Jin Kim MontclarePolytechnic Institute of NYU

Department of Chemical and Biological Sciences

March 22, 2010

Engineered Protein Biomaterials

Protein Interactions: Intra- and Extracellular Matrix

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In vivo incorporation of unnatural amino acids

Amino acid analogs incorporated into E. coli proteins

Wagner G. et al. Nature 1999, 400, 86; Poux, A. N. and Marmorstein R. Biochemistry 2003, 42, 14366; Treivel, R.C., Li, F.-Y. and Marmorstein, R. Anal. Biochem. 2000, 287, 319.

Histone acetyltransferase tGcn5

C-terminus

specificity

N-terminus

affinity

10 Phe’s distributed

Question: How does fluorinated phenylalanines influence stability, function

and selectivity of tGcn5?

Histone acetylation or Transcription factor acetylation--> birth defects, cancers, heart disorders

Histone Acetyltransferases (HATs)

Transcription factor p53

Histone

Variants with altered substrate specificity

K. Mehta

Two variants pFF-F112R and pFF-112R-Q114L exhibit a preference for p53 with no measurable activity for target histone H3.

H3

p53

Kca

t/km (

mM

-1s-1

)K

cat/k

m (

mM

-1s-1

)

Selectivity switch by integrating chemical and genetic diversity

Combination of unnatural amino acid incorporation with mutagenesis modulates substrate specificity.

Transcription factor p53

HistonepFF-F112R

pFF-F112R

pFF-F112R

X

Protein Interactions: Intra- and Extracellular Matrix

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• Mono-dispersity

Proteins

(PLGAs)

Synthetic Polymer

(PMMA)

I. Chen and B. Gao Anal. Chem., 1997, 69, 4399

• Well defined secondary structures

[(AG)xEG]n

Krejchi et.al. Science, 1994, 265, 1427

Chain length, sequence and stereochemistry

Biopolymer Synthesis

Block polymers of 2 different self-assembling domains (SADs)

Understand self-assembly,

control hierarchical organization

Integrate signaling molecules

Control binding of

small molecules:

delivery

Tunable smart materials

Controltemperatur

e-dependent assembly

Orientation of SADs influenceStructure and

assembly

Vitamin D3

Cartilage oligomeric matrix protein coiled coil (COMPcc)

Ozbeck, S., Engel, J., Stetefeld, J. EMBO J. 2002, 21, 5960.

Comprised of homopentamer of coiled coils

Hydrophobic pore 7.3 nm long and 0.2-0.6 nm diameter

Binds the hormone 1,25-dihydroxy (vitamin D3)

Comprised of pentapeptide repeat (GVPXP)n

Exhibits lower critical solution temperature (LCST) depends on identity of X and number of

repeatsUrry, D.W. and Parker, T.M. J. Muscle Res Cell Mot. 2002, 23, 543.

Elastin polypeptide

Secondary structure and stability characterization

The orientation of fusion does make a difference on overall structure of di-blocks

The number of blocks play an important role in overall conformation and temperature dependent behavior of block polymers

Influence of vit D on the polymer structure and assembly is dependent on block orientation and composition

J. Haghpanah, C. Yuvienco

EC CE

ECE

6.9oC -11.6oC1.3oC

TEM Analysis of Block Polymers: Particle-Fiber Switch

EC and ECE look to have similar features with slightly larger sizes when compared to CE, consistent with DLS

While ECE is larger in molecular weight, the article sizes are slightly smaller than EC.

J. Haghpanah, E. Roth

EC CE

ECE

33.8-40.1 nm 26.9-29.8.1 nm 31.5-39.2 nm

Block Polymer Binding of ATR and Ccm via Fluorescence

J. Haghpanah, H. Barra

CE: binds best to ATR and Ccm indicating importance of N-terminal C domain

ECE and EC: additional C-terminal E domain improves binding

Microrheology of Block Polymers

J. Haghpanah, R. Tu

9.6 mg/ mL

4.8 mg/ mL

EC CE

ECE

EC: elastic, CE: viscous and ECE: viscoelastic--orientation and blocknumber important

Future collaborations