
• PDMEAMA
• MW: 26 kDa
• PDI: 1.01
UV lamp at 365nm
Since GFP typically glows
when it is correctly folded,
this indicates that both pure
protein and bioconjugate
samples contain correctly
folded GFP.
For
bioconjugate
solutions
For
separated
solutions

5. Results, Conclusions, and Future Work
AcknowledgmentsReferences
This work was sponsored by the Amgen Foundation and facilitated by the
Massachusetts Institute of Technology UROP Program. I would like to thank
Amgen, MIT, Dr. Bradley Olsen, Dr. Sieun Kim, and the rest of the Olsen Lab
for their support and mentorship.
1. Obermeyer, Allie C., et al. "Complex coacervation of supercharged proteins with polyelectrolytes." Soft matter 12.15
(2016): 3570-3581.
2. Lam, Christopher N., Helen Yao, and Bradley D. Olsen. "The Effect of Protein Electrostatic Interactions on Globular
Protein–Polymer Block Copolymer Self-Assembly." Biomacromolecules 17.9 (2016): 2820-2829.
3. Chang, Dongsook, et al. "Effect of polymer chemistry on globular protein–polymer block copolymer self-
assembly." Polymer Chemistry 5.17 (2014): 4884-4895.
Turbidity Results Future Work
1. Successfully expressed GFP proteins with varied charge distributions
2. Various CTAs for RAFT polymerization were synthesized
3. Polymerized model polymers by RAFT polymerization with low
polydispersity (PDI: ~1.1)
4. Bioconjugated mutated GFP proteins with PNIPAM, showing expected
molecular weight and preserved protein folding
5. This study will show the effects of protein charge patch size on various
intermolecular interactions
DLS1
Different patch size of
proteins may show various
electrostatic interactions
between molecules
Turbidity can
elucidate the
extent that
proteins form
aggregates or
coacervates with
polymers and
polyelectrolytes 100 80 60 40 20 0
0.0
0.2
0.4
0.6
0.8
1.0
Absat750nm
Weight fraction, PEG
C(-20)
Janus
Homogeneous
100 80 60 40 20 0
0.0
0.2
0.4
0.6
0.8
1.0
Absat750nm Weight fraction, qPDMAEMA
C(-20)
Janus
Homogeneous
100 80 60 40 20 0
0.0
0.2
0.4
0.6
0.8
1.0
Absat750nm
Weight fraction, PAA
C(-20)
Janus
Homogeneous
Preliminary data: GFP (-20) and Janus GFP show high turbidity in
solution with the strong polycation
Neutral: PEG Cationic: qPDMAEMA Anionic: PAA
Protein
Modeling
Conclusions