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

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7.3 Hemoglobin Structures Demonstrate

the

Consilient Mechanism

265

hydrophobic dominance, separate

reorient

a way to

minimize AGap.

The

separated

and

reoriented charged groups

now

reach

out for

more hydration, such that

the

oxygen-binding

event

at one

heme decreases

the

hydrophobic-

ity

(the

amount

hydrophobic hydration)

the second heme

at the

other

end of the

crevasse

and

facilitates

the

binding oxygen

the second heme.

The

decrease

hydropho-

bicity

of the

second heme

due to

oxygen

binding

to the

first heme uniquely becomes

the consilient mechanism's explanation

for the

sigmoid binding curve, that is,

for the

effective

oxygen transport function

hemoglobin.

In short,

propose that first polar oxygen,

its

approach

to the

heme binding site

hemoglobin, experiences repulsion

due to the

hydrophobicity

of the

heme

and its

environ-

ment. The barrier results from

the

loss

hydra-

tion available

to the

oxygen molecule

as it

approaches

the

hydrophobic hydration

of the

exposed heme

and its

surrounding hydrophobic

groups. Once

the

first polar oxygen molecule

binds,

however,

the

resulting decrease

hydrophobicity transmits through

the

water-

filled crevasse,

reoriented, more competitive

charged groups,

to the

second heme

and

facili-

tates binding

of the

second oxygen. Structural

justifications

for

this view follow.

7.3.3 Examination

the Structural

Changes Resulting from

Oxygen Binding

7.3.3.1

General Appearance

of the

Crystal

Structures

Deoxyhemoglobin

and Oxyhemoglobin

7.3.3.1.1

The

Size

of the

Aqueous Core

of the

T State

Hemoglobin

A space-filling representation. Figure 7.11A,B

presents stereo views

deoxyHb

and

oxyHb(T) from

the

perspective

of the

twofold

(diad) axis.

first point

note with this per-

spective

that

gives

the

most minimal size

the aqueous core

of the

hemoglobin molecule.

Looking

stereo view into

the

aqueous core

at several slight angles from

the

diad axis,

one

sees

much larger water-filled

core.

Thus,

water

not

only accessible from

the

outside

of the

molecule

to the

areas

association between

subunits,

but

water

also available

to the

qua-

ternary structure from

the

inside.

fact,

measured from slabs taken perpendicular

the twofold axis,

the

water-filled core, which

waters would constitute part

of the

"waters

Thales," approaches 40%

of the

total diameter.

The thickness

the wall

on one

side

can be

less

than

the

width

of the

aqueous channel.

7.3.3.1.2

The

Limited External Differences

Between deoxyHb

and

oxyHb(T)

From

the

perspective

Figure 7.11, small

dif-

ferences

are

seen between

the

P-chains

and

between

the

a-chains within

the

same tetramer.

Surprisingly, these small intramolecular

dif-

ferences appear

to be as

great

as the

inter-

molecular differences between deoxyHb

and

oxyHb(T). Attempts

oxygenate crystalline

deoxyHb

room temperature destroy crys-

talline order. However, crystals

deoxyHb

formed

4° C, carefully changed

to a

40% poly-

ethylene glycol-water solution,

and

oxygenated

4°C

survive with oxygen bound

at all

four

hemes.^^

The

result

T-state oxyhemoglobin,

that

is,

oxyHb(T).

The

protocol

for

forming

oxyHb(T)

is a

prescription

for

reigning

in the

consilient mechanism certainly

at the

level

changing quaternary structure. Nonetheless,

choose

utilize oxyHb(T) because,

oxygen

binding does decrease

the

hydrophobicity

the

heme group, evidence

this should

be in the

heme environment

long

there does remain

adequate water

in the

crystal

evidence

the

competition

for

hydration.

7.3.3.1.3 Looking

to a

Molecular Basis

for the

Coherence

Phenomena

The phenomenological observations detailed

above

section

7.2.4

clearly exhibit

coher-

ence

phenomena with

the

consiUent mecha-

nism.

Our

objective, therefore,

is to

determine

the particular expressions

of the

consilient

mechanism

at the

molecular level.

As a

starting

point, we look from

the

aqueous medium

to the

heme group

in its

protein setting

and

make

gross comparison between

the

heme environ-

ment

myoglobin

and the

environments

of the

hemes

hemoglobin.

In our

view,

the

first clue

266

Biology Thrives Near a Movable Cusp of Insolubility

FIGURE

7.11.

Stereo view of the human hemoglobin

tetramer from the perspective of the twofold (diad)

axis using space-filling representation with a-chains

in white and ^-chains in gray and with the light dots

for the propionate oxygens of heme ligands within

to the molecular basis for differences in oxygen

binding, represented in Figures 7.5,7.6, and 7.7,

gains from such a gross perspective.

7.3.3,2

Structure and Disposition of Heme

Groups Before and After Oxygen Binding

7.3.3.2.1 Gaining Perspective of the

Heme Group

A key aspect of our consilient view^ of oxygen

transport by hemoglobin, derived as it has been

the upper right and lower left quadrants

(A) Deoxy-

hemoglobin (deoxyHb).^^ (Protein Data Bank,

Structure File, 1A3N.) (B) Oxyhemoglobin

in the T state, oxyHb(T).^^ (Protein Data Bank,

Structure File, IGZX.)

from the inverse temperature transition for

hydrophobic association, places the heme

group in the unique position of being the most

hydrophobic component of hemoglobin. Fur-

thermore, our general experience has been that

the addition of a polar group like the oxygen

molecule would significantly decrease heme

hydrophobicity. A sense of the heme group

begins with Figure 7.12, which presents in ball

and stick representation a stereo view of the

structures of the heme (ferroprotoporphyrin

7.3 Hemoglobin Structures Demonstrate the Consilient Mechanism

267

IX) group, as found in the binding sites of the

a-chain for deoxyHb in Figure 7.12A and

oxyHb(T) in Figure 7.12B.

Figure 7.13 provides some sense of the rela-

tive size and disposition of the heme group in

situ within the p^-chain. The structures without

and with bound oxygen are shown in stereo

view in a space-filling representation for the

hemes and stick representation for the p^-chain.

Figure 7.13A presents the heme for deoxyHb

from the side where histidine binds the central

iron (upper part) and from the side to which

oxygen binds (lower part). Figure 7.13B simi-

larly presents the heme for oxyHb(T) where

the bound oxygen molecule appears in its lower

part.

With the P^-chain in stick representation, the

heme groups can still be seen and yet retain

FIGURE 7.12. Stereo view of P-chain heme group of

human hemoglobin tetramer in ball and stick repre-

sentation with an iron atom in the center surrounded

by protoporphyrin IX. See text for further descrip-

tion. (A) Deoxyhemoglobin (deoxyHb).^^ (Protein

Data Bank, Structure File, 1A3N.) (B) Oxyhemoglo-

bin in the T state, oxyHb(T), showing the diatomic

oxygen atom bound to the central heme iron.^^

(Protein Data Bank, Structure File, IGZX.)

some (albeit a somewhat exaggerated) sense of

the relative size of heme to that of the protein

and its aromatic side chains. The heme is basi-

cally a very large hydrophobic (aromatic)

group moderated by two attached polar propi-

onate groups, -CH-CH-COO~, that are equiv-

alent to the side chain of glutamic acid (Glu, E).

Also the iron at the heme center presents a

binding site, one side for the aromatic His

Ugand and the other side for the very polar

oxygen molecule. The heme ring structure

extends further due to the substitution at its

edge of two vinyl groups, -CH=CH2, and four

methyl groups, -CH3, which further increase its

hydrophobicity.

7.3.3.2.2 Views from Aqueous Solution of

the Heme in Myoglobin and the Hemes

in Hemoglobin

Figure 7.14 presents from solution a stereo view

of the crystal structure of myoglobin^^ in space-

fiUing representation with the protein chain in

white, except for the most hydrophobic aro-

matic residues shown in black, and with the

heme in gray and its propionate oxygens as

light atoms on gray heme. Figure 7.14A gives

the structure of deoxymyoglobin, deoxyMb,

and Figure 7.14B presents the structure of

oxymyoglobin, oxyMb. Also included in both

myoglobin crystal structures are two sulfate

ions at the upper right hand edge.

In similar fashion. Figure 7.15A presents the

deoxyhemoglobin a^p^ dimer and Figure 7.15B

the oxyhemoglobin a^p^ dimer with the view in

both cases being taken from the p^-heme to the

a^-heme. Strikingly, the hemes of hemoglobin

reside in sites that are much more hydro-

phobic than those of the heme of myoglobin,

as readily seen by the black nonhistidine-

most hydrophobic aromatic residues. In our

view, this feature alone ensures that the affinity

for oxygen would be lower for Hb than for

Mb.

By the apolar-polar repulsive free

energy of hydration, AGap, a polar oxygen mol-

ecule would experience greater repulsion on

approaching the heme binding sites in hemo-

globin than the heme site in myoglobin.

The question now advances to the issue of

FIGURE

7.13. Stereo views of the

heme (ferroprotoporphyrin IX) in

space-filhng representation as it

appears in the p^-subunit of human

hemoglobin. The P^-chain is given in

stick representation in order not to

obscure the heme and in order

to gain some perspective of rela-

tive size. (A) Deoxyhemoglobin

(deoxyHb).^^ The upper part shows

the side at which the His imidazole

remains bound, and the lower part

shows the side to which oxygen

binds.

(Protein Data Bank, Structure

File,

1A3N.) (B) Oxyhemoglobin in

the T state, oxyHb(T).^^ The upper

part shows the side at which the His

imidazole remains bound, and the

lower part shows the bound oxygen.

(Protein Data Bank, Structure File,

IGZX.)

FIGURE

7.14. Stereo view of the crystal structure of

myoglobin^^ in space-filling representation with a

white protein chain, except for black non-His

aromatic residues, and gray heme ligand with

propionate oxygens shown as Ught atoms on

gray heme at upper center of structure. (A)

Deoxymyoglobin (Protein Data Bank, Structure

File,

1A6N). (B) Oxymyoglobin (Protein Data Bank,

Structure File, lA6M).The crystal structure for both

states contains two identically positioned sulfate ions

on the surface. Note the limited exposure of aromatic

residues to the surface, particularly in the area occu-

pied by the heme group, and compare it with that

observed in hemoglobin in Figures 7.15 and 7.16.

7.3 Hemoglobin Structures Demonstrate the Consilient Mechanism

269

FIGURE

7.14.

Continued

how the binding of oxygen at a first heme

enhances oxygen affinity at a second heme.

Based on the view in Figure 7.16 from the a^-

heme to the p^-heme, the approach to the

answer gains from an expanded view of the a^P^

heme pair.

7.3.3.2.3 The Presence of a Hydrophobic

Crevasse on the Protein Surface That

Connects the P^a^-heme Pair

Figure 7.17 presents an expanded stereo view of

Figure 7.16 and discloses details of a crevasse

FIGURE 7.15. Stereo view of the hemoglobin a^p^

dimer in space-filling representation with the protein

chain in

white,

except for non-His aromatics in black,

and with the light gray heme showing propionate

oxygens as light atoms on two gray hemes starting at

center left and upper center and oriented toward

upper right of the structure. The view is taken from

the P^-heme to the a^-heme. Note the most

hydrophobic aromatic residues connecting the two

hemes.

(A) Deoxyhemoglobin (deoxyHb).^^ (Protein

Data Bank, Structure File, 1A3N.) (B) Oxyhemoglo-

bin in the T state, oxyHb(T).^^ (Protein Data Bank,

Structure File, IGZX.)

270

Biology Thrives Near a Movable Cusp of Insolubility

FIGURE 7.16. Stereo view of the

hemoglobin a^P^ dimer in space-

filling representation with the

protein chain in white, except for

non-His aromatics in black, and

with the hght-gray heme showing

propionate oxygens as light atoms

on two gray hemes starting from

center and upper center and tilted

toward the upper right of the

structure. This view is taken from

the a^-heme to the P^-heme. Also

note the presence of the very

hydrophobic aromatic residues

between the two hemes. (A)

Deoxyhemoglobin (deoxyHb).^^

(Protein Data Bank, Structure

File,

1A3N.) (B) Oxyhemoglobin

in the T state, oxyHb(T).^^

(Protein Data Bank, Structure

File,

IGZX.)

connecting the a^P^ heme pair. Whether in the

deoxyHb or the oxyHb(T) state, the exposed

hydrophobic hemes form the ends of the

crevasse that is lined with very hydrophobic

(black) groups. In particular, contributed from

the P^-chain, there are the hydrophobic side

chains of

F45,

F42,

F41,

H92, L88,

L91,

L96, and

H97,

and contributed from the a^-chain there

are Y72, H45, F46, H58,

L91,

L86, and L83.

In addition, when looking from the a^-heme

to the base of the P^-heme, at the very bottom

of the crevasse, abutting the P^-heme in Figure

7.17, resides the V98 residue, and an enlarged

view of the crevasse in Figure 7.15 shows the

similarly positioned V93 residue at the base of

the a^-heme. As shown in Figure 7.8, these

residues participate in hydrogen bonds with

residues Y145 and Y140, respectively, that are

disrupted on oxygen binding. Most significantly,

a widened view of Figure 7.17, but taken from

directly above the crevasse, is included below in

Figures 7.19 and 7.20. It shows the key residues

Y145 and H146 of the p^-chain and Y140 and

R141 of the a^-chain. Therefore, all of the crit-

ical interactions in Figure 7.8 that change on

oxygen binding reside within the area of influ-

ence of these hemes. Thus, the happenings

within the interheme crevasse become a means

7.3 Hemoglobin Structures Demonstrate the Consilient Mechanism

271

of communication not only between the hemes

of the particular crevasse but also provide com-

munication between the a^p^-heme pair and the

a^p^-heme pair.

Returning to the interheme crevasse, the

unique grouping of hydrophobic residues

associated with the a^p^-heme pair and with

the equivalent a^p^-heme pair constitutes two

remarkably hydrophobic domains for exposure

at an aqueous surface. It is true, however, that

much of the hydrophobicity Ues in the depths

of the water-filled crevasse. These are circum-

stances under which the consilient mechanism

dictates an acute competition for hydration

between the hydrophobic residues of each

crevasse and its associated surface charged

groups. Such a competition gives rise to a repul-

sion between hydrophobic and charged groups

as each requires water unperturbed by the

other to lower their free energy. Based on the

studies reviewed in Chapter 5, this, of course, is

what we have called an apolar-polar repulsive

free energy of hydration and have indicated by

AGap. As the hydrophobic groups are held as

a more rigid part of the protein structure, it

would seem to be left to the charged side chains

at the ends of their aUphatic chains with their

greater range of motion to reflect the repulsion.

7.3.3.2.4 The Disposition of Charged Side

Chains Associated with the Crevasse

Connecting the P^- and a^-Heme Groups

for deoxyHb

There are basically two ways in which charged

side chains of amino acid residues can lower

their free energy when confronted with a

hydrophobic domain. One way is to form ion

pairs.

The second way, when possible, is to move

toward locations more remote from the most

hydrophobic surface. Both examples are shown

in the crevasse that connects the

and a^ heme

groups as well as in the equivalent crevasse con-

necting the a^p^-heme groups.

As shown in Figure 7.17A, there are seven

charged residues in the immediate vicinity of

the P^a^ crevasse. Starting at six o'clock and

reading clockwise, they are

K61,

K90, R92, R40,

E43,

K66, and K95. Interestingly, this means six

positively charged and one negatively charged

side chains. However, the two heme groups add

four more negatively charged groups associated

with each crevasse. Those residues in position

for ion pairing are K61, R92, E43, and K66.

Despite literally being bathed in water, ion

pairs form. The positively charged side chains

of K90, R40, and K95 are expected to have

moved to their positions of lowest accessible

free energy, that is, to minimize repulsion

coming from the apolar crevasse. Thus these

charges are expected to be pointing in the

direction of lower hydrophobicity. This finding

indicates that the region of the a-heme is

less hydrophobic than that of the p-heme.

This observation is consistent with a number of

reports that the T state exhibits a fivefold

greater binding at the a-heme than at the

P-heme.^^^2

7.3.3.2.5 The Effect of Oxygen Binding on the

Disposition of Charged Side Chains Proximal

to the Crevasse Connecting the p^- and

a^-Heme Groups

As shown in Figure 7.17B, oxygen binding

effects a remarkable change in the disposition

of the seven charged side chains. The four side

chains positioned for ion paring separate from

their ion paired positions, and the charges of

the three positively charged, non-ion-paired

residues, K90, R40, and K95, reorient their

charges. Yet the forces that effect these changes

cannot have come through the protein struc-

ture.

In our view, the driving force for these

oxygenation-effected side chain reorientations

came through the "waters of Thales" of the

hydrophobic crevasse. We beUeve this demon-

strates that oxygen binding, even though it

occurs on the other side of the hemes, decreases

the hydrophobicity of the hydrophobic crevasse

sufficiently to allow improved hydration of

these charged species. Thus, we arrive at a spe-

cific set of structural changes on oxygenation

consistent with the consilient mechanism.

7.3.4 Consideration of

Communication Between the a^p^-

and a^p^-Heme Pairs

The consilient mechanism provides a means of

communication between the pair of hemes of

272

Biology Thrives Near a Movable Cusp of Insolubility

FIGURE 7.17. Crevasse on hemoglobin surface con-

necting a^- and p^-hemes: Stereo view showing a

space-filling representation in which the residues are

shaded with aromatics in black, other hydrophobics

in gray, neutrals in light gray, charged in white, and

with the heme in a lighter gray with propionate

oxygens shown as Hght atoms. This is a magnified

view taken from the a^-heme to the P^-heme as in

Figure 7.16 in order to define the disposition of the

charged groups. (A) Deoxyhemoglobin (deoxyHb).^^

(Protein Data Bank, Structure File, 1A3N.) (B)

Oxyhemoglobin in the T state, oxyHb(T).^^ On the

binding of oxygen, the charged residues reorient in

a manner indicating a decrease in hydrophobicity

in the inter-heme crevasse. See text for discussion.

(Protein Data Bank, Structure File, IGZX.)

7.3 Hemoglobin Structures Demonstrate the Consilient Mechanism

273

the a^p^ crevasse and equivalently of the a^p^

crevasse. However, can the consilient mecha-

nism be directly responsible for the communi-

cation between the a^p^- and a^p^-heme pairs?

We begin by consideration of the a^P^ interface,

which is equivalent to the a^p^ interface.

7.3.4.1

Changes in the Interface Between

the o^fi^ and df^ Dimers Due to

Oxygen Binding

There is general appreciation that the principal

quaternary structural changes giving rise to

cooperative oxygen binding occur at the a^p^

(and a^p^) interfaces. For example, "The qua-

ternary structural change preserves hemoglo-

bin's exact two-fold symmetry and takes place

entirely across its d-f^ (and o^-pf) interface.

The d-P^ (and o?-/?) contact is unchanged.

.. .'"^^ The hydrophobic crevasse discussed

above constitutes part of that interface.

7.3.4.1.1 Estimates of the Hydrophobicities of

the deoxyHb a^P^ Interface

With the representation of the interface given

in Figure 7.18, it becomes possible to ap-

proximate, AGHA, the Gibbs free energy for

hydrophobic association of the a^p^-a^p^ inter-

faces.

The calculation uses the hydrophobicity

scale of Table 5.3, estimates the area of the

residue exposed to the interface, and reduces

the effect of charge by one-half when the

charged residue is ion paired, as was the expe-

rience from ion pairing between complemen-

tary chains of elastic protein-based polymers.

The estimated value of

AGHA

was -9kcal/mole-

face for the a-face and -24kcal/mole-face

for the P-face. These appear to be reasonable

approximations, but do not take into consider-

ation the fact that some of the residues, such as

K99 and R104, lie on the aqueous core of the

twofold axis shown in Figure 7.11.

7.3.4.1.2 Locating the Ion-pair Interactions

Between Interfaces and Visualizing the

Overlay of the a^p^ and a^p^ Dimers and the

Change on Oxygen Binding

From Figure

7.8,

important ion pair interactions

in deoxyHb that orient the a^p^ dimer with

a-face

FIGURE

7.18.

Human deoxyhemoglobin^^

showing,

space-filling representation, the interface between

ap dimers in which the residues are shaded with aro-

matics in black, other hydrophobics in gray, neutrals

in light gray, charged in white, and with the two

hemes (indicated by He at each propionate) in a

lighter gray with attached propionate oxygens seen

as light atoms. Key residues are indicated. The circled

numbers indicate ion-pairing sites between inter-

faces,

and the black dot in the lower left quadrant

gives the point of rotation between interfaces. See

text for discussion. (Protein Data Bank, Structure

File,

1A3N.)

respect to the a^p^ dimer are indicated by the

circled numbers 1 and 2, as defined in Figure

7.18, which represents the face of the a^P^

dimer. A view of the interaction of the two

dimers becomes possible by making a trans-

parency of Figure 7.18. The transparent copy is

placed exactly over the original, turned over,

rotated by 90 degrees clockwise, and adjusted

until the two circles with 2 in them exactly

overlay. The transparency now becomes the

a^P^ dimer. This arrangement approximates the

association between the two faces for deoxyhe-

moglobin. Note that H97 of the p-face aligns

between residues P44 and T41 of the a-face of

the a^p^ dimer. On going from the T state of

deoxyhemoglobin to the R state of oxyhemo-

globin, a rotation occurs, centered at the black

274

Biology Thrives Near a Movable Cusp of Insolubility

dot, such that H97 now aligns between residues

T41 and T38. This rotation, otherwise indicated

in Figure 7.10, represents the quaternary struc-

tural change between the a^p^ and a^(3^ dimers

that results from oxygen binding.

7.3.4.1.3 Visualizing the Relationship

Between the a^p^- and a^P^-heme Pairs

Before and After Oxygen Binding

The He labels on Figure 7.18, located at heme

propionates, indicate the widths of the a^- and

P^-hemes. With the transparency, representing

the a^P^ dimer properly placed over the a^p^

dimer as indicated above, the relationship

between the o?- and P^-hemes is shown at the

upperleft and the relationship between the a^-

and P^-hemes is shown at the lower right. Per-

forming the above indicated rotation increases

the distance between the a^p^-hemes and the

nearly equivalent a^P^-hemes. The relationship

between the a^P^ hemes is shown in Figures

7.15A and 7.16A.

7.3.4.2

Looking Directly into the o^pf

(of^) Interheme Crevasse also to See the

Classic Intersubunit Interactions That

Change on Oxygen Binding

Having gained a sense of the three-dimensional

relationship between the hemes and realizing

that the quaternary structural changes are

principally due to disruption of the ion pairs

involving the H146 and R141 C-terminal

carboxylates, we now look for the relationship

of these functional groups to the a^P^ (oc^P^)

interheme hydrophobic crevasse. For that

purpose. Figure 7.19 provides a look directly

into the a^p^ crevasse for the full width of that

interfacial interaction with a few of the critical

residues labeled.

7.3.4.2.1 Are the Critical Ion Pairs for

Quaternary Structural Change Within Reach

of the Heme-based Consilient Mechanism?

Importantly, the H146-K40 ion pair that oc-

curs between the a^p^ interface, located by the

circled '2's in Figure 7.18, is readily seen in the

expanded view of the a^P^ interheme crevasse

of Figure 7.19A. Also, the C-terminal carboxy-

late of the o? R141 is visible that contributes its

negative charge to the ion pair with a^ K127

(the circled 'I's interactions in Figure 7.18). The

question now becomes, can oxygenation of

a heme in the a^P^ (or the a^P^) interheme

hydrophobic crevasse weaken these ion pairs

that are thought to trigger the quaternary

structural changes considered responsible for

the interaction between a^P^- and a^P^-heme

pairs? To do so by the consilient mechanism

would seem to require intervening "waters of

Thales."

7.3.4.2.2 Demonstrating the Presence of the

"Waters of Thales"

Figure 7.19B shows the observed "waters of

Thales" when the chains are given in space-

filling representation. The observed water mol-

ecules in a crystallographic structure represent

a minimal number of water molecules actually

present, as many more waters are too mobile to

be positioned by X-ray diffraction. Conversion

of the chains in Figure 7.19 to the stick repre-

sentation in Figure 7.20 allows delineation of

the unseen residues in Figure 7.19. Further-

more, the stick representation allows for

three-dimensional visualization of all of the

dif-

fraction-detected waters in this region. From

Figure 7.20B with the detected water mole-

cules,

it is not unthinkable that the consilient

mechanism might play a role in the relaxation

of these critical ion pairs and thereby con-

tribute to the communication between a^P^-

and a^P^-heme pairs that facilitates oxygen

binding. After all, formation of oxyhemoglobin

occurs with the uptake of water, in our view, as

ion pairs and hydrophobic regions dissociate.

Figure 7.21 gives an expanded view of the part

of the p^-chain that undergoes disruption of

ion pairs and hydrogen bond. Figure 7.21 can

also facilitate identification of residues in

Figure 7.20 without the distraction of so many

labels.