Impact of Fluorinated Amino Acids on Artificial Protein

Block Copolymers of Two Self-assembling DomainsCarlo Yuvienco1, Jennifer S. Haghpanah1, Richard Hwang and Jin Kim Montclare1,2

Chemical & Biological Sciences, Polytechnic Institute of NYU, Brooklyn, NY, 112011

Department of Biochemistry, SUNY Downstate Medical Center, Brooklyn, NY, 11203 2

ACKNOWLEDGEMENTS

WE WOULD LIKE TO THANK POLYTECHNIC UNIVERSITY START-UP FUNDS, THE OTHMER INTITUTE, THE WECHLER

AWARD, AIR FORCE OFFICE OF SCIENTIFIC RESEARCH, SOCIETY OF PLASTIC ENGINEERS, ACS CHEMISTRY

INSITUTE, ACS ENVIORNMENTAL CHEMISTRY DIVISION, UNILEVER, THE NATIONAL SCIENCE FOUNDATION GK-12

FELLOWS GRANT DGE-0741714

Abstract

The requirement for smart protein-derived biomaterials to change in

macromolecular structure in response to external stimuli necessitates

the design of controllable modes of self-assembly. The recent

advances in unnatural amino acid incorporation enables the

integration of chemical diversity into such proteins, further expanding

the level of control and materials properties. In particular, fluorinated

amino acids have been `of the biomaterials. Specifically, we have

incorporated para-fluorophenylalanine and trifluoroleucine into three

block polymers that consist of a β-spiral elatin-mimetic protein (E)

and an α-helical coiled-coil region of cartilage-oligomeric matrix

protein (C). These proteins, synthesized as the block sequences –

EC, CE, and ECE – are chosen for their distinct structures, functions,

and modes of self-assembly. We demonstrate successful

incorporation of the non-natural amino acids as well as

characterization emphasizing their structural and functional distinction

relative to the non-fluorinated constructs.

1 Expression7

Conclusions & Future Work

• Successful incorporation of pFF into elastin/COMPcc fusions

•Difference in melting curves apparent in fluorinated constructs

•Increase in CE cooperativity, demonstrating a dependence on

block orientation when comparing fluorinated proteins

•Increase in ECE cooperativity but decrease in Tm, suggesting

an accelerated hydrophobic effect

•We will continue to incorporate other non-canonical aa into the

fusions, including trifluoroLeu, photoLeu and p-azidoPhe

•Microscopy studies of protein solutions to investigate

supramolecular assembly structures

8 Purification of pFF Constructs

OH

OH

HO

1,25-dihydroxyvitamin D3 O

OH

all-trans retinoic acid

O

OH

all-trans retinoic acid

O

OH

elaidic acid

cyclohexane

Cartilage Oligomeric Matrix Protein

Coiled-Coil (C)

2

V. N. Malashkevich, R. A. Kammerer, V. P. Efimov, T. Schulthess, and J. Engel, Science, (1996) 274, 761-765.

Suat Ozbek, Jürgen Engel, Jörg Stetefeld, EMBO J 2002, 21, 5960-8.

B. Li, V. Daggett, J Muscle Res Cell Motil 2002, 23, 561-73.

Bumjoon Kim, Ashutosh Chilkoti, J Am Chem Soc 2008, 130, 17867-73.

Elastin (E)3

-14

-12

-10

-8

-6

-4

-2

0

2

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6

8

10

190 200 210 220 230 240 250

[θ] m

rw(x

10

3d

eg

· c

m2

· d

mo

l-1)

EC

-14

-12

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-8

-6

-4

-2

0

2

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6

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190 200 210 220 230 240 250

Wavelength, nm

CE

-14

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-8

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-2

0

2

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190 200 210 220 230 240 250

ECE

4

5

Protein Block Polymers

MRGSH6GSKPIAASA–Elastin–LEGSELA(AT)6AACG–COMPcc–LQA(AT)6AVDLQPS

MRGSH6GSACELA(AT)6AACG–COMPcc–LQA(AT)6AVDKPIAASA–Elastin–LEGSGTGAKL

MRGSH6GSKPIAASA–Elastin–LEGSELA(AT)6AACG–COMPcc–LQA(AT)6AVDKPIAASA–Elastin–LEGSGTGAKL

Elastin = [(VPGVG)2VPGFG(VPGVG)2]5VP

COMPcc = DLAPQMLRELQETNAALQDVRELLRQQVKEITFLKNTVMESDASG

6

•Different structure vs. EC

•More a-helical at 4C

9

Comparison of Melting Curves of Wild-type

and Fluorinated Constructs

•Negligible effect on melting temperature for EC and CE diblocks

•Notable shift in Tm for ECE triblock

•Enhanced cooperativity of ECE with incorporated fluorinated Phe

•Though melt temperatures are the same as wild-type, the CE shows an increase in

transition cooperativity versus the EC diblock upon incorporation

10

EC

4C 55C

CE

4C 55C

•Random-like

structure at low T

•Shift to a-helical

11

65

55

45

35

30

25

20

15

10

4

Temperature Scale, °C

Hydrophobic pore

73 Å long

2-6 Å wide

Self-assembles into a pentameric

bouquet-like structure

α-Helical secondary structure

10.9 kDa

(Val/Ile)-Pro-Xaa-Yaa-Gly

Non-canonical Amino Acids

Residue-specific incorporation of non-natural

amino acids

740 790 840 m/z

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/Bruker/OmniFLEX/Users/User1/Carlo/090611_ECwt/1Lin/pdata/1 Administrator Thu Jun 11 16:17:06 2009

740 790 840 m/z

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/Bruker/OmniFLEX/Users/User1/Carlo/090611_ECpff/1Lin/pdata/1 Administrator Thu Jun 11 16:15:30 2009

740 790 840 m/z

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750 770 790 810 m/z

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/Bruker/OmniFLEX/Users/User1/Carlo/090611_ECEpff/1SLin/pdata/1 Administrator Thu Jun 11 16:20:54 2009

L pre 19aa wt pFF pre 19aa wt TFL

p-fluoroPhe trifluoroLeu

25 kD

20 kD

15 kD

37 kD

50 kD

0.66 0.60 1.78 1.56 0.86 1.44 2.56 1.74

OD600

25 kD

20 kD

15 kD

37 kD

50 kD

0.60 0.66 1.90 1.58 0.68 1.20 2.34 1.76

OD600

37 kD

25 kD

20 kD

50 kD

75 kD

0.60 0.64 1.72 1.26 0.84 0.94 2.00 1.66

OD600

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-8

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6

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190 200 210 220 230 240 250

[θ] m

rw(x

10

3d

eg

· c

m2

· d

mo

l-1)

EC pFF

-14

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190 200 210 220 230 240 250

Wavelength, nm

CE pFF

-14

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190 200 210 220 230 240 250

ECE pFF

L pre 19aa wt pFF pre 19aa wt TFL

p-fluoroPhe trifluoroLeu

L pre 19aa wt pFF pre 19aa wt TFL

p-fluoroPhe trifluoroLeu

•Small-scale(5 mL) and large-scale (100 mL) expressions of

•Phe auxotrophic AFIQ cells

•Leu auxotrophic LAM1000 cells

•Residual natural amino acids removed prior to induction with IPTG by washing with 2

cycles of washing with 0.9% NaCl

•All lanes in gels below normalize to OD600=1.00

•Purification of whole cell lysate under denatured conditions (6M urea)

•Co2+ affinity chromatrography using a 5mL HiTrap IMAC FF column (GE)

•Gels showing elution fractions from purification

•MALDI-TOF analysis confirming non-canonical amino acid incorporation

EC pFF CE pFF ECE pFF

37 kD

25 kD

20 kD

50 kD

75 kD

25 kD

20 kD

15 kD

37 kD

50 kD

25 kD

20 kD

15 kD

37 kD

50 kD

768 Da

768 Da

750 Da

772 Da

EC wt

768 Da

750 Da

Rela

tive Inte

nsity

CD of pFF Constructs

Tang, Y. et al. Fluorinated Coiled-Coil Proteins Prepared In Vivo Display Enhanced Thermal and Chemical Stability.

Angew. Chem. Int. Ed. Engl 40, 1494-1496(2001).

[1]A James Link, David A Tirrell, Methods 2005, 36, 291-298.

mRNA

Cells washed of residual Phe

AFIQ Pheauxotroph

E. coli

Introduction of non-canonical amino acid

upon induction

•Incorporated non-canonical amino

acids have been shown to enhance

structural stability some proteins

•Photo-crosslinking possible with the

incorporation of p-azidoPhe

•We hypothesize that the

incorporation of fluorinated amino

acids into our fusion sequences will

result in an alteration of temperature-

sensitive behavior

•Separate incorporation of p-fluoroPhe

and trifluoroLeu

•Different wavelength scan

•Initial structure at 4C

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 10 20 30 40 50 60 70 80 90

Fra

cti

on

Fo

lded

EC pFF and wt

p-fluoroPhe

wild-type

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 10 20 30 40 50 60 70 80 90

Temperature (°C)

CE pFF and wt

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 10 20 30 40 50 60 70 80 90

ECE pFF and wt

Tm = 47C 33CTm = 44C 45CTm = 33C 34C

•Temperature-sensitive aggregation

and self-assembly

•Coupling to other functional domains

has been shown to affect both

aggregation and temperature-

sensitivity

•Focus of potential use lies in drug

(small molecule) delivery and release

Lower criticial solution temperature

(LCST) – from liquid to solid by

adding heat

Binding small molecules