1.  Preparation of modified peptide solutions:
‒ Preheated (80⁰C) for 20min, kept at 37⁰C.
Cell culture: Mouse fibroblast (NIH 3T3) cells were seeded and incubated with
several peptides [15uM, preheated] for 1 hr in different temperature.
Flow cytometer (Cell Lab Quanta SC MPL, Beckman Coulter): Cells were
washed, trypsinized and analyzed.
 Circular dichromism (CD) spectroscopy: CD provided conformational data of
peptides at different temperatures.
Confocal microscopy (Olympus Fluoview 1000): Confocal imaging was used to
investigate peptide internalization.
Peptide Internalization Triggered by Temperature
Myungeun Oh, Chloe Hu, Katarzyna Slowinska, Ph.D.
Department of Chemistry and Biochemistry, California State University Long Beach
Cell penetrating peptides (CPPs) are known for their ability to carry molecules
across cell membrane. CPPs are efficient carriers of molecular cargo in micro-
molar concentration and penetrate cell membrane without damage to the cell.
Yet, CPP carriers are not selective and are not capable of targeted delivery
unless specific modification is added into CPP structure. This modification is
usually attachment of small molecule or peptide sequence targeting specific
molecular receptor. Here we report the attempt to design a protocol for
selective uptake of CPP that is based on peptide conformation. This protocol
opens a possibility of targeting cells without molecular targets. Here we
modified CPPs with collagen-like domain that allows peptide to fold into triple
helix conformation. This folding process is reversible and temperature
dependent. With confocal microscopy and flow cytometry we show the
controlled internalization of CPP, dependent on peptide folding that is
controlled with temperature. This type of controlled delivery may open the
possibility of active targeting cells that does not have known molecular targets.
Peptide Sequence Tm (⁰C)
V2R FITC-Aβ-GG- GPPOOGPGGGPOOPGOOPGGOOPP- R6 -
FL8V1 FITC-Aβ-GG-(POG)8- (RRG)2 48.8
FL6V1 FITC-Aβ-GG-(POG)6- (RRG)2 15.33
FL7V1 FlTC-Aβ-GG-(POG)7-(RRG)2 16.29
CPPs are well known for their ability to penetrate cell membrane and their
potential application as drug carriers. Yet their indiscrimination of cellular
membrane penetration hinders their potential clinical use. Here, we showed
the controlled localization of modified peptides using their conformational
change triggered by temperature. Result consistently indicated that the
peptides were internalized at temperature lower than their Tm. We concluded
that the cellular localization of peptides can be manipulated by temperature.
1. Bakota, E. , Sensoy, O. , Ozgur, B. , Sayar, M. , & Hartgerink, J. (2013). Self-
assembling multidomain peptide fibers with aromatic
cores.Biomacromolecules, 14(5), 1370-1378.
2.Fretz, M. , Penning, N. , Al-Taei, S. , Futaki, S. , Takeuchi, T. , et al. (2007).
Temperature-, concentration- and cholesterol-dependent translocation of l- and d-
octa-arginine across the plasma and nuclear membrane of cd34+ leukaemia
cells. The Biochemical Journal, 403(2), 335-342
3.Madani, F. , Lindberg, S. , Langel, U. , Futaki, S. , & Gräslund, A. (2011). Mechanisms
of cellular uptake of cell-penetrating peptides. Journal of Biophysics (Hindawi
Publishing Corporation : Online), 2011, 414729
4.Shinde, A., Feher, K.M., Hu, C., Slowinska, K. (2014).Folding short arginine-rice
sequences into triple helix enables efficient internalization. [submitted]
Introduction
Conclusion
Result
Methods and Materials
References
4⁰C
1 hr
Both Brightfield FITC
37⁰C
1 hr
4⁰C
1 hr
Both Brightfield FITC
37⁰C
1 hr
4⁰C
1 hr
Both Brightfield FITC
37⁰C
1 hr
Flow
4⁰C
1 hr
Both Brightfield FITC
37⁰C
1 hr
Flow
A B
Figure 2. Diagram of methods. (A) Procedure for taking confocal image with cell-seeded MatTek
dish. (B) Setup for dynamic temperature experiment.
We used confocal imaging and flow cytometer to investigate the
internalization of peptides by conformational change due to temperature
difference. Resulting data (Fig. 3) showed peptide internalization at
temperature lower than Tm for the peptides that have collagen mimetic
sequence. No internalization was observed for peptide at temperature
higher than Tm.
In order to look at peptide internalization controlled by temperature,
apparatus was designed which involved temperature gradient. This tubing
apparatus (Fig. 4) was set up inside the incubator to keep the cells at body
temperature (37⁰C). Fountain pump was used to circulate cold water
through the tube. One end of the coverslip with cells was put on top of the
tube to give low temperature (8 ⁰C -13 ⁰C). The other end of the coverslip
was put on top of a piece of tube to level out. This created the temperature
gradient on the coverslip which ranged from 8 ⁰C to 25 ⁰C. Using this
temperature gradient, dynamic peptide internalization can be investigated.
Contacts: Chloe Hu
Chloe.hu@student.csulb.edu
Katarzyna Slowinska
Katarzyna.slowinska@csulb.edu
Website: http://slowinskalab.weebly.com/research.html
Merlyn Arostegui
Merlyn.arostegui@gmail.com
Myungeun Oh
Myungeun.oh@student.csulb.edu
 This project was supported by National Institutes of Health.
 Special thanks to Dr. Slowinska and all the members of Slowinska lab .
Acknowledgements:
Figure 1. Diagram of methods. (A) Structure of modified peptide. (B) CD curve showing transition
temperature (Tm) and peptide conformation of FL7V1 peptide.
Table 1. Modified peptides information
Figure 3. Images from confocal microscopy (Both, Brightfield, and FITC) and flow cytometer. All flow data
represent peptides at 4⁰C and 37⁰C except for FL6V1 (15 ⁰C and 37⁰C).
A
Flow
Flow
Figure 4. Experiment looking at dynamic internalization of peptide. (A) Control coverslip sitting
inside incubator shelf. Edge of the coverslip is marked with red tape. (B) Coverslip with warm end
(bottom) and cold end (top, water tube running underneath). Edge of the coverslip is drawn on
screen with black line.
B
Related posters:
 ORGM 414: Circular dichroism studies of hybrid cell penetrating–collagen peptides
Author: Chloe Hu [8:00-10:00pm Mar 24, Hall C – Colorado Convention Center]
 BIOT 256: Feasibility of targeting cells without unique molecular targets
Author: Katarzyna Slowinska [6:00-9:00pm Mar 24, Imperial Ballroom – Grand Hyatt Denver]