GFP: Research Presentation

Anthony Baldridge

Georgia Institute of Technology

Laren Tolbert Group

What is GFP?First noted in 1955

Found in multiple organisms but cloned from Aequorea victoria

Protein of 238 amino acids

Chromophore formed by intramolecularautocatalytic cyclization

Resulting chromophore responsible for the green fluorescence

Photos: http://www.rpc.msoe.eduhttp://chemistry.nrl.navy.mil

http://technovelgy.com

Used extensively in biology

Protein/protein interactions

Cell studies

Advantages

What is the GFP chromophore?

Photo: http://www.rpc.msoe.edu

NN

O

H O

Wild-type GFP (wt-GFP)Absorptions: 398 nm and 477 nm

Fluorescence Emission: 508 nm

Quantum yield: 0.79

p-HBDIAbsorption: 370 nm

Fluorescence Emission: 440 nm

Quantum Yield: 0.0001

Why the gap in Φ?

Z (cis) form = fluorescent

E (trans) form = non-fluorescent

Excited-state cis/trans isomerizationMain non-radiative decay pathway

Ultrafast (<1 ps) deactivation to recover ground state.

Internal conversion faster than fluorescence and low quantum yield is observed.

NN

O

H O N

N

H O O

hv

Z (cis) E (trans)

Restricting conformational freedom

By restricting conformational freedom then Φ should increase.

Blocking main deactivation pathway

Provide a rigid structure

Similar conditions found in β-barrel (wt-GFP).

In addition to rigidity, complexation may produce planar structure.

N NN

O

XN N

N

O

X M +

M +

Proof of ConceptN N

N

O

PDI-Metal complex

PDI-Metal complex

Fluorescence Peak Change at 428 nm as PDI-ZnCl2 Formation

0

200000

400000

600000

800000

1000000

1200000

1400000

0.00E+00 5.00E-01 1.00E+00 1.50E+00 2.00E+00 2.50E+00 3.00E+00 3.50E+00

ZnCl2 Concentration (mM)

Peak

Inte

nsity

Hula-TwistMechanism of isomerizationunknown.

Derivatives synthesized that maximize interaction with carbonyl of imidazolone.

Preliminary results of excited state twisting.

 

NN

O

R

H

O

H

NN

O

R

H

O

HN

N

O

R

HO

H

NN

O

R

OH

HNN

O

R

H

OH

OBT-φ

T2-B

HT

OBT-τ

τφ

Erlenmeyer Synthesis

Most common technique to synthesize imidazolonesfound in literature

Unsuccessful for 2-pyridine derivatives

A r C H O + A cHN C O 2H A c 2O, A cON a

re fluxA r

NO

O

R N H2

E tOH , ref lu xA r

NN

O

Erlenmeyer, E. Justus Liebigs Ann. Chem. 1893, 275, 1-8.

Imidazolone Coupling Rxn.

Knoevenagel Condensation

First reproducible successful method for 2-pyridine derivatives.

Allows for use of any aromatic aldehyde.Schiff base is difficult to synthesize

CHO

N

N

O

piperidineN

N

O

+R

R

Combining the two

Combinatorial Approach

Any aromatic aldehyde

Readily can do substitutions

Synthetic Progress

October 2008

8 compounds

July 2009

75 compounds

CollaborationsProtein Docking

Database Screening

Solid-State Studies

Octa-acid Encapsulation

Encapsulation by DNA

Aptamers

CollaborationsDr. V. Ramamurthy – Miami

Encapsulation of chromophore to mimic protein environment

Dr. Young Tae Chang – Singapore

Screening methods to identify activity of chromophores

Dr. Dipaktar Sen – Simon Frazier

Binding chromophores to RNA Aptamers

Dr. Pance Naumov – Osaka

Solid-state studies of dimerization

Hula-twist studies

AcknowledgementsDr. Laren Tolbert

Dr. Janusz Kowalik

Dr. Kyril Solntsev

Dr. Arthur J. Ragauskas

CollaboratorsDr. V. Ramamurthy – Miami

Dr. Young Tae Chang – Singapore

Dr. Dipaktar Sen – Simon Frazier

Dr. Pance Naumov – Osaka