“Harvesting Naturally Occuring And Engineered Proteins For Use in Synthetic

Systems” :Biotransporter Based ” Devices – From Sensors to

Actuators to Fuel Cell Membranes

:Engineering membranes and transporters: Structure and function of membranes and membrane transporters

• John Cuppoletti, University of Cincinnati

• Supported by Army Research Office MURI and DARPA

John Cuppoletti Department of Physiology University of Cincinnati

Mechanical Behavior of MaterialsSynthetic Active Transport MURI

(University of Cincinnati)OBJECTIVE:

To produce synthetic flexible membranes containing biological transport proteins that can utilize energy for the selective uptake, concentration and release of ions and molecules in an organized manner. The effort includes production of both macroscopic membranes and nanostructures containing transport proteins with vectorial transport function.

APPROACH:

• Reconstitute biological transport proteins into synthetic flexible membranes in functional form

• Investigate the structural determinants of regulatory regions in native and specifically modified or engineered proteins

• Identify and engineer, using guidance from computational modeling, transport systems with the capability to transport other substances

RESEARCH TEAM:

University of CincinnatiJohn Cuppoletti (Physiology and Biophysics)T.L. Beck (Computational+Theoretical Chem.)J. Boerio (Materials Science and Engineering)J.Y.S. Lin (Chemical Engineering)P.R. Rosevear (Biochemistry and Microbiology)

University of PittsburghR. Coalson (Computational Chemistry+Physics)Virginia Tech Don Leo

John Cuppoletti Dept of Physiology Univ of Cincinnati

Pumps, Carriers and Channels

• Pumps use chemical energy to create gradients

• Carriers use gradients to transport substances. These do not have open pores.

• Channels have closed and open states to transport ions and water.

John Cuppoletti Department of Physiology University of Cincinnati

hClC-2 model (model3_07AB_BL020001)

Homology models of eukaryotic channels

pH Sensor loop

ClC-2 ClC-2

Bacterial StructurepH Sensor loop

John Cuppoletti Department of Physiology University of Cincinnati

Membrane Based Anthrax Detector

0 20 40 60 80 100

0

-5

-10

-15

-20

B

A

n=9n=9

n=9

n=9

n=9

n=9

n=9

n=9cu

rren

t (n

A)

[PA63] (ng/ml)

John Cuppoletti Department of Physiology University of Cincinnati

SOLID SUPPORTED MEMBRANES

K+

LIPID

ION CHANNEL BASED SENSORS

John Cuppoletti Department of Physiology University of Cincinnati

-- --0

20

40

60

80

Water Transport

mM

Wat

er/c

m2 /1

8 h

r

Actuators

John Cuppoletti Department of Physiology University of Cincinnati

Proton Transport at 116oC, I M HCl BIOLOGICAL TRANSPORTERS CAN BE VERY STABLE

John Cuppoletti Department of Physiology University of Cincinnati

Summary• Biological transporters can be used to prepare useful devices.

Examples of successes include a membrane based anthrax detector based on protective antigen, a water transporting actuator based on sucrose transporters, high throughput screening devices based on composite membranes containing ion channels. Evidence was presented that some transporters can function at temperatures greater than 100 degrees Centigrade.

• Solid supported membranes and porous membranes can be formed by self assembly.

• It is possible to engineer both proteins and devices.• Examples studied include high throughput screening, actuators,

detectors and fuel cells.• Biological engineering may mean something different in future

years. • Supported by ARO MURI, DARPA, and NIH

John Cuppoletti Department of Physiology University of Cincinnati