Urry D.W. (Ed.) What Sustains Life? : Consilient Mechanisms for Protein-Based Machines and Materials

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2.6 Progression to Biology's Machines from Model Protein Machines

Waters of

Hydrophobic

Hydration

Waters of

Hydrophobic

Hydration

PO4

beginning

of folding

destabilizes

phosphate

0 represents COO

FIGURE

2.15. A primitive swinging cross-bridge/

sUding filament model of muscle contraction is intro-

duced for the purpose of providing a conceptual

bridge from the simple oil-like dissociation/associa-

tion for the opening/closing of a clam-shaped globu-

lar protein toward the more complex structural

aspects of muscle contraction. Major limitations in

filaments

slide

spontaneously

because

transition

temperature

below body

temperature

represents

COO"

Ca"^^COO"

the drawing include the attachment-detachment

process and the required forward movement of the

cross-bridge to achieve attachment at the next actin

binding site. These issues are considered in Chapter

where a twisting and untwisting of the cross-bridge

is implicated.

cell membrane. ATP-consuming protein-based

machines travel along nucleic acids, correcting

the structures in a number of ways or identify-

ing a sight to be cleaved. ATP produces

energetically equivalent GTP, and GTPases

constitute a family of molecular switches. Thus,

ATPases are recognized as the workhorse

protein-based machines that perform the day-

to-day chores of living organisms.

Putting the ATPase motor to work can begin

with attachment to a surface, as shown in

Figure 2.16. In this figure the simple

attachment/detachment cycle begins with

attachment. In the absence of ATP, an oil-like

domain of the surface associates with an oil-like

domain of the ATPase. Transient dissociations

occur with opening of a cleft. The presence of

complementary charges on each hydrophobic

surface facilitates association as well as dissoci-

ation. More pKa-shifted charged groups, as in

the series of model proteins in Figures 1.2 and

1.3 with increasing numbers of more oil-like

phenylalanine (Phe, F) residues, favor associa-

tion of oil-like domains with ion pairing.

Limited hydration of the charged groups, due

to competition with oil-like groups for hydra-

tion, facilitates ion-pair formation. Despite the

presence of charges and because of their

complementary positions, dissociation in the

absence of ATP results in the formation of too

much oil-like hydration such that the equilib-

rium is shifted toward association, as indicated

by the length of the arrows connecting the two

states in Figure 2.16A.

Bound ATP, with its highly charged tripep-

tide tail at the base of the cleft, destructures oil-

like hydration by orienting water molecules for

its own hydration, as shown in Figure 2.16B.

The decreased amount of oil-like hydration

becomes insufficient to maintain association,

and the equilibrium shifts toward the dissoci-

ated state. The situation shown in Figure 2.16

parallels that of the protein-based motor of

skeletal muscle with positive charges on the

ATPase and complementary negative charges

at the actin binding site. Again, to begin con-

sideration of the more complex case, the lower

part below the cleft recedes somewhat on dis-

sociation to suggest further structural changes

that can accompany association and dissocia-

What Sustains Life? An Overview

FIGURE 2.16. An initial step in converting the

"idUng"

ATPase motor into a motor capable of per-

forming work involves attachment and detachment

as between components of actin and myosin

involved in the sUding filaments of muscle contrac-

tion. A The cartoon depicts an equilibrium between

states of oil-like association and dissociation with

fluctuating cleft openings, but in the absence of

ATP,

the equilibrium markedly shifts toward association.

B Binding of MgATP with its negative charges at the

base of the cleft destroys the hydration around

oil-Uke groups in the process of achieving its own

hydration and in so doing shifts the equilibrium

dramatically toward dissociation. This represents the

first step of muscle contraction involving dissociation

of the cross-bridge of the myosin II motor from the

actin filament.

tion. There can also be structural changes

within the ATPase that, as the result of

phosphate release, develop important elastic

forces on attachment to the surface and con-

traction at fixed length.

2.6.6 Scallop Muscle and the Clam-

shaped Globular Protein Motif

The scallop adductor muscle, of course, is that

tasty seafood delicacy that can be prepared

2.6 Progression to Biology's Machines from Model Protein Machines

fried, baked, or even broiled. For the scallop it

functions to open and close the two halves of

this beautiful classic bivalve shell. Importantly,

the crystal structure data have been obtained of

the cross-bridge of scallop muscle from the

myosin filament to the site for actin binding,

and the cross-bridge has been obtained in two

different states, a near rigor state and a state

with an ATP

analogue.^^'^^

Somewhat ironically,

the structure of the cross-bridge provides an

actual analogue of the clam-shaped globular

protein in Figure 2.14A.

The crystal structure is of the SI cross-bridge

that contains the globular head, the a-helical

lever arm, and its wrappings, the essential

(ELC) and regulatory (RLC) hght chains,

which seem to contribute by strengthening the

single helical rod. For our purposes here, the

light chains will be removed in order to make

apparent the changes that occur when going

between contracted and ATP bound states. As

we will see, ATP binding opens the hydropho-

bic association between key components of

the cross-bridge, which bears analogy to the

binding of a single phosphate to open the

hydrophobically associated clam-shaped globu-

lar protein.

tion, that is, causes opening of the hydrophobi-

cally closed association.

2.6.6.1.2 Structural Reorientation on

Dephosphorylation

The long limb in Figure 2.17 is the a-helical

lever arm that in part connects the myosin fila-

ment to the globular part of the cross-bridge.

The stump or pedicle-like structure is the N-

terminal domain. In Figure 2.17A,C, the view is

in a direction along the myosin filament,

whereas in Figure 2.17B,D, the view is approx-

imately perpendicular to the myosin filament as

may be judged from the foot-like terminus that

is taken as being directed parallel to the myosin

filament.

Thus,

dephosphorylation results in a

rotation of the cross-bridge around the head of

the lever arm to tuck it under the stump of the

N-terminal domain. As expected from the con-

sihent mechanism, loss of the negatively

charged phosphate results in association of oil-

like domains. As considered in the text associ-

ated with Figures 8.47 through 8.57, this

hydrophobic association provides the power

stroke of muscle contraction.

2.6.6.1 Binding of ATP Analogue to

Cross-bridge ''Opens'' the Oil-like

Associated State as Occurs with the Clam-

shaped Globular Protein Motif

2.6.6.1.1 The Process of Muscle Contraction

Itself Can Be Viewed as a Variation on the

Clam-Shaped Globular Protein Theme

Figure 2.17 gives stereo views obtained from

the crystal structure of the cross-bridge of the

scallop myosin II motor in the contracted near-

rigor state in A (from the side of the ATP

binding site) and C (from the side opposite the

ATP binding site) and in the bound ATP ana-

logue state in B (from the side of the ATP

binding site) and in D (from the side opposite

the ATP binding site). Incredibly, as a direct

analogy to the hydrophobically closed clam-

shaped globular protein in Figure 2.14A, the

binding of the negatively charged ATP with its

triphosphate tail causes hydrophobic dissocia-

2.6.6.2 The Clefts Within Which the ATP

Resides Act as Conduits to Direct

the Forces Emanating from the

Thirsty Phosphates

The cleft at which the ATP resides runs in two

directions, with the y-phosphate positioned to

direct its thirst for water toward the actin

binding site and toward the open clam-like

region between upper lever arm and pedicle of

the N-terminal domain. Seen from the front, the

actin binding

side,

the ATP site resides in a cleft

under an overhang with its y-phosphate in the

depths at the aqueous edge of the cleft. The

cleft runs in two directions. In one direction in

Figure 2.17B the cleft runs outward and upward

at about 10 o'clock to the junction of the

myosin head with the actin filament, such that

ATP binding disrupts attachment to the actin

filament by the repulsion between oil-like and

charged species. In the other direction the cleft

runs to the oil-like association between the

head of the lever arm and the N-terminal

What Sustains Life? An Overview

FIGURE 2.17. Cross-eye stereo views show a space-

fiUing representation of scallop muscle cross-bridge

(SI).

The lever arm is present, but essential and

regulatory light chains are removed with neutral

residues Hght-gray, aromatics black, other hydropho-

bics gray, and charged residues white. A The near-

rigor state shows a hydrophobic association between

the N-terminal domain and the upper part of the

lever arm with view looking approximately along the

axis of the myosin filament (Protein Data Bank,

Structure File 1KK7). B The ATP analogue state

shows hydrophobic dissociation between the N-ter-

minal domain and the upper part of the lever arm.

The view is now approximately perpendicular to the

myosin

filament,

that

is,

the lever arm rotates with an

untwisting on hydrophobic dissociation. Protein

Data Bank, Structure File 1KK8 C,D The backside

of a cross-bridge, that

is,

the side opposite from ATP

binding site is shown. These parts were prepared with

the crystallographic results of Himmel et al.^^ as

obtained from the Protein Data Bank, Structure

Files 1KK7 and 1KK8.

2.7 Consequences of Protein Machines Based on the Inverse Temperature Transitions

domain to cause the association to "open", that

is,

to dissociate. The cleft acts like a conduit

directing the thirst of the phosphate for hydra-

tion in both directions to disrupt hydrophobic

associations. The cleft is shown from several

dif-

ferent vantage points in Figures 8.58 and

8.59 and further discussed in their associated

text.

In Chapter

more structural background and

molecular details of contraction exhibited by the

linear myosin II motor are considered after, in

Chapter 5, the physical basis for the apolar

(oil-like)-polar (vinegar-like) repulsive energy

that controls hydrophobic association is experi-

mentally and analytically developed. The crystal

structures of the cross-bridge of scallop muscle

provide remarkable examples of the consilient

mechanism functioning in this protein-based

machine!

2.7 Consequences of Protein

Machines Based on the Inverse

Temperature Transitions

We again draw comparison by quoting from

Schrodinger's What is Life! as published in

1944:

"What I wish to make clear in this last

chapter is, in short, that from all that we have

learnt about the structure of living matter, we

must be prepared to find it working in a manner

that cannot be reduced to the ordinary laws of

physics."^^ In the present volume, our founda-

tion

is,

of course, the somewhat counterintuitive

inverse temperature transition. It is by means

of the inverse temperature transition that the

energies essential to sustain Life can, in fact, be

accessed. This efficient consilient mechanism,

however, requires no new laws of physics. In

fact, the seeds of this mechanism are found in

a 1937 report of Butler,^^ in which he analyzed

the thermodynamic elements of the solubility

of oil-like groups in water.

The essential aspect of the capacity of the

inverse temperature transition to achieve

diverse energy conversions resides within large

chain molecules, which were just becoming

known when the first edition of Schrodinger's

book appeared. As we have sketched above, the

functional properties of the model protein-

based machines result from the interaction

between hydrophobic (oil-like) and hydrophilic

(vinegar-like) groups constrained to occur in

unique sequence along the chain molecule,

which combines with the capacity of vinegar-

like functional groups to shift between being

more vinegar-like and more oil-like.

The special effectiveness of proteins com-

pared with all other known chain molecules

arises from biology's capacity accurately to

produce diverse sequences. As for accuracy, the

error in placing the correct amino acid residue

at the specified sequence position depends on

the ability to delineate one R-group from

another at the stage of attachment to t-RNA

(see Chapter 4).^^ In general, the error is

remarkably small. It is also very important that

all of the residues be of the correct optical con-

figuration,"^^ as random mixing of mirror images

of amino acid residues would destroy structure

with the consequence of eliminating reliable

function (see the paragraphs on Pasteur and

mirror image molecules in section 3.1.6 of

Chapter 3).

These features of protein polymers result in

regular, nonrandom structures, which, as will be

shown later, are the reason for efficient energy

conversion. As for diversity, theoretically each

position in the sequence can be any one of 20

different amino acid residues. Again very sig-

nificantly, the protein sequence is specified

by the nucleic acid sequence and not by the

energetics within the sequence of a particular

protein. In this way, a sequence that would

normally be highly improbable becomes just as

probable as any other sequence. (Improbabili-

ties would otherwise arise by virtue of being a

single sequential arrangement of amino acids

among an inordinate number of possible

arrangements of the same collection of amino

acids and because of unfavorable interaction

energies between the R-groups and thus being

of high energy.) Of course, much energy has

been spent in producing the "improbable"

sequence, as discussed further in Chapter 4.

Production of unique and improbable

sequences might have been one place where

the far-from-equilibrium arguments of Pri-

gogine could have come into play. Order is

indeed achieved out of chaos, but it occurs at a

What Sustains Life? An Overview

high price in energy—in the energy required to

copy the parent chain of DNA, in the energy

required to transcribe the DNA sequence into

RNA, and in the energy required to translate

the nucleic acid sequence into a protein

sequence.

the best of our present knowledge,

however, each of the many steps in the proce-

dure can be treated and studied in the test

tube as an equiUbrium process. Thus, once the

machinery of the cell is in place, the individual

steps within the cell appear to be reversible and

explicable in terms of the thermodynamics

of Boltzmann and Gibbs. However, as noted

below and treated in more detail in Chapter 4,

biology has employed a throw-away energy; the

pyrophosphate product of each step of the

assembly essential for reversibiUty is broken

down by the interesting enzyme pyrophos-

phatase to ensure unidirectionality to both

nucleic acid and protein synthesis.

Now, because it can cost just as much energy

to produce an inefficient and more limited

protein-based machine as it can to produce a

more efficient and/or a new machine that can

access a new energy source, obviously with

natural selection the arrow of time for biology

is toward greater complexity and diversity func-

tion (see Chapter 6).

2.7.1 Protein-based Machines as

Catalysts for Energy Conversion

When an energy input causes an inverse tem-

perature transition of hydrophobic folding, a

protein converts part of the energy into its own

folding and ordering; this constitutes one aspect

of a decrease in entropy. On completing a cycle

by returning to the unfolded and disordered

state,

the net entropy change, however, be-

comes zero for this cyclic folding and unfolding.

When in the process of cycling—mechanical,

chemical, electrical, or some other work—has

been catalyzed, then there results a useful

decrease in entropy resulting from the energy

input. A productive negative entropy change

does occur when, in the process of folding, the

protein picks up protons and deposits them at

a higher concentration, that is, pumps protons.

or the protein lifts a weight, that is, pumps iron,

for some useful purpose, or the protein phos-

phorylates ADP to form ATP. In each case

movement is away from disorder; a beneficial

increase in order, that is, a decrease in entropy,

has been achieved. Proteins that hydrophobi-

cally fold as the result of an energy input to

result in mechanical work, chemical work, or

some other energy output could reasonably be

classified as negative entropy machines, but this

is an incomplete characterization. An adequate

characterization would be to view protein-

based machines simply as catalysts of energy

conversion.

It is now possible to design and prepare

elastic-contractile model proteins capable of

interconverting the energies that sustain Life.

The set of six energies interconverted by living

organisms are represented symbolically in

Figure 2.18. Indicated by the bold intercon-

necting arrows are the pairwise energy conver-

sions,

directly demonstrated by designed model

proteins. Additional energy conversions have

been demonstrated less directly. These model

proteins are biomolecular machines of further

interest as advanced materials for medical and

nonmedical applications to enhance quality of

Life and to assist in sustaining an ever larger,

more complex and more productive society.

2.7.2 Efficiency and the Definition of

Chemical Energy

The efficiency, r|, of a process can be described

as the work achieved divided by the energy

input, that

is,

work performed/energy input.

The work performed (w) can be the lifting of a

weight, which is the mass (m) of the object

times the force of gravity (g) times the height

(h) the object was raised, that is, w = mgh, as

indicated in Figure

2.5.

The energy input can be

due to the increase in concentration of protons,

which is proportional to the size of the step

along the horizontal axis in Figure 2.6B. In

Figure 2.6B, the size of step required to go com-

pletely from COO" to COOH for a less oil-like

protein with a broader sigmoid curve is greater

than for the more oil-like protein with its nar-

rower sigmoid

curve.

As the energy input comes

2.7 Consequences of Protein Machines Based on the Inverse Temperature Transitions

CHEMICAL

ENERGY

ELECTRICAL

ENERGY

FIGURE 2.18. Energies are shown that can be inter-

converted by means of elastic-contractile model

proteins capable of exhibiting inverse temperature

transitions functioning by means of the competition

for hydration between oil-like and charged groups

called an apolar-polar repulsive free energy of

hydration. See Chapter

for a more complete devel-

opment of the phenomenology and physical basis

and Chapter 8 for details of the molecular process.

in the denominator for the expression of effi-

ciency, this means that energy conversion is

more efficient for the more oil-like proteins.

Another expression of relative efficiency, intro-

duced in Chapter 1, is the Hill coefficient that

quantifies positive cooperativity. As considered

below, when it is possible to compare directly

the apolar-polar repulsive free energy of

hydration with the more commonly considered

electrostatic mechanism of charge-charge

repulsion in water, the former wins by several

factors of ten in calculated efficiency (see

Chapter 5 and, specifically. Figure 5.36). The

efficiency, with which proteins can convert avail-

able energy by means of the inverse temperature

transitions to those energies required to sustain

Life is one argument for the significance of this

mechanism in biology.

2.7.3 Cooperativity and Survival of

Efficient Mechanisms

The steeper acid-base titration curves (of

Figures 1.2 and 2.6B, that result from more oil-

like proteins) express positive cooperativity,

which is to say that the binding of a first proton

facilitates the binding of the second proton and

the initial two cooperate to facilitate binding of

the third, and so forth, (see Figures 1.4 and

5.31). Another mechanism for energy conver-

sion results from the repulsion of like charges

much as static electricity causes hair to stand

out, preventing it from assembling on the head

as one might like.

The charge-charge repulsion mechanism can

be used to drive extension of a chain molecule.

Then the chemical energy required to remove

repulsion, relax extension, achieves the

mechanical work of lifting a weight. Because

the first charge repels the addition of the

second charge, and so forth, however, the

curves as represented in Figure 2.6B would

all be much broader. As a consequence, much

more energy input would be required to

achieve the same amount of mechanical work.

The electrostatic (charge-charge) repulsion

mechanism is much less efficient than the

mechanism based on water-mediated repulsion

between apolar and polar groups that operates

for inverse temperature transitions.

Calculation of the data in Figure 5.36 provides

quantitative comparison of the contractile effi-

ciencies of two cross-linked matrices where, in

both cases, protonation of -COO" to form-

COOH drives contraction. The polymer using

the charge-charge repulsion mechanism is

poly(methacrylic acid), [-CHs-CHsCCOOH-]^,

and the model protein using the apolar-polar

(oil-like/vinegar-like) repulsion mechanism

of the inverse temperature transition is

poly(GVGVP GVGFP GEGFP GVGVP

GVGFP GVGFP), where E stands for the

glutamic acid residue with the R-group,

-CH2-CH2-COOH. Both matrices are loaded

with a force, f = mg, of a 2-gram weight, and the

What Sustains Life? An Overview

contraction, that

is,

the decrease in length (AL)

as the pH is lowered, occurs on addition of

acid. Now efficiency can be written as r| = fAL/

(2.3RTApH)An, where R is the gas constant

(1.987

cal/mol-deg),

T is the temperature in

degrees K (centigrade degrees +

273),

and An is

the number of protons

used.

Based on the steep-

est slope for each curve in Figure 5.36, the ratio

of the efficiencies of the inverse temperature

transition mechanism,

r|ap,

to the charge-charge

repulsion mechanism, r|cc, is given by r|ap/r|cc =

[ApHAn]pMA/[ApHAn]E4F. On substitution of the

experimental numbers, r|ap/r|cc = [(2.24)(6.86 x

10-0]/[(0.59)(6.12

10-^)] «

40.

The mechanism

based on the inverse temperature transition of

the designed elastic-contractile model proteins

is more than 40 times more efficient. The elec-

trostatic mechanism in water is only a few

percent as effective as the mechanism that uses

the inverse temperature transition.

Due to the struggle to survive under circum-

stances of limited food

supply,

organisms evolve

to use the most efficient mechanism available to

their composition. The most efficient mecha-

nism available to the proteins that sustain Life

would seem to be the apolar-polar repulsive

free energy of hydration as observed for the

inverse temperature transitions for hydropho-

bic association. The efficiency of designed

elastic-contractile protein-based machines

and a number of additional properties make

designed protein-based materials of substantial

promise for the marketplace of the future.

2.8 "What Sustains Life?"

Becomes "What Sustains

Society?" (Biomolecular

Machines as Advanced Materials

for the Future)

Advances in physics, chemistry, and biology

spawned technologies that now support larger

populations at higher standards of living; they

have provided fossil fuel-powered, hydro-

powered, nuclear-powered, and chemical-

based energy sources (e.g., electricity, fuels,

explosives); they have given us electronics

and communications, agricultural enhancement

methods and products, medicines, new materi-

als such as plastics, and related medical de-

vices.

Now, fertile technologies, spawned from

advances in molecular biology and molecular

biophysics, promise opportunities to continue

to sustain society at higher population levels

with higher standards of living.

Here we utilize insights gained from the

capacity of living organisms to access and trans-

form energy and apply these protein-based

machines to problems of consequence to an

ever larger, more complex

society.

Three major

concerns of our society are an unsustainable

rise in medical care costs, the drug addiction

problem that drives an increasing crime rate

and supports terrorism, and pollution of the

environment.

The preceding design of model proteins that

emulate energy conversions of living organisms

constitutes useful knowledge for the design of

model proteins for medical and nonmedical

use.

In this brief overview, we mention three

appUcations that relate to the above-noted

problems in our

society.

Two

are medical appli-

cations, tissue reconstruction and drug delivery,

and the third is primarily a nonmedical appU-

cation of (bio)degradable plastics. These are

treated more extensively in Chapter 9.

The chosen three examples result from our

newly developed understanding of protein-

catalyzed energy conversion. This brings us to

the modern-day situation so well-stated by J.

Bronowski,"^^ "In effect, the modern problem is

no longer to design a structure from the mate-

rials,

but to design the materials for a structure."

For medical applications the biocompatibil-

ity of the model proteins must, of course, be

established; this has been done for three basic

compositions, but verification of nontoxicity

must be established for each specific composi-

tion designed for a particular human or veteri-

narian use.

The ultimate limitation to the utilization of

model proteins as advanced materials for the

future resides in low-cost, reliable production.

Proteins are unique polymers in that they can

be prepared by genetic engineering, allowing

for fidelity and diversity of sequence not pos-

sible for any other known chain molecules.

An overview would be incomplete without a

2.8 "What Sustains Life?" Becomes "What Sustains Society?"

few comments on this capacity as presently

demonstrated.

2.8.1 Genetic Engineering of

Model Proteins

On chemically synthesizing the first very long

chains of poly(GVGVP) early in 1984 with the

support from the Army Medical Research

Office, our peptide chemist, K. U. Prasad, sug-

gested that it would make a very nice chewing

gum, an idea dismissed with a laugh for one

stick of gum would have cost a few hundred

dollars. As the result of an Accelerated

Research Initiative for producing elastomers of

repeating peptide sequences by genetic engi-

neering, which was announced by the Office of

Naval Research in

1985,

the current anticipated

cost for an edible stick of gum could became as

little as a few pennies. This cost comparison

between chemical synthesis in the research lab-

oratory and microbial biosynthesis illustrates

the expansion of applications possible with

the recent success of recombinant DNA tech-

nology for producing (GVGVP)25/^ and other

protein-based polymers.

As discussed in subsequent chapters, the

error-free preparation resulting from microbial

biosynthesis is just as important for function as

lowering production cost is for expansion of

applications. The basic gene design and con-

struction occur with essentially no limitations

on sequence, and expression of the protein-

based polymer proceeds with few constraints

on sequence. The basic (monomer) gene is

polymerized enzymatically to provide genes for

the entire possible range of exact polymer

chain lengths. To date, model proteins con-

taining as many as 4,000 residues have been

produced with remarkably high quantities

prepared per volume of the fermentor. An

example of the microbe E. coli producing

(GVGVP)i2i is shown in Figure 2.19.^^ Later

chapters contain further details of the genetic

engineering approaches.

2.8.2 Tissue Reconstruction

Within a tissue such as skin, artery, or lung, cells

attach to proteins of their extracellular matrix

by means of short specific sequences of amino

acid residues in extracellular proteins. Within

the cells, cytoskeletal fibers, much like muscle

fibers, span between attachment

sites.

When the

tissue is stretched, so too are the intracellular

cytoskeletal fibers. This mechanical stretching

of the cytoskeletal fibers and their attachments

results in chemical signals that instruct the

nucleus to express those genes for producing

the proteins and other extracellular macromol-

ecules required to sustain the deforming forces.

In recent years this has been called the tenseg-

rity

principle f'^

FIGURE

2.19. Example of the production of the

elastic-contractile model protein, (GVGVP)i2i, by

genetic engineering of E. coli. A Transformed E. coli

shows large inclusion bodies of product having phase

separated intracellularly. B Nontransformed E. coli

shows no such inclusion bodies. (Reproduced with

permission from Urry et al."^^)

What Sustains Life? An Overview

An equivalent conversion of mechanical

energy into chemical energy occurs with the

elastic model proteins. Stretching of properly

designed and cross-linked elastic model pro-

teins,

such as the one in Figure 2.20B, increases

affinity for protons and removes protons from

the surrounding solution. Equivalently, we

believe that stretching could energize an

attached phosphate to phosphorylate another

molecule as part of the chemical signal to the

nucleus.

As a test of this perspective, we prepared

elastic model proteins with and without cell

attachment sequences and cross-linked them to

form elastic sheets called bioelastic matrices.

Cells do not adhere to these matrices without

the cell attachment sequences; cells do adhere

to the bioelastic matrices containing the cell

attachment sequences, as depicted in Figure

2.20. Furthermore, the cells spread out and

grew to completely cover the surface. Perhaps

even more significantly, cyclic stretching of the

Effect of Stretching on a Cell Attached to an Elastic Substrate

B X^"-poly[40(GVGVP),(GRGDSP)]

C Unstretched Matrix

cytoskeletal

. . fibers

integrin

attachment

sites

D Stretched Matrix

stretched

cytoskeletal fibers

stretched

integrin

Cross-section Cross-Section

integrin

_^^N^

attachment sites

stretched

integrins

Top View

I I Bioelastic Matrices Containing Cell Attachment Sequences

e.g. X^^-poly[20(GVGVP),(GRGDSP)]

FIGURE 2.20. Elastic-contractile model proteins are

used as temporary functional scaffolding for soft

tissue restoration by means of an attached normal

cell capable of mechanochemical transduction to

restore a natural tissue. A Elastic matrices without

cell attachment sequences show no attachment of

human fibroblasts. B Elastic matrix with cell attach-

ment sequences show human fibroblasts having

attached, spread, and with capacity to grow to con-

fluence. C, D Simultaneous stretching of matrix and

cell causes cellular mechanochemical transduction.

See text and Chapter 9 for further discussion.

(Adapted from Urry."^^)