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

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3.1 Initial Glimpses

it no longer rotated the plane of polarized light

after heating for several hours.^^ He learned

this after having found that crystals of the inac-

tive (paratartaric or racemic acid) product of

heating existed as left-handed and right-handed

mirror images and that solutions of the sepa-

rated mirror image crystals rotated the plane of

polarized light in opposite directions.

Most significantly for understanding the

products and composition of living organisms,

when Pasteur set the racemic mixture of mirror

image crystals to ferment, the living organisms

of the ferment consumed only the right-handed

(dextro-rotatory) acid molecules leaving

behind the left-handed (levo-rotatory) acid

molecules. In every other physical and chemi-

cal property these molecules were identical,

yet living organisms were able to distinguish

between them, consuming one and leaving the

other.

In the words of Pasteur, "Thus we find intro-

duced into physiological principles and in-

vestigations the idea of the influence of the

molecular asymmetry of natural organic prod-

ucts,

of this great character which establishes

perhaps the only well marked line of demarca-

tion that can at present be drawn between the

chemistry of dead matter and the chemistry

of living matter."^ In fact, we now know that

for each asymmetrical carbon atom (a carbon

atom with four different chemical groups

attached) there are two optical isomers. Thus,

for molecules such as glucose, with four asym-

metrical carbon atoms, there would be 2^

optical isomers. Biological molecules are com-

prised of but one of the possible optical

isomers. This fact is fundamental to the success

and efficiency with which proteins function as

molecular machines accessing available energy

and converting it to biologically essential

functions.

Thus,

in Httle more than three decades after

Wohler's accidental synthesis of urea, it was

learned that living organisms exhibited a

unique capacity to distinguish a molecule from

its mirror image. So profoundly indicative of

Life is the handedness of carbon-containing

molecules that a search for the existence of

Life in outer space once centered on finding

such optically active molecules in matter-

like meteorites. Although hydrocarbons have

been reported in meteorites, the occurrence

of molecules in meteorites that rotate the

plane of polarized hght has yet to be proven.

With the heating that a meteorite sustains on

entering the earth's atmosphere, it is not

unlikely that racemization would occur as it

did for the tartaric acid of Pasteur's original

observations.

3.1.7 The Carbon Atom Key to

Mirror Image Molecules

The mirror images made possible by the carbon

atom result from four different substituents

bound to a single carbon atom. As shown in

Figure 3.3B, the carbon atom is at the center of

a tetrahedron with a different substituent at

each of the four apices. One of the two back-

bone carbon atoms of the amino acid residues

shown in Figure 2.1 A has four different sub-

stituents, the H atom, the C=0, the NH, and the

R-group. Therefore, amino acid residues occur

with mirror images. The substituents are

"placed at the summits of an irregular tetrahe-

dron" as had been anticipated by Pasteur^^ and

as shown in Figure 3.3B.

As the surface of a protein interacts with an

L-amino acid residue in Figure 3.3B, for

example, the correct orientation of chemical

contacts must occur on the protein surface in

order to identify and associate with any three

points of contact with the residue. Regardless

of the side of approach to the tetrahedron with

four different substituents, the three approach-

able substituents are arranged in a clockwise

order in one case and a counterclockwise order

for the mirror image. Therefore, different

surfaces are required for proper fitting much

as the handshake between two right hands

fits whereas that between a left hand and a

right hand does not.

In a protein sequence, the selection of only

one of the two possible mirror image amino

acid residues at each position is responsible for

the regular folding and assembly of protein,

such as the regular formation and association

of helices shown in Figure 2.1C,D. Utilization

of but one mirror image molecule makes

possible the regular folding and assembly of

The Pilgrimage

proteins, which is responsible for the effective

energy conversions to be discussed in sub-

sequent chapters. The effect on folding and

assembly of the inclusion of a single incorrect

mirror image amino acid residue is depicted in

Figure 3.3C, which disrupts the regularly folded

and assembled structure depicted in Figure

2.1C,D. The random inclusion of very few

mirror image monomers disrupts the formation

of regular structures on which the efficiency of

Life's energy conversions depends.

3.2 Determination of Biological

Structures and Their Syntheses

Since the time of Wohler (1828), the usual

approach has been experimentally to deter-

mine the structure of a biological product by

combination of chemical and physical methods

and then to synthesize the product chemically

as proof of structure. For the last half century,

however, proof of structure has usually been

achieved by determination of the crystal struc-

ture using X-ray diffraction methods, whereby

a complete three-dimensional image of the

molecule can be obtained.

3.2.1 Structure and Synthesis

of Carbohydrate

The structure of the most common carbohy-

drate, the simple sugar D-glucose, was deter-

mined by the renowned chemist, Emil Fischer,

in 1891 to be as shown in Figure 3.4. Viewing

the six carbon atoms in the linear represen-

tation of glucose, four of the carbons con-

tain four different substituents so that there

are 16 (2^^) different optical isomers. On cycliz-

ing to form the pyranoses, a fifth optically active

carbon results with 64% forming the a-

anomer and 36% the p-anomer for D-glucose.

Fischer^^ synthesized each of the 16 optical

isomers of glucose and demonstrated the

biological form to be D-glucose, the one shown

in Figure 3.4.

On combining into chain molecules with

dif-

ferent attachments between the repeating units,

D-glucose forms several well-known carbo-

hydrates of remarkably different structural

properties. There is cellulose, the primary

component of wood with an estimated 10 tril-

lion tons being produced annually through pho-

tosynthesis in

trees.

There are the plant starches

(amylose and amylopectin) of cooking and

nutrition note, and the animal starch glycogen,

the stored source of energy in muscle.

6CH2OH

u 5C Q

H OH

^/ OH

a-D-Glucopyranose

i9^

^-2?-

-0-

H-C-OH

CH2OH

6CH2OH

>9 \ OH

\9H l/\

P-D-Glucopyranose

D-Glucose

(linear form)

FIGURE 3.4. Structural representations of glucose,

the major sugar and the primary source of energy of

higher animals that gives rise to 36 ATP molecules

during its oxidation to carbon dioxide and water. In

the Fischer projection, the central structure, carbon

atoms 2, 3, 4, and 5 each contains four different

substituents, making them asymmetrical centers.

Because each asymmetrical center can exist as either

one of two mirror images, this means that there are

2"^

= 16 distinguishable optical isomers. The single

structure shown here is the structure of biological

origin. Interactions with the active sites of enzymes

distinguish this structure from the other 15.

3.2 Determination of Biological Structures and Their Syntheses

Each glucose monomer derived from glyco-

gen gives rise to

ATP molecules^^ to provide

the immediate energy source for functions such

as muscle contraction, nucleic acid syntheses,

protein synthesis, and maintaining the proper

intracellular environment and extracellular

matrix.

3.2.2 Limiting Optical Isomers

(Mirror Images) Essential to

Effective Function

Because D-glucose is for living organisms the

pivotal molecular source of energy, its concen-

tration in the organism must be carefully regu-

lated. The result of inadequate regulation of

glucose in humans is the disease diabetes mel-

Utus.

The diagnosis of diabetes mellitus—once

nearly a certain death sentence arising from

complications of cardiovascular disease, kidney

disease, blindness, and frequent infection—

transformed into the prognosis of a long and

healthy life with the discovery of insulin.

The small protein insulin maintains the

correct blood level of D-glucose, and the accu-

rate recognition of a sugar such as D-glucose

requires an intimate association of the sugar

with a protein at each of

its

many sites of action.

The intimacy of this interaction is such that

a left-hand structure is distinguishable from a

right-hand structure, again as apparent in a

handshake. If there were not strict control of

the optical isomers, one could imagine that

control of glucose levels and the avoidance of

disease would require

different

systems;

such

a situation would be very cumbersome, ineffi-

cient, and more prone to disease by the factor

raised to the power of the number of sites

of interaction. It is difficult to imagine a suc-

cessful organism under such circumstances.

3.2.3 Structures of Fats, Oils,

and Membranes

The chemical syntheses of biological fats and

oils do not lend themselves to such identifiable

historic moments, but their preparation and

reconstitution into functional membranes con-

stitute achievements central to an understand-

ing of cell function.

Commonly categorized as lipids, biological

fats and oils are comprised of components such

as fatty acids, glycerol, triglycerides, phospho-

lipids,

sterols, and steroids. They are of interest

in energy conversion for two main reasons.

First, nutritionally their metabolism provides

two and one-half times more energy (calories)

per gram than do carbohydrates and proteins.

Second, they form the membranes that sur-

round the living cell and that delimit internal

organelles.

All cellular nutrients, excretory products, and

components of the extracellular matrix must

pass through the cell membrane. In addition,

ions are pumped, certain ions into the cell and

certain other ions out of the cell, to provide the

required internal environment and to set the

stage for response to stimuli. The ion pumps

within the cell membrane constitute a very

demanding aspect of energy consumption

required to sustain the cell.

3.2.3.1

Fatty

Acids

Fatty acids are simply the acetic acid molecules

of vinegar, CH3COOH, with long oil-like tails

formed by the addition of hydrocarbons such

as repeating saturated -CH2-CH2- and unsatu-

rated -HC=CH- groups. A general example of

saturated fats is CH3(CH2)nCOOH, where n

can vary from 10 to 22 (even values of n only).

When n is 16, it is the primary fatty acid of red

meats,

called stearic

acid.

A common example

of an unsaturated fatty acid is oleic acid,

CH3(CH2)7HC=CH(CH2)7COOH, the primary

and beneficial component of olive oil.

3.2.3.2 Triglycerides

The attachment of three fatty acids to the three

OH groups of glycerol, CH2OH-CHOH-

CH2OH as shown in Figure 3.5A, constitutes a

triglyceride. This is the primary storage mole-

cule of fat cells, and it is one of the lipids

monitored during blood lipid chemistry deter-

minations to assess risk of heart disease.

3.2.3.3 Phospholipids and Membranes

In phospholipids, two of the hydroxyls of the

glycerol have attached fatty acids, whereas

The Pilgrimage

attached to the third hydroxyl is a hydrophilic,

polar, or charged group. A common phospho-

lipid, lecithin, shown in Figure 3.5B, forms the

primary component of cell membranes. These

molecules form so-called lipid bilayer mem-

branes that constitute the membrane surround-

ing the living cell. They do so by the same

separation of oil from vinegar and of oil from

water described for proteins in the Chapter 2

overview and given a physical basis in Chapter

Once the lipid tail is long enough, there forms

so much hydration around the oil-like moieties

that the transition for assembly is below body

temperature and the separation from water

occurs with the geometry of the lipid bilayer

membrane as the result. The relative size of the

polar head group and the lipid tail determines

whether the resulting structure is a near planar

membrane or a sphere with an oily core and

polar surface called a micelle.

The polar and charged phosphocholine part

tries to stay in a watery environment, and the

oil-like hydrocarbon chains of the fatty acids try

to remain distant from the polar part and sep-

arated from water. The result is the assembly of

the lipid bilayer membrane as an integral part

of the cell membrane represented in Figure 3.6

with the oily tails of the lipids buried within and

forming the oily membrane and the polar

charged groups at the surface interacting with

water. This involves the same forces of separa-

tion and segregation of oil-like groups from

vinegar-like groups and oil from water that is

central to the consilient mechanism of protein-

A^CH

0 o

1 I

Ci=0 Ci=0

CH.

I ^

CHo

I ^

CH2

I ^

CH^

I ~

CH^

I '-

CH2

l|9

CH2

6CH3

CH2

l|9

CH2

II12^

CH2

I ^

CH2

oCH

3 1

CH2

CHo

I "

CH2

I ^

CH2

I ^

CH2

I ^

CH^

I "

CH2

I ^

CH2

I ^

CH2

,CH3

CH^

H3C-N^-CH3

CHo

I "

CH2

-o-p=o

"^CH^-^C 'CH

" I I •

c=o

(CH2)7 (CH2)i6

C —H CH3

1 -Palinitoleoyl-2-linoleoyl-

3-stearoyl-glycerol

C —H

(CH2)7

CH3

l-Stearoyl-2-oleoyl-3-phosphatidylcholine

FIGURE

3.5.

A A specific triglyceride comprised of a

glycerol molecule, carbon atoms 1, 2, and 3, the

hydroxyl functions of which are attached to three

fatty

acids.

In this case it is three different fatty acids,

two with different unsaturated, double bonds, and

the third is the saturated fatty acid common in red

meat. Ba A specific lecithin wherein the glycerol

molecule has a charged phosphocholine group at

carbon 3 and two different fatty acids, one saturated

and the other unsaturated at carbons 1 and 2. Bb

Space-filling model of the lecithin in (b) showing the

effect of the double bond to be the introduction of a

kink in an otherwise essentially linear hydrocarbon

chain. (B Courtesy of Richard

Pastor, FDA, Wash-

ington D.C. A and B from Biochemistry, Second

Edition,

Voet and

1995,

John

Wiley &

Sons,

New

York.

Reprinted with permission

of John Wiley & Sons, Inc.)

3.2 Determination of Biological Structures and Their Syntheses

FIGURE 3.6. Cartoon of a cell membrane composed

primarily of a lipid bilayer membrane, mostly con-

taining the phospholipid lecithin, and decorated with

occasional cholesterol molecules. Fundamental to

the cell membrane function are integral membrane

proteins that span from one side to the other of a cell

membrane and commonly function in transmem-

brane transport and

signaling,

in cell recognition, and

so forth. (From Biochemistry, Second Edition, D.

Sons,

New York. Reprinted with permission of John

Wiley & Sons, Inc.)

based machines and, as argued here,

the primary driving forces for changes in

protein structure that determine function.

3.2.4 The Essential Role of Protein

Machines Within the Cell Membrane

As with proteins, the distribution of oil-like and

vinegar-like groups dictates membrane struc-

ture.

Furthermore, it is structure, and changes

in structure, that dictate function, because

charged species like protons, sodium ions, and

potassium ions cannot pass through an intact

oily lipid bilayer membrane of the cell without

the cell membrane being specially modified by

proteins called integral membrane proteins

(see Figure 3.6). Integral membrane protein

machines, therefore, control the passage of

charged and other polar species into and out

of the cell. The Fo-motor of ATP synthase re-

presents perhaps the most important protein

machine integral to the cell membrane, as it

uses positively charged protons to produce

some 90% of the ATP whereby living organ-

isms function.

3.2.4.1 Protein Machines Pump Ions into

and out of the Cell

With ATP as the source of energy, a particular

protein in the cell membrane pumps two potas-

sium ions, K^, into the cell and three sodium

ions,

Na^, out of the cell; another protein pumps

calcium ions, Ca^^, away from the contractile

apparatus of muscle cells. The result is a polar-

ized membrane with more negative charge

inside than outside of the cell.

3.2.4.2 Protein Channels in Cell

Membranes Open and Close to Allow

Excitatory Flows of Ions

Still other integral membrane proteins, most

common in the cell membrane of nerve cells,

contain transmembrane channels that open

with the proper stimuli, letting sodium ions rush

in (depolarizing the membrane) as an excita-

tory action that activates neural networks

required for thought processes. Additionally,

nerve cell processes impinging on muscle cells

depolarize and in turn activate channel

The Pilgrimage

openings in muscle cell membranes and ulti-

mately trigger the rush of calcium ions into the

muscle cell to initiate muscle contraction.

3,2.4.3 Protein-based Machines

in the Thylakoid and Inner

Mitochondrial Membranes

A flow of electrons in a series of cell mem-

brane-associated oxidation and reduction

cycles provides the energy source in the energy-

converting thylakoid membranes of plants and

inner mitochondrial membranes of plants and

animals. In the process of this electron flow,

membrane proteins pump protons across a cell

membrane to increase the concentration of

protons on one side of the membrane (to be

considered in Chapter 8). Another protein-

based machine uses the high concentration of

protons on one side of a cell membrane to drive

the formation of ATP as the protons pass to the

low concentration side of the membrane (also

to be considered in Chapter 8).

While defining the cell's outer boundary, the

viable membrane, that is, one that contains the

required complement of proteins functioning in

the lipid bilayer membrane surrounding the

cell, represents an essential component of

the fundamental unit of Life. Life begins with

the cell, and the cell no longer lives when the

cell membrane has been rendered irreparably

nonfunctional.

There is significance in the knowledge that

man has synthesized the biological fats and oils;

he has made the biological fats and oils into

membranes, and he has incorporated proteins

into the membranes with reconstitution of inte-

gral membrane protein function. As discussed

below, man has also chemically synthesized

protein. This completes the conceptual capacity

to reconstitute the critical functional mem-

brane component of the viable cell.

3.2.5 Syntheses of Sterols

and Steroids

The total chemical synthesis of cholesterol (see

Figure 3.7), the chief sterol of vertebrates and

the essential precursor of the steroid hormones,

was achieved in 1951 by R.B. Woodward, along

CH,

18 I 2 2 2

CH3 I -\

CH.

Cholesterol

FIGURE 3.7. The structure of cholesterol of blood

lipid chemistry noted for its relevance to heart

disease A bond and B spacefilling representations

cholesterol,

noted for its relevance to heart disease

steroid hormones. (B Courtesy of Richard

Pastor,

FDA, Washington D.C. A and B from Biochemistry,

1995,

John Wiley & Sons, New York. Reprinted with

and for its precursor role in the biosynthesis of permission of John Wiley & Sons, Inc.)

3.2 Determination of Biological Structures and Their Syntheses

with the synthesis of cortisone, the anti-

inflammatory steroid often obtained in creams

to prevent itch, decrease redness, and enhance

healing. In addition, steroids and their ana-

logues are well known for their uses as oral con-

traceptives (estrogens and gestogens for birth

control), as hormone replacement therapy for

postmenopausal women, and as bodybuilding

and athletic prowess enhancement factors

(anaboUc steroids, the androgens). Addition-

ally, of course, blood levels of cholesterol signify

predisposition to heart disease.

Clearly, the molecules that we can now

produce by modern chemistry and the chemi-

cal energy that they represent have profound

effects on humans and other living organisms

of all levels of complexity. This is no longer

an issue for debate as it was as recently

as several decades ago. Instead, the debate is

how to ensure the appropriate use of this

capacity. For example, the pressure for athletes

to use performance-enhancing steroid and

peptide hormones and other drugs poses

serious concerns for the International

Olympic Committee, other sports regulatory

efforts, and the overall long-term health of the

athlete.

3.2.6 Animal and Plant Waxes

The wool of sheep repels water due to a coating

of a wax called lanolin; it is a combination of

sterols with long chain fatty acids and is often

utilized as an ointment base in cosmetic and

skin care formulations. Beeswax, perhaps the

most commonly known animal wax, derives

from the honeycombs of bees; it is the combi-

nation of straight long chain alcohols esterified

to straight chain fatty acids. For example, the

straight chain alcohol, H3C(CH2)34CH20H and

the long chain fatty acid HOOC(CH2)34CH3

combine with the loss of water, H2O, to form

the ester H3C(CH2)34COOC(CH2)34CH3, which

is the longest chain molecule of beeswax; it is

used in candles, cosmetics, modeling artificial

fruits and flowers, and shoe poUsh.

A familiar plant wax is the wax coating of

polished apples; it is a cuticle wax seen most

often on the upper surfaces of leaves. Well-

known cuticle waxes are carnauba wax from the

fronds of a Brazihan palm tree and candelilla

wax from the wild candelilla plants of Mexico

and Texas. Carnauba is a hard, high-poHsh wax

for automobile, floor, and high quaUty shoe pol-

ishes.

These plant and animal waxes should be

distinguished from the similarly utilized

paraf-

fin waxes and petrolatum, which originate from

petroleum. If biological origins of petroleum

are considered, however, that distinction loses

much of its significance.

3.2.7 Vitamins

It was appreciated in the early part of the twen-

tieth century that a diet containing the correct

amounts of carbohydrate, fats, and protein was

insufficient for good health. Other "accessory

food factors" were required. The initially iden-

tified "accessory substances" were amines, and

so these "vital amines" were called vitamines.

When it became clear that not all accessory

substances were amines, the name became

vitamins.

For humans, there are 13 essential vitamins,

broadly classified as water soluble (or vinegar-

like) and fat soluble (or oil-like). They often

function as coenzymes or cofactors of meta-

bolic reactions, where they commonly change

their oil-like or vinegar-like character, or they

bring about a change in a protein to a more oil-

like or vinegar-like state. For example, vitamin

B3 (niacin, the ring structure used to form

nicotinamide as shown in Figure 3.8A) and

vitamin B2 (riboflavin, which becomes the

flavins as shown in Figure 3.8B) are the princi-

ple coenzymes that receive electrons to form

their more oil-like reduced state or give up

electrons to form a more vinegar-like oxidized

state.

A change in the oxidative state of nicoti-

namide and flavin causes functional changes in

the propensity of the protein, to which they are

attached, to associate hydrophobically. Though

beyond the scope of this effort, it would be of

interest to assess, in general, the extent to which

all vitamins, by effecting a change in oil-like

character, drive folding and unfolding in

accomplishing protein function.

In the early part of this century many vitamin

deficiency diseases were identified and cured.

Vitamin D cured rickets. Vitamin C (ascorbic

The Pilgrimage

K<^

Nicotinamide

(niacinamide)

NH2

K-^

Nicotinic acid

(niacin)

FIGURE

3.8.

Structures

vitamins

vitamin-

derived molecules that function

oxidation-

reduction reactions.

The

oxidation

these redox

groups

in the

inner mitochondrial membrane con-

tributes

to the

electron transport chain that carries

electrons from

the

oxidation

glucose

oxygen

and

in the

process pumps protons from one side

the other

the inner mitochondrial membrane (see

Chapter

8 for

details).

The

proton gradient thus

formed is used

phosphorylate ADP

form 32 of

the

ATPs resulting from

the

oxidation

of one

glucose molecule

six CO2 and six H2O molecules.

A Vitamin B3, also called niacin

nicotinic

acid,

becomes converted to the amide (nicotinamide) and

dressed up with a ribose sugar.

Then,

a manner like

that

riboflavin

in B

becomes phosphorylated

form nicotinamide mononucleotide

(NMN) or

further reacted with

the

addition

adenosine

monophosphate

(AMP) to

form nicotinamide

adenine dinucleotide (NAD).

Vitamin

B2,

also

known as

riboflavin,

shown converted to the forms

involved in redox reactions such as those of the elec-

tron transport chain. (From Biochemistry, Second

Edition,

Voet and

1995,

John

Wiley &

Sons,

New

York.

Reprinted with permission

of John Wiley & Sons, Inc.)

CH2OH

H-C-OH

CH,

ATP

ADP

Riboflavin

flavokinase

CH2-0-P()f

H-C-OH

Flavin mononucleotide (FMN)

\ pyrophosphorylase

NH.

CH2-O- P-O- P-OCH2

H-C-OH

O — I /

H-C-OH

H>—TH

H-C-OH

CH,

OH OH

H3C^^N^^"

Flavin adenine dinucleotide (FAD)

acid, discovered

by the

same Albert Szent-

Gyorgyi

of the

early muscle contraction

studies) cured scurvy. Vitamin

cured

beriberi. Pernicious anemia, which like diabetes

mellitus once meant certain death, was cured by

vitamin

B12,

cyanocobalamine.

A large collaborative team

chemists

headed by the same Woodward who had earlier

synthesized cholesterol

and

cortisone, chemi-

cally synthesized vitamin B12.

fact,

all of the

13 essential vitamins

for

humans have been

chemically synthesized. Thus continues

the

pilgrimage from proscription

successful

preparation.

3.2.8 Structure

and

Chemical

Synthesis

Natural Proteins

Emil Fischer achieved

the

first chemical syn-

thesis

of a

protein-like fragment

1907, when

he assembled

peptide linkage

chain

of 18

amino acids

and

showed that

the

chain

was

degraded

proteases, proteins functioning

enzymes

degrade protein.

Not surprisingly, the history

protein struc-

ture determination

and

synthesis involved

proteins with

key

roles

energy conversion.

Insulin, essential

to the

proper metabolism

of glucose,

was the

first protein sequenced.

3.2 Determination of Biological Structures and Their Syntheses

Determination of the amino acid sequence of

insulin by Sanger in 1953 provided the first

demonstration that proteins do indeed have a

specified sequence. Figure 3.9A gives the

chain structure of preproinsulin that becomes

proinsulin (Figure 3.10) on secretion from the

cell with cleavage of the amino terminal signal

sequence. The signal peptide is required for

secretion of the protein through the oily lipid

bilayer of the cell membrane (note in Figure

3.6) and in the process is cleaved off to give

proinsulin. Then proinsulin becomes active

insulin on excision of the C chain. Figure 3.9B

gives the crystal structure, and Figure 3.9C gives

the band or ribbon representation of the insulin

molecule. The amino acid sequence of proin-

sulin is given in Figure 3.10, and the single-

residue and mean-residue hydrophobicity plots

for proinsulin are seen in Figure 3.11, which is

a type of plot to be described and used in

Chapter 7 for noting changes in composition and

for identification of oil-like domains of special

relevance to protein structure and function.

The first chemical synthesis of a natural

protein was also that of insulin. Furthermore,

the first three-dimensional protein structures

determined were those of myoglobin, the

oxygen storage/transport protein of muscle, and

hemoglobin, the structurally related oxygen

transport protein of blood. The structure and

function of hemoglobins are considered in

Chapter 7.

Chemical synthesis of insulin^^ occurred in

the laboratories of three different research

groups in the early

1960s.

The protein hormone

insulin is made up of two chains, one of approx-

Preproinsulin

Proinsulin

NH,

Mature

insulin

C A

coo-

Signal

sequence

H,N

NH,

B chain

COO"

Signal sequence

FIGURE 3.9. A Steps in the conversion

of the gene product for insulin from

preproinsulin to active (mature)

insulin. B Space-filling representation

of the active insulin molecule, as

obtained from the crystal structure. C

The ribbon model of active insulin

showing the helical and extended

regions of the A and B chains and the

disulfide bridges that hold the A and B

chains together after removal of the C

peptide. (Reproduced with permission

from Lehninger,

Nelson,

and

Cox,

Prin-

ciples

of Biochemistry, Second Edition,

Worth Publishers.)

The Pilgrimage

C Chain

B Chain

FIGURE 3.10. Primary structure (sequence of amino

acid residues) of proinsulin, including location of the

disulfide bridges with identification of the A, B, and

C chains and locations of the cleavage sites for con-

version to active (mature) insulin. (Reprinted with

permission from R.E. Chance, R.M. Ellis and W.W.

1968 AAAS.)

imately 20 and the second of approximately 30

amino acid residues. Johannes Meienhofer

at the Technischen Hochschule in Aachen,

Ching-i Niu at the Institute of Biochemistry in

Shanghai, and P. G. Katsoyannis at the Univer-

sity of Pittsburgh with their colleagues each

synthesized insulin. They determined the prod-

ucts of the different chemical syntheses to be

active by determining biological activity and to

be crystallizable with the formation of the same

crystals as the natural product.

Classic chemical synthesis in solution, how-

ever, was never a practical way to produce

commercial quantities of insulin for therapeu-

tic use to control diabetes. On the other hand,

Merrifield developed an alternative approach

in the 1960s and 1970s. In this chemical method

the protein chain was grown on a solid support,

one residue at a

time.

The process used an auto-

mated machine to proceed through the many

chemical steps required to add each amino acid

residue. This solid phase chemical synthesis has

been used to synthesize small proteins like

insulin and interferon for commercial use. Now

the most effective means of producing proteins

of therapeutic value is to use the biological

synthetic apparatus

itself,

by means of recom-

binant deoxyribonucleic acid (DNA) technol-

ogy, also referred to as genetic engineering.

Human insulin, marketed as Humulin by Eli

Lilly, became the first commercial protein phar-

maceutical produced by recombinant (DNA)

technology.

3.2.9 Chemical Synthesis and

Structure of Elastic-contractile

Model Proteins

Prepared initially by chemical syntheses, de

novo-designed model proteins were found to

contract and produce motion and, in general, to

perform mechanical and other kinds of work

in response to chemical and other stimuli.

In fact, the chemically synthesized elastic-

contractile model proteins were found, just as

their design had intended, to perform the kinds

of energy conversions that sustain Life.

The demonstrations of these energy conver-

sions (Chapter 5), of the physical basis for the

energy conversions (Chapter 5), of the rele-

vance of the mechanism to naturally occurring

proteins and protein-based machines (Chapters

7 and 8), and of their potential as materials for

medical and nonmedical applications (Chapter

9) constitute the central messages of this

volume.

A brief description of the chemical synthesis

of elastic-contractile proteins immediately fol-

lows in the context of the general message of

the chemical synthesis of natural biological

products.

3.2.9.1 Chemical Synthesis of

Elastic-contractile Model Proteins

Both classical solution and the Merrifield solid

phase synthetic methods were used to prepare