Protein-Based Micro Power Generator

Y. C. Lee
Department of Mechanical Engineering
NSF Center for Advanced Manufacturing and Packaging
of Microwave, Optical and Digital Electronics
University of Colorado, Boulder, CO 80309-0427
(303)492-3393 (phone), (303)492-3498 (FAX)
leeyc@colorado.edu

March 29, 2005

 Contents
• Molecular Biology, Micro/Nano-Scale Engineering
• Homeworks
• Energy Conversion:
- Fatty acid-powered microsystems
- Solar cells using Bacteriorhodopsin
- Proton engine
- Protein stability
- Thermal management
NSF workshop on Control and System Integration
of Micro- and Nano-Scale Systems

• 9:20 – 9:40am MEMS Design/Fabrication, Devices, and Systems: Research

Directions in MEMS, Martin Schmidt, MIT
• 9:40-10:00am Nano Fabrication: A Review of Nanofabrication Efforts and
Challenges, Jun Jiao, Portland State University.
• 10:15-10:35 BioMEMS and/or nanobiotechnological Systems: MEMS for
Biomedical Applications, Bill Tang, Irvine.
• 10:35 – 10:55 Biological (or Biomolecular or Biochemical) and Chemical Systems
on the Micro- and Nano-Length Scales:Challenges and Opportunities in Biological
Systems Analysis and Design, Costas Maranas, Penn State.
• 10:55 – 11:15 Control Systems with a MEMS and/or Nano Perspective: Dynamics
and Control of Material Microstructure and Biological Networks, Panagiotis
Christofides, UCLA
• 11:15 – 11:35 Measurement, Modeling, and Model Validation at the
Micro/Nanoscale: Modeling Transport in Micro and Nanofluidic Devices, Terry
Conlisk, Ohio State University
Control: Macro Æ Micro Æ Nano?

Voltage as
Capacitance as input
output
Emergent Integrated Circuit of the Cell

Sensors, actuators, noise and forces Æ Paradigm Shift in Control

 Contents
• Molecular Biology, Micro/Nano-Scale Engineering
• Homeworks
• Energy Conversion:
- Fatty acid-powered microsystems
- Solar cells using Bacteriorhodopsin
- Proton engine
- Protein stability
- Thermal management
ATP Synthase
 Molecular
Shuttles:
Two
Options

H. Hess and V. Vogel, Rev. Mol. Biotechnol. 67(2001)

 Some Numbers
• 0.5 µm/second
• 6 to 8 pN force for a single motor
• nN per µm2 cumulative forces
• MT: 25 nm in diameter and many µm long.

Speculation vs. Feasible Approach

Myosin II Bipolar
Thick Filament
 Muscle
Contraction
ATP-to-Mechanical Movements
Muscle
 Contents
• Molecular Biology, Micro/Nano-Scale Engineering
• Homeworks
• Energy Conversion:
- Fatty acid-powered microsystems
- Solar cells using Bacteriorhodopsin
- Proton engine
- Protein stability
- Thermal management
Mitochondrion
Mitochondrion

1000’s of Mitochondria in each cell.

 Fatty acid Pyruvate

Fatty Acid or
Pyruvate-to-
NADH or
FADH2
 Fatty Acid Æ Electricity
Fatty Acid In Membrane with protein complexes

H+ H+
III
I IV I III IV H+
II II

Substrate with Immobilized Enzymes

Proton Engine High H+

Low and H+;
H+ Generator 100 – 200 mV

Electricity Out
Protein Complexes?
 Frank Frerman, Ph.D.
We are investigating the catalytic mechanism of
glutary-CoA dehydrogenase (GCD), a tetrameric
flavoprotein dehydrogenase that catalyzes the
oxidation of glutaryl-CoA, an intermediate in the
oxidation of lysine. The electron acceptor for all nine
dehydrogenases is electron transfer flavoprotein (ETF)
which transfers electrons to a membrane-bound iron-
sulfur flavoprotein, ETF-ubiquinone oxidoreductase
(ETF-QO). The investigations are driven by the fact
that inherited defects in these proteins cause metabolic
diseases that are often fatal. Our approach is to
identify patients’ mutations in the proteins, and then
express and purify the mutant proteins.
Intact Membranes

Known-Good Membranes
Isolation of Specific Membranes
• Cellular disruption and low speed centrifugation
(600xg) to remove unbroken cells and nuclei.
• Sedimentation at 10,000xg for 30 min will
sediment mitochondria and intact lysoomes
• Sedimentation at 100,000xg for 60-90 min will
sediment essentially all membranes.
• Velocity sedimentation using sucrose gradients to
isolate specific membranes of interest.
 Self assembly of covalently anchored phospholipid supported
membranes by use of DODA-Suc-NHS-lipids
G. Brink *, L. Schmitt, R. Tamp, E. Sackmann, 1994

N-[(hydroxysuccinimidyl)-N-
succinyl]dioctadec-
ylamine (DODA-Suc-NHS) which serves
as a anchor and covalently binds the
phospholipid membrane to the surface.
 Contents
• Molecular Biology, Micro/Nano-Scale Engineering
• Homeworks
• Energy Conversion:
- Fatty acid-powered microsystems
- Solar cells using Bacteriorhodopsin
- Proton engine
- Protein stability
- Thermal management
Photon 100 - 200 mV

Bacteriorhodopsin for Proton Pumping

Purification and
Reconstitution
 Solar Energy In

H+ in steam
Membrane with BR

H+ H+ H+

Substrate H+

Proton Engine High

Low and H+;
H+ Generator 100 – 200 mV

Electricity Out
 Charging e-
Hydride Formed
V H+ H+
Difficult to e- H+
Convert
(Good; NEC battery)
Proton
Gradients into Protons Charging
generated
Electricity by photons
H+ H+
through BR
H+
(BAD)
Complex IV: Cytochrome c Oxidase

Tfr's e- from cyt c to

O2
13 subunits, some
coded by mt DNA,
some by nuclear DNA
O2 to H2O = 4 e-
reduction:
one at a time, cyt c =
single e- carrier.
 BR + COX
Solar Energy In

H+
H+ H+ H+ H+
H+ H+ H+

Electrons Out

H+ H+ H+ H+ H+ H+ H+ H+

Dean Ho, B. Chu, H. Lee and Carlo Montemagno, Nanotechnology, 15(2004) 1084-1094
 13 Different Soluble Proteins

Streptavidin
Biotin

Charge-neutral
SAM covering
all other area
exposed

Ni (II) ions chelated

Streptavidin over NTA-SAM Biotin
 Contents
• Molecular Biology, Micro/Nano-Scale Engineering
• Homeworks
• Energy Conversion:
- Fatty acid-powered microsystems
- Solar cells using Bacteriorhodopsin
- Proton engine
- Protein stability
- Thermal management
ATP Synthase

Show Movie: ATP Synthase

 ATP Synthase Æ Solutions
-
-
- C C
C -
C
- C
C
C C - +
- +
- H+ +

H+ H+
H+
H+

Electrostatic mechanism to be finalized Æ Electrostatic

Proton Engine Modulated by H+ Valve
High H+;
100 – 200 mV Low H+

H+ Valve
Vacuum
Electrode with H+
N N

S S
Electro-magnetic plate Flexure/Spring
 Proton
Transport
(new)
 H+ Valve
Hydrophobic surface
Flexure/Spring stopping proton flow
to Low H+ reservoir

Low H+
Valve Element Electrode

High H+
Electrical Actuation On

Hydrophilic bridging surface enabling

proton flow to charge electrode from High H+ reservoir

Slow charging/discharging process?

Electrostatic Actuator Driven by 0.2 V?
High H+;
100 – 200 mV Low H+

H+ Valve
Vacuum
Electrode with H+
N N

S S
Electro-magnetic plate Flexure/Spring
ATP-to-Mechanical Movements
 Comb Drive Actuator
Generated
Electrostatic Forces

N=18

After W. C. Tang, et.al, Sensor & Actuator, vol.2, pp.25-32, 1989.

 Comb Drive Actuator
• For the case of interdigital finger arrangement of the comb actuator we get,

n=2

After V. P. Jaecklin, et.al, J. of Micromech. Microeng., vol.2, pp.250-255, 1992.

Path of Electrons through Three Complexes
Electron Transfer Chain
 Solar Energy
Harvester
(1 protein type)
Micro Power Generator Solar Energy
(17 protein types)

Future Large Scale Integration Pacemaker

Artificial Muscle

Dog Bird

Fish
 Contents
• Molecular Biology, Micro/Nano-Scale Engineering
• Homeworks
• Energy Conversion:
- Fatty acid-powered microsystems
- Solar cells using Bacteriorhodopsin
- Proton engine
- Protein stability
- Thermal management
 Killer Issues
• Integration of proteins (enzymes)
- long-term stability
- system design and analysis
- processing
- thermal management
• Proton-to-electricity conversion
- design of right proton engines
- efficiency
Thermophilic bacteria and energy transduction
•All use a chemiosmotic mechanism, proton
gradients across the membrane, for energy
production.
•Hyperthermophiles 75-110oC
organotrophs - use a modified Entner-Duodoroff pathway to
oxidize organic compounds for energy (oxygen or sulfur serves as
final electron acceptor)

Thermococcus, Thermoproteus, Desulfurococcus, Thermofilum,

Pyrococcus (organic compound + S Æ H2S + CO2 )
Sulfolobus (organic compound + O2 Æ H2O + CO2 )
Staphylothermus (organic compound Æ CO2 + fatty acids
Pyrococcus (organic compound Æ CO2 + H2)
 Protonmotive force generating
enzymes from thermophiles

• NADH-II (A. ambivalens )

• Bo3 type oxidase (T. sulfolobus)
• C1aa3 type oxidase (T. thermophilus)
• Baa3 oxidase (T. thermophilus)
Denaturation and Inactivation

Fields, 2001
A Measurement Example

Fields, 2001
Protein Structure
 Protein Structure
• Primary structure: In proteins, the linear arrangement
(sequence) of amino acids and the location of covalent
(mostly disulfide) bonds within a polypeptide chain.
• Secondary structure: In proteins, local folding of a
polypeptide chain into regular structures including the
a helix, b sheet, and U-shaped turns and loops.
• Tertiary structure: In proteins, overall three-
dimensional form of a polypeptide chain, which is
stabilized by multiple noncovalent interactions between
side chains.
 Proteins for Extremophiles
• No new amino acids, covalent modifications or
structural motifs that explain the ability of these
molecules to function in such harsh environments.
• Rather, subtle redistributions of the same
intramolecular interactions required for protein
stabilization at moderate temperatures are
sufficient to maintain structural integrity at hot or
cold extremes.

Psychrophile, Mesophile and Thermophile

Fatty acids
Protein Conformation
Leucine Replaced by Alanine

Stability Reduced

Cavity formed
Uncharged Polar Æ Non-polar Residue

Stability Increased

Non-polar Æ Hydrophobic
Non-charged Æ Charged Residue

Stability Increased
Amino Acid
The Helix Increase or decrease stability

-
 Mesophile Æ Thermophile
• A decrease in uncharged polar residues, mainly in favor of
non-polar amino acids that participate in hydrophobic
interactions.
• An increase in charged residues, which may be involved in
larger numbers of stabilizing ion pairs and networks, although
location of ion pairs within molecular structures also appears
to be important in determining stability
• An increase in residue hydrophobicity, which is expected to
stabilize warm-adapted proteins as the hydrophobic effect
becomes stronger with in creasing temperature.
• Increased residue volume, which may enhance stability in the
thermophilic proteins by more efficient packing and reduced
rotational entropy of the unfolded form.
 Mesophile Æ Phychrophilic
• Few ionic interactions
• Less number of intramolecular hydrogen
bonds
• Reduction in hydrophobicity index (due to
low temperature)
• Increased number of polar or charged
groups to increase hydrophilicity
 Molecular Stability vs.
Structural Flexibility
• Stability is needed to ensure the appropriate
geometry for ligand binding, as well as to
avoid denaturation.
• Flexibility is necessary to allow catalysis at
a metabolically appropriate rate.
 Protein function at thermal extremes:
balancing stability and flexibility
• No organism can survive across the entire
temperature range found in the biosphere.
• A given species can rarely support active
metabolism across more than a few tens of oC.
• Nevertheless, life can be maintained at
surprisingly extreme temperatures, from below -
50 to over 110oC.

Fields, 2001
Eye lens crystalline from a variety of vertebrate species
correlated to habitat temperature

The denaturation temperature Tm:

temperature at which 50% of the
secondary structure has been lost.
Enzyme, muscle-type lactate dehydrogenase A4 -LDH ,
from organisms occupying a range of thermal habitats
kcat: turnover number
- the rate at which one
enzyme active site
converts substrate to product
 A4 - LDH Michaelis Constants

Km measures the binding affinity of enzyme for substrate and can also be used
as an indirect measure of the inherent flexibility of an enzyme molecule.
Km at 10 oC or Habitat Temperature
 Protein Stability
• Defining and measuring protein stability
• Deleterious reactions and stabilizing interactions
in proteins
• Enzyme behaviour in organic solvents
• Thermophilic proteins
• Protein engineering
• Chemical modification
• Use of additives
Immobilization!
Fagain, 1995
 Contents
• Molecular Biology, Micro/Nano-Scale Engineering
• Homeworks
• Energy Conversion:
- Fatty acid-powered microsystems
- Solar cells using Bacteriorhodopsin
- Proton engine
- Protein stability
- Thermal management
Chip-Scale Integration for Atomic Clocks
Epoxy / Solder / Spacer 1/4 waveplate
Cs cell (NIST) on MEMS
Heater/Sensor
MicroMirror
(MEMS)
n
i l ico
S MicroMirror
Ball Lens or
(MEMS)
Microlens
Bias
Lens holder
(MEMS) Photodetector
VCSEL ic
ra m
Ce
RF output
a n ge
e Ch al
as ri
Bipolar Ph Mate
Junction ic
r a m
Transistor Ce
(BJT) DC input
Decoupling Microstrip
Capacitor Resonator
Chip-Scale Integration for Atomic Clocks
Cs cell with a local heater
VCSEL with a local heater 73 oC (0 to 6.5 mWatt)
46-50 oC (2 to 9 mWatt)
0 to 6.5 mWatt
n
i l ico
S
Epoxy/
27 oC/6.5 mWatt Spacer

a
Silic
se d
Fu
46 oC/30 mWatt (solid) m ic-
e ra a n ge
C Ch al
~ 0 oC/30 mWatt (liquid) as e ri
17 to 30 mWatt Ph Mate
m ic
r a
Bipolar Ce
Junction
Transistor
(BJT)
Case: 0 to 50 oC variation
 Biology
Developmental
Cellular

Molecular

10 nm
Nano-machines

Micro-machines System Integration

Engineering
 Summary
• Molecular Biology, Micro/Nano-Scale
Engineering
• Independent Projects
• Energy Conversion:
- Solar cells using Bacteriorhodopsin
- Proton engine and subharmonic resonance
- Fatty acid-powered microsystems
- Protein stability
- Thermal management
Energy conversion from nutrients in the blood stream?