DESIGN, SYNTHESIS AND ACTIVITY STUDY OF

ANTIFREEZE PEPTIDES DERIVED FROM

SHORTHORN SCULPIN TYPE 1 AFP

Supervisory Committee:
Prof. Dr. Mohd Basyaruddin Abdul Rahman
Dr. Bimo Ario Tejo
Presenting by:
Azren Aida Binti Asmawi
GS31420
Outline
Research Background &
Literature Review
Objectives
Research Methodology
Results & Discussions
Conclusions & Future Works
Question & Answer
 Why antifreeze proteins?

Fresh ice cream

The same ice cream after 7 weeks in

the freezer

Ice crystals burst open meat tissue. Damaged tissue causes meat to
have less juice and bad texture

AFPs  edible antifreeze with less or zero toxicity 3

 Food-grade antifreeze agent
 Propylene glycol (PG) is the most
common antifreeze agent used in
food and other consumer products.

Frozen food

PG is used everywhere…

Classified as “generally
recognized as safe” (GRAS) by
FDA

A new, edible antifreeze agent

 GRAS status was given based on
derived from natural sources is
common use of PG in foods
needed!
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 Antifreeze protein (AFP)

 Antifreeze proteins were first discovered in Antarctic

nototheniidae by Eastment and DeVries over 30 years
ago.
 AFPs play protecting role as inhibitor toward ice
crystal formation.
 Since the discovery of AFPs in fish several other AFPs
have been discovered in other organisms such as
bacteria, fungi, plants, insects, and vertebrates.

Eastman, J. T., & DeVries, A. L. (1986). Renal glomerular evolution in Antarctic notothenioid fishes. Journal of Fish Biology, 29(6), 649–662
Jia, Z., DeLuca, C.L., Chao, H., & Davies, P.L. (1996). Structural basis for the binding of a globular antifreeze protein to ice. Nature. 384:285-288
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 Antifreeze mechanism of AFP

Hydrophilic surface binds

to disordered water
molecules

Prism faces Hydrophobic

surface binds to
AFPs well-ordered
water molecules

ice ice ice

Disrupted water structure is protected from

being overgrown
Ice crystal growth
Antifreeze protein
a) in a solution without AFPs;
b) in a dilute solution of AFPs which is absorbed to the
prism face, forming a crystal that is hexagonal in
shape;
c) in a concentrated solution of AFPs, shaping an ice Ice nucleus An antifreeze protein adsorbed on ice surface
crystal into a hexagonal bipyramidal. inhibits ice crystal growth

Nutt, D. R., & Smith, J. C. (2008). Dual function of the hydration layer around an antifreeze protein revealed by atomistic molecular dynamics simulations. Journal of the American
Chemical Society, 130(39), 13066–13073
Hassas-Roudsari, M., & Goff, H. D. (2012). Ice structuring proteins from plants: Mechanism of action and food application. Food Research International, 46, 425–436
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 Design of α-helical antifreeze peptide

DOWNSIZING THE PROTEINS

(a) (b)
Key elements for short AFPs:

a) Short peptides derived from large protein can be

Respiratory Syncytial Virus
(RSV) F protein, α-helix-
rich structure
fixed in highly α-helical structures in water via (i,
i+4) or (i, i+7) lactam bridges. These constrained
compounds have similar biological activities as
native proteins.
(c) b) Structure of all type I AFPs exhibit a hydrophobic
face which is an alanine rich surface while
hydrophilic residues vary.
c) The efficacy of antifreeze peptides corresponding to
the helicity and relatively flat of binding site of the
peptide.

Harding, M. M., Ward, L. G., & Haymet, A. D. J. (1999). Type I ` antifreeze ’ proteins Structure - activity studies and mechanisms of ice growth inhibition. European Journal of
Biochemistry, 264, 653–665
Harrison, R. S., Shepherd, N. E., Hoang, H. N., Ruiz-Gómez, G., Hill, T. A., & Driver, R. W. (2010). Downsizing human, bacteriail, and viral proteins to short water-stable alpha
helices that maintain biological potency. Proceedings of the National Academy of Sciences of the United States of America, 107(26), 11686–11691
Garner, J., & Harding, M. M. (2007). Design and synthesis of alpha-helical peptides and mimetics. Organic and Biomolecular Chemistry, 5(22), 3577–3585
7
1. To design and synthesize antifreeze peptides derived from different
segment of Type-I shorthorn sculpin AFP, SS-3.

2. To determine antifreeze activity of designed peptides by thermal hysteresis

(TH) and ice recrystallization inhibition (IRI) assays.

3. To elucidate secondary structure of peptides using infrared spectroscopy

(FTIR) and circular dichroism (CD).

4. To evaluate the antifreeze ability of peptides on ice cream.

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9
 Design of antifreeze peptides

A recombinant form of the type I shorthorn sculpin antifreeze protein (PDB reference IY03)

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 Design of antifreeze peptides
(A) (B) (C) (D) (E)

Salt bridge

Salt bridge

The cartoon representation of predicted 3D conformation of the designed peptides using PEP FOLD and
PyMOL. Both modified peptides are believed to form better in hydrophobicity distribution and high in helicity
content, subsequently exhibit higher antifreeze activity.

(A) peptide SC-N, (B) peptide SC-M, (C) peptide SC-C, (D) peptide SC-MM, (E) peptide SC-NM
Group coloring key: Nonpolar (yellow), polar/uncharged (green), acidic (red), basic (blue)
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 Synthesis of antifreeze peptides

(a) (b)

HPLC analysis of peptide SC-N. (a) crude peptide (b) purified peptide.
Amblard, M., Fehrentz, J. A., Martinez, J., & Subra, G. (2006). Methods and protocols of modern solid phase peptide synthesis. Molecular Biotechnology, 33(3), 239–254
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 Molecular weight analysis

+MS Chip Scan (8.925-9.070 min, 10 Scans) Frag=175.0V SCN-10ppm-Pos-15_30.d Subtract

x10 6
Peak List
m/z z Abund
1.5 562.6315
562.6315 3 1506575.8
1.25 563.2998 3 759058.7 m = 562.6315
1 843.4451 2 526072.5
844.4466 2 259044
z=3
0.75
0.5 843.4451
Thus, (562.6315 x 3) – 3
0.25 = 1684.8945 g/mol
0 200 400 600 800 10001124.9285
1200 1451.7692
1400 1600 1800 2000 2200 2400
Counts vs. Mass-to-Charge (m/z)

(SC-N) Expected mass: 1684.8678, experimental mass: 1684.8945

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 Ice crystal morphology

All designed peptides able to

modify ice crystal shape.

Peptide SC-MM and SC-NM

show significant hexagonal
shape similar as low
concentration of SS3 AFP 1.

Ice crystal morphology start from a single ~10µm ice crystal and was slowly grown in a solution of (A)
unbuffered water solution at pH 5 without peptide, (B) Scrambled peptide, (C) SC-C, (D) SC-M, (E) SC-N, (F)
SC-NM, (G) SC-MM, (H) SS3 AFP 1 at 1 mM concentration and (I) SS3 AFP 1 at 10 Mm concentration.

Harding, M. M., Ward, L. G., & Haymet, A. D. J. (1999). Type I ` antifreeze ’ proteins Structure - activity studies and mechanisms of ice growth inhibition. European Journal of
Biochemistry, 264, 653–665 14
 Ice recrystallization inhibition (IRI)
(a) (b)

(c)

…initial ice crystal (-6oC)

(d) (e) Antifreeze peptides

without peptide with peptide SC-MM inhibit ice growth

…after 3 hours (-6oC)

The growth of ice crystal in the presence of 10 mM peptides, that was observed after 3 h of incubation at the
temperature of -6 oC.
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 FT-IR analysis

Amide I
Amide II

α-helix
1545 cm-1 (Amide II)
1650 - 1654 cm-1  (Amide I)

β-turn
1625 - 1640 cm-1 (Amide II)
1520 - 1530 cm-1  (Amide I)

unordered
1528 cm-1 (Amide II)
1640 - 1651 cm-1  (Amide I)

Charpentier, T. V., Neville, A., Millner, P., Hewson, R., & Morina, A. (2013). An investigation of freezing of supercooled water on anti-freeze protein modified surfaces. Journal of
Bionic Engineering, 10, 139–147 16
 Circular dichroism analysis

 At 4 °C, peptide SC-C, SC-M,

SC-MM and SC-NM spectra
display two minima bands at
about λ ≈ 221–223 nm and 202-
206 nm while maxima band can
be found at 190–195 nm
wavelength regions.

• This maxima and minima values

demonstrate the presence of α-
helical conformation.

Raussens, V., Ruysschaert, J. M., & Goormaghtigh, E. (2003). Protein concentration is not an absolute prerequisite for the determination of secondary structure from circular
dichroism spectra: A new scaling method. Analytical Biochemistry, 319(1), 114–121 17
 Cytotoxicity analysis

From screening of peptide SC-NM, SC-MM, SC-C, SC-M and SC-N in 3T3 normal cells, these peptides
were not cytotoxic towards this cell line, as indicated by their respective IC 50 value which is more than 100
µg/mL.
Yang, Q. Z., Wang, C., Lang, L., Zhou, Y., Wang, H., & Shang, D. J. (2013). Design of potent, non-toxic anticancer peptides based on the structure of the antimicrobial peptide, temporin-
1CEa. Archives of Pharmacal Research, 36, 1302–1310 18
 Antifreeze peptides in ice cream

before thermal cycles (a) (b)

( -40°C)

Ice cream + Ice structuring activity

Peptide determination (c) (d) (e)

The addition of designed peptides able to suppress

the growth of ice crystal in an ice cream mix after
seven thermal cycles at -14 to -12°C.
(a) negative control (without peptide),
(b) positive control (SS-3 AFP),
(f) (g)
(c) peptide SC-C,
(d) peptide SC-N,
(e) peptide SC-M
(f) peptide SC-NM,
(g) peptide SC-MM.

Damodaran, S. (2007). Inhibition of ice crystal growth in ice cream mix by gelatin hydrolysate. Journal of Agricultural and Food Chemistry, 55, 10918–10923
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 Short peptides with less
than 25 amino acids still
able to exhibit antifreeze
activity and may have
significant technological
application.

Ice binding affinity of

Replacing Pro with Ala
modified peptides (SC-
residue in peptide SC-N
NM and SC-MM) is
sequence able to
improved by
increase helicity of the
modification of better
peptide, which can have
hydrophobic and
significant antifreeze
hydrophilic surface in
activity.
their structure.

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Future works
Molecular dynamics (MD) simulation to investigate the
possible interaction between peptide and ice interaction

Further modify peptides to improve their affinity to

ice, perhaps stronger IRI activity could be obtained

Conformation study using NMR spectroscopy for precise

secondary structure determination

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Paper
• Shah, S. H. H., Kar, R. K., Asmawi, A. A, Rahman, M. B. A., Murad, A. M. A., Mahadi, N. M., Basri, M., Rahman, R. N. Z. R. A.,
Salleh, A. B., Chatterjee, S., Tejo, B. A., & Bhunia, A. (2012). Solution structures, dynamics, and ice growth inhibitory activity of
peptide fragments derived from an antarctic yeast protein. PloS ONE, 7(11), 1–16.

• Abdul Rahman, M. B., Asmawi, A.A., Abdmalek. E., Salleh, A. B., & Tejo, B. A. (2015). Tailoring peptidomimetics antifreeze
protein from exotic antarctic marine. Malaysian Journal of Analytical Science. (in press).
 
Proceeding
• Azren Aida Asmawi, Ahmad Omar Abdelazim Warrad, Mohd Basyaruddin Abdul Rahman, Abu Bakar Salleh, Abdul Munir Abdul
Murad, Bimo Ario Tejo (2012). Synthesis and Activity of Short Antifreeze Peptides Derived from Type I Shorthorn Sculpin
Antifreeze Protein. 17th Malaysian Chemical Congress (17MCC), 15th – 17th October 2012, Putra World Trade Centre, Kuala
Lumpur.
 
• Azren Aida Asmawi, Mohd Basyaruddin Abdul Rahman, Abu Bakar Salleh, Abdul Munir Abdul Murad, Bimo Ario Tejo (2013).
Ice Recrystallization Inhibition of Short Antifreeze Peptides Derived from Type I Shorthorn Sculpin Antifreeze Protein.
Fundamental Science Congress 2013 (FSC 2013), 20th – 21st August 2013, Universiti Putra Malaysia, Serdang, Selangor.

• Azren Aida Asmawi, Mohd Basyaruddin Abdul Rahman, Abu Bakar Salleh, Abdul Munir Abdul Murad, Bimo Ario Tejo (2013).
Structure-activity Relationship and Function of Ice Structuring Peptides from Type 1 Shorthorn Sculpin. The 26 th Regional
Symposium of Malaysia Analytical Sciences (SKAM26), 4th – 5th December 2013, Kuching, Sarawak.

• Azren Aida Asmawi, Mohd Basyaruddin Abdul Rahman, Abu Bakar Salleh, Abdul Munir Abdul Murad, Bimo Ario Tejo (2015).
Rapid Structural Characterisation of Ice Structuring Peptides from Type 1 Shorthorn Sculpin by FTIR Spectroscopic. The 28 th
Regional Symposium of Malaysia Analytical Sciences (SKAM28), 17th – 20th August 2015, Ipoh, Perak.

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Thank You