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Considerations on the Tertiarv Structure of Proteins
H. K. SCHACHMAN
Biochemistry and Virus Laboratory, University of California, Berkeley, California
INTRODUCTION
tion that many of the larger protein molecules are
composed of two or more polypeptide chains linked
in some cases by covalent bonds and in others noncovalently. In general there is a correlation between the number of"active sites" and the number
of subunits (or chains) in the protein, but in at
least one enzyme (aldolase) the cooperative folding
of three polypeptide chains is apparently required
to make one "active site".
In this communication an attempt is made to
survey the present state of knowledge of the gross
morphology of proteins along with the nature of
the forces responsible for the maintenance of the
specific architecture. The diversity of proteins will
be illustrated with a few specific, well studied
systems. Finally, attention is drawn toward the
problem of the acquisition of the three dimensional
structure of the polypeptide chains in those proteins composed of more than one subunit. Owing
to space limitations in this volume, the discussion
of various problems will be brief and references to
the pertinent literature are included for more
detailed accounts.
Current research in molecular genetics and protein biosynthesis has stimulated renewed interest
in investigations of the folding of disorganized
polypeptide chains to produce biologically active
protein molecules with unique speeificities and
three-dimensional conformations. According to
present views the characteristic secondary and
tertiary structures of native protein molecules are
the direct consequence of the sequential arrangement of the amino acids in the polypeptide chains.
In support of this view, examples can be cited
where the disorganized polypeptide chains produced
by the denaturation of proteins were transformed
readily into biologically active macromolecules
having the properties and architecture characteristic of the native proteins.
With many proteins, however, the attempts to
convert the disordered, and perhaps randomly
coiled, polypeptide chains into the specific conformations existing in the naturally occurring
maeromolecules have been unsuccessful. These
latter experiences coupled with the seeming improbability ofeffecting in vitro the specific refolding
of random chains have led to the view that protein
denaturation is an irreversible process. In order to
account for the apparent irreversibility of denaturation of many proteins, it is imperative that both
kinetic and thermodynamic barriers to the reversal
be considered and distinguished. It may be, for
example, that the reformation of the native structure is feasible on thermodynamic grounds but
the rates of the proper refolding reactions are
interminably slow. Accordingly, it is important to
evaluate, on theoretical grounds, whether the
native conformation of a protein represents the
thermodynamically stable form of lowest free
energy.
Determining the relative stabilities of the multitudinous conformations of polypeptide chains in
water is a formidable task, and it is not likely that
we will soon have a satisfactory quantitative theory
that takes into account all the various types of
interactions which are involved. But meanwhile,
considerable progress has been made in the development of a qualitative treatment of protein stability.
Attempts at formulating an all-embracing theory
have been complicated further by the demonstra-
THE MORPHOLOGY OF PROTEINS
IN SOLUTION
For many years it has been common practice to
treat hydrodynamic data for solutions of macromolecules in terms of three types of models. The
first of these represented by the so-called globular
proteins considers the kinetic units as rigid, impenetrable ellipsoids (prolate or oblate) of revolution having modest axial ratios. Solutions of such
macromolecules are only slightly more viscous than
the pure solvent. But other proteins and biological
macromolecules when dissolved in water cause a
very large increase in the viscosity of the liquid
and they experience much greater frictional resistance (per unit weight) during their movement
(both rotationally and in a translational sense)
through a viscous fluid. For these apparently rigid
particles a rod-like model frequently has been
employed. The experience of polymer chemists
with the physical chemical behavior of synthetic
polymers has led to the introduction of an additional model known as the random coil. With these
materials neither the attractive forces responsible
409
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410
SCHACHMAN
TABLE 1. ~NTRI~SIC VISCOSITIES OF MACROMOLECULES
Compact globular particles
Mol.Wt.
Randomly coiled chains
Mol.Wt.
[~]
cc/g
Polystyrene Latex
Particles
Ribonuclease
Lysozyme
Myoglobin
fl.Lactoglobulin
10~
13,700
14,400
1%000
35,000
2.4
3.3
3.0
3.1
3.4
[y]
Rod.like particles
Mol.Wt.
ce/g
Polystyrene in Toluene
Polystyrene in Toluene
Reduced Ribonuclease
Oxidized Ribonuelease
Oxidized Ribonuclease in
45,000
70,000
13,700
14,100
14,100
28
37
14.4
11.6
13.9
44,000
66,000
34
22
66,000
53
200,000
93
1.5 • 10e
100
5 • 10e
150
[~]
ce/g
Fibrinogen
Collagen
Myosin
DNA
TMV
330,000
27
345,000 1150
620,000 230
5 x 10e 5000
4 • 10T
29
Urea
Ovalbumin
Serum Albumin
Hemoglobin
44,000
65,000
67,000
4.0
3.7
3.6
Liver Alcohol
Dehydrogenase
Hemerythrin
83,000
10%000
4.0
3.6
Aldolase
142,000
3.8
Ribosomes (Yeast)
Bushy Stunt Virus
Ovalbumin in Urea
Serum Albumin in Urea
Reduced Serum Albumin
in Urea
Myosin in Guanidinehydroehloride
RNA
Heat-Denatured DNA
3.5 x 10s 5.0
8.9 x l0 s 4.0
for the compactness of the globular particles nor
the special structural features which confer rigidity
on the elongated units are present, and the flexible
chain-like molecules coil almost at random.
To illustrate the behavior of different materials,
viscosity data for a variety of macromolecules are
assembled in Table 1 and grouped in the three
categories outlined above. It is clear that most of
the enzymes and many other biologically active
proteins and particles exist in aqueous solution as
compact particles almost spherical in shape. This
is true for proteins as small as ribonuclease of
molecular weight 13,700, and as large as aldolase
of molecular weight 142,000. As expected theoretically on the basis of the Einstein viscosity equation, the intrinsic viscosity is independent of
molecular weight even for large particles such as
the yeast 80S ribosomes and bushy stunt Virus.
For the rod-like particles represented by the
fibrous proteins, fibrinogen, collagen, and myosin
the intrinsic viscosity varies considerably since it
is a function of the square of the axial ratio of the
hydrodynamic units. In between these two classes
of rigid particles are listed a group of materials
with hydrodynamic properties characteristic of
flexible, randomly coiled chains. For these, the
intrinsic viscosity is dependent on the molecular
weight in a manner which varies slightly with the
degree of rigidity (or flexibility) and permeability
of the coils.
According to the Einstein equation, the intrinsic
viscosity of solid, spherical particles placed in a
continuous medium is 2.5. This requires that the
concentration of the particles be expressed on the
basis of volume fraction. Converting the figures in
Table 1 to this basis (except for the latex particles)
is hazardous because of the lack of knowledge of the
effective hydrodynamic volumes of the macromolecules. Nonetheless, it is clear that the volume intrinsic viscosities of the globular particles are only
about twice the Einstein value. This could be
interpreted in either of two ways. Either the kinetic
units are slightly anisometric with axial ratios of
about 3 to 1; or, alternatively, the particles are
essentially spherical but swollen slightly with
solvent to give a volume slightly greater than that
which would result if the polypeptide chains were
folded into tight balls containing no solvent. For
aldolase the intrinsic viscosity, if interpreted on the
basis of a spherical model, indicates that the radius
of the hydrodynamic unit is only 25% greater than
that for a solid, uniform sphere of the same weight
and density.
When the constraint of the folding of the polypeptide chains is removed by the action of denaturing agents like urea, the intrinsic viscosity of
globular proteins increases to values similar to
those found for random coils of corresponding
molecular weight. Evidence for the constraint imposed on the polypeptide chain is illustrated by
the viscosity changes for serum albumin. In the
native molecules both non-covalent and covalent
bonding lead to a compact structure having an
intrinsic viscosity of 3.7 cc]g. Rupturing the noncovalent crosslinking bonds with urea leads to an
increase to 22 cc/g. But a further increase in the
intrinsic viscosity results if the residual constraint
is removed by the rupture of the many disulfide
bonds linking remote regions of the polypeptide
chain. This is seen by the substantial increase to
53 cc/g when cysteine is added to the protein in
urea (Kauzmann, 1954). Similar results are found
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CONSIDERATIONS ON THE TERTIARY STRUCTURE OF PROTEINS
with ribonuclease (Harrington and Schellman,
1956; Ginsburg and Schachman, 1960).
It should be noted that the rigid elongated
particles represented as rod-like particles in Table
1 collapse into more random macromolecules of
much lower intrinsic viscosity when the inter- and
intra-chain forces are destroyed by denaturing
agents. This is shown both with myosin (Young
et al., 1962) and DNA (Dekker and Schachman,
1954).
LEVELS OF ORGANIZATION
Proteins are generally considered to have three
levels of structure--primary, secondary, and
tertiary--although it is recognized that for some
proteins no clear distinction or demarcation can be
made between the latter categories. Nonetheless,
this division (Linderstrom-Lang, 1952; Lumry and
Eyring, 1954), even if vague or arbitrary for
certain proteins, is of value conceptually in discussions not on]y of the structure and stability of
proteins but also of the progressive stages in the
biosynthesis of active protein molecules.
The first level of organization and biosynthesis,
termed primary structure, deals with the sequential
arrangement of amino acid residues in the polypeptide chains. Elucidation of this structure involves the determination of nearest neighbor
relationships. In the folding of polypeptide chains
to form the three dimensional structures characteristic of native proteins, near neighbors in the
chain (such as the 1st and the 4th, the 2nd and the
6th, the 27th and the 30th, and so on) frequently
bear a fixed spatial orientation and location relative
to one another. This level of organization, termed
secondary structure, can be accounted for in terms
of bond distances, bond angles, the effect of
restricted rotation, and non-covalent bonds. Such
intra-ehain structure is illustrated best by the
a-helix (Pauling et al., 1951) which is present in
many fibrous proteins, polypeptides in organic
solvents, and in some proteins in crystalline form
and in aqueous solutions. To account for the folding
of polypeptide chains into compact, globular
molecules, it is necessary to consider another level
of organization termed tertiary structure. This
deals with the spatial relationships among remote
segments of the same polypeptide chain or even
different chains and can be considered as depicting
inter-chain structure. Intra-chain structure involving large loops would also be classified as tertiary
structure. It should be recognized that the formation of a stable tertiary structure may preclude
the existence of local secondary structure in some
cases; whereas in other systems the forces responsible for the secondary and tertiary structures may
411
reinforce one another to give added stability to
both.
According to this formulation of the various
aspects of the structure of proteins, no restrictions
need be made about the nature of the forces responsible for the stability of the secondary and
tertiary structures. Covalent bonds as well as noncovalent bonds may be involved. Among the
former only disulfide bonds have been shown to
be of importance. Often this bond is responsible,
in part, for erosslinking remote regions of a polypeptide chain as in ribonuelease, lysozyme, trypsin,
bovine serum albumin, alkaline phosphatase, and
?-globulin; but in addition it serves as a link
between different polypeptide chains as in insulin
and 7-globulin. For these proteins the disulfide
bond contributes to the tertiary structure. In ~,globulin there appear to be many intra-chain
disulfide bonds and several inter-chain bonds
(Porter, 1962). The A chain of insulin also contains
a disulfide bond linking the half cystine residues,
6 and 11 (Sanger, 1956). In this case the disulfide
bond may be considered part of the secondary
structure; it should be noted that the constraint
imposed on the polypeptide chain by that covalent
bond probably precludes the formation of an ahelix in that region of the chain. Evidence for the
existence of other types of covalent crosslinks, such
as ester bonds, peptide bonds, and phosphodiester
bonds, is so meagre that they seldom are considered
of importance in proteins. It should be noted, however, that their presence (if they exist at all) is likely
to be difficult to detect because of their rarity.
No information is available which would permit
distinguishing separate phases in the folding of
newly synthesized polypeptide chains to give first
a secondary structure followed by the tertiary
structure. Indeed, some folding may even be occurring before the entire chain is synthesized.
Despite the rapidity of the fluctuations in the conformation of the disorganized chain it is not unreasonable to assume that interactions among
neighboring regions of the chain cause local order
such as the :c-helix, and that the folding back of
the chains or their association to give the tertiary
structure is done subsequently. In this respect it
is of interest to note that the pairing of the eight
half cystine residues in ribonuclease to form the
four disulfide bonds indicates that the formation
of these bonds occurred only after the complete
polypeptide chain was synthesized. Also, folding
back of the chain must have occurred in several
places in order for the appropriate sulfhydryl
groups (reading from the N-terminal end of the
chain 1 and 6, 2 and 7, 3 and 8, 4 and 5) to be
located in the proper spatial orientation for disulfide bond formation.
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412
SCHACHMAN
The findings regarding the structure of hemoglobin (Perutz, 1962) are also relevant in terms of
speculations about the sequence of the folding
processes. Both the cr and /~ chains within intact
hemoglobin molecules exist in conformations
remarkably similar to the polypeptide chain in
myoglobin (Kendrew, 1962). This occurs even
though there are substantial differences in amino
acid composition and sequence among the three
types of chains. Despite the similarity in the local
folding (secondary structure) in the chains of hemoglobin and myoglobin, the hemoglobin molecule
assumes a tertiary structure with two ~ chains and
two fl chains packed into a compact unit while
myoglobin exists in solution predominantly as
single chain molecules. It is as if the single folded
myoglobin chains are best satisfied (thermodynamically) by interacting with the solvent, whereas the
folded :r and fl chains of hemoglobin have a surface
topography which favors chain association to
produce stable four chain molecules.
Other examples illustrating a progression of
folding events stem from reconstitution studies on
phosphorylase (Madsen and Gurd, 1956) and aldolase (Stellwagen and Schachman, 1962; Deal et al.,
1963). From these preliminary investigations it
appears as if local folding of the individual separate
polypeptide chains is followed by association of the
chains (four in the case of phosphorylase and three
in aldolase) and then a final stage involving an
annealing process leading to stable enzymically
active molecules.
Since many of the compact globular proteins
enumerated in Table 1 do not possess disulfide
bonds, their folding into stable conformations must
be attributed to non-covalent bonds. The status of
knowledge regarding such bonds is summarized in
the next section with particular emphasis on the
evaluation of the thermodynamic stability of
native proteins as compared to other chain
conformations.
NON-COVALENT ATTRACTIVE FORCES
AND T H E STABILITY OF PROTEINS
After several decades of absolute reliance on the
peptide hydrogen bond as the ~ine qua n o n of
native proteins, the pendulum has now swung so
far that this bond is now being discounted as an
important force contributing to the stability of
folded polypeptide chains in globular proteins.
Taking its place as the principal (non-covalent)
attractive force stabilizing the folded chains is the
apolar or hydrophobic bond. These bonds, as well
as side chain hydrogen bonds and ionic bonds, are
considered briefly in this section along with a
qualitative treatment of the thermodynamic sta-
bility of folded polypeptide chains in native
proteins. For detailed discussions of these problems
the reader should consult recent reviews emphasizing different points of view (Kauzmann, 1959a;
Klotz, 1960; Tanford, 1962a; Ndmethy and
Scheraga, 1962c). Although there have been rapid
strides both theoretically and experimentally, our
understanding of the forces responsible for the
stability of proteins is still woefully inadequate.
The results with model compounds or individual
proteins lead frequently to the temptation to
ascribe the strategic role in stabilizing protein
conformations to one or another type of noncovalent bond. Despite the appeal of individual
arguments, it seems prudent at this juncture to
consider all types of bonds, individually weak as
they are, as acting cooperatively to stabilize the
folded polypeptide chains.
HYDROGEN BONDS
Peptide hydrogen bonds between the amide
hydrogen of one residue and the carbonyl oxygen
of another are the only types of non-covalent bonds
that can be formed in very large numbers (two per
amino acid residue) to produce folded structures
with extensive regularity. Since the energy of an
N - - H . . . . O ~ C hydrogen bond was estimated to
be of the order of 8 kcal/mole, it was taken for
granted in early considerations of the folding of
polypeptide chains in proteins that virtually all
peptide hydrogen and oxygen atoms would participate in hydrogen bonding (Pauling et al., 1951).
Otherwise the coiled chains could not be stable in
solution. Indeed, in experiments with synthetic
polypeptides in non-polar solvents it is found that
stable hydrogen bonded structures of the type
described by Pauling et al. (1951) do form spontaneously and do exhibit considerable stability.
But reservations must be attached to any conclusions regarding the crucial role of hydrogen bonds
in proteins (in aqueous media ) unless the effect of
the water is explicitly accounted for in estimates
of the value for the intrinsic strength of the
hydrogen bond. As Kauzmann (1954) pointed out,
the energy required to rupture interpeptide hydrogen bonds in water is much smaller than 8 kcal/
mole, since the process involves an exchange
reaction in which water-water hydrogen bonds are
also ruptured and two water molecules participate
in forming two new hydrogen bonds with the N - - H
and C ~ O groups.
The first reasonably satisfactory value for the
heat of formation of a peptide hydrogen bond in
water came from the theoretical studies of Schellman (1955b) aimed at evaluating the stability of
hydrogen bonded polypeptide helices in aqueous
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CONSIDERATIONS
ON
THE
TERTIARY
HYDROGEN BONDING IN AGGREGATION OF N-METHYLACE'[AMIDE
IC
l
OE
Q(
a
0.4
0.2
QOt
O~
-2
-I
MOLARITY NMA
kO
IO
0
I
LOG (NMA)
FIGURE 1. V a r i a t i o n of degree of association, :r of Nm e t h y l a c e t a m i d e (NMA) as a f u n c t i o n of c o n c e n t r a t i o n in
t h e solvents, c a r b o n tetrachloride, dioxane, a n d water. T h e
degree of association w a s calculated f r o m t h e infra-red
a b s o r p t i o n at 1.48/~ ( F r o m K l o t z a n d F r a n z e n , 1962).
solutions. From an analysis of the thermodynamic
properties of urea solutions, Schellman (1955a)
calculated the heat required to rupture a hydrogen
bond to be only 1.5 kcal/mole, a value substantially
less than that used in the earlier considerations.
But even this value, though far more reliable than
the previous estimate, is not entirely satisfactory
since it is based on indirect calculations requiring
hazardous assumptions.
Recently an attempt has been made to determine
more directly the heat of formation of a peptide
hydrogen bond from studies of the aggregation of
the model substance, N-methylacetamide, in water
and other solvents. Upon the formation of intermolecular hydrogen bonds there was a shift in the
near infra-red absorption spectrum which permitted
Klotz and Franzen (1962) to calculate the amount
of interamide hydrogen bonded aggregates as a
function of concentration. Their results, presented
in Fig. l, show that aggregation occurs readily in
carbon tetraehloride even at low concentrations of
N-methylaeetamide. In dioxane the tendency for
the solute to form aggregates is much less; and in
water hydrogen bonded aggregates form only in
extremely concentrated solutions. These results
show clearly the crucial role of solvent molecules in
competing for the hydrogen bonding groups in the
solute molecules. Even when there are only about
four water molecules for each N-methylacetamide
molecule the bulk of the solute molecules are not
aggregated. From spectral studies at different temperatures, Klotz and Franzen were able to estimate
the intrinsic strength of the hydrogen bond as
measured by the enthalpy change (AH) on the
formation of the bond. For N-methylacetamide in
carbon tetraehloride AH for hydrogen bond formation was --4.2 kcal/mole in agreement with other
determinations for this particular compound in
benzene solutions. The value of AH for the dioxane
solutions was -0.8 kcal/mole; and in water AH
STRUCTURE
OF
PROTEINS
413
for hydrogen bond formation between N-methylacetamide molecules was virtually zero.
Although the calculations of Schellman, and
more particularly the experimental results of Klotz
and Franzen, indicate that the intrinsic strength of
interpeptide hydrogen bonds in water is extremely
small, it is not appropriate to conclude that hydrogen bonds do not contribute significantly to the
stability of the secondary and tertiary structures
of proteins dissolved in water. As Klotz and Franzen
(1962) and N~methy et al. (1963) point out, the
interior of a protein molecule is likely to have a
high concentration of hydrocarbon-like residues
and differ substantially from the aqueous environment experienced by the model compounds. If the
hydrogen bonding groups, both in the bonded and
unbonded states, are in regions of low dielectric
constant and relatively free of water, then the
strength of the interpeptide hydrogen bond would
be greatly enhanced. Under these circumstances
marked stabilization would accrue from the formation of such bonds. Moreover, the local concentration of peptide bonds in the interior of protein
molecules is very high (Klotz, 1960). This would
tend to favor hydrogen bond formation.
In summary, the status of the concept that the
hydrogen bond is the dominant non-covalent force
stabilizing folded protein structures has been shaken
during the past few years. But it would be shortsighted indeed to dismiss it as unimportant. Myoglobin and hemoglobin in crystalline form as well as
in aqueous solutions contain large amounts of helical regions involving intra-chain peptide hydrogen
bonds. Other proteins such as lysozyme, chymotrypsinogen, and ribonuelease apparently do not. It is
clear that other factors must be reckoned with and
that the wide diversity in structures among protein
molecules must be attributed to cooperative interactions involving different types of non-covalent
bonds rather than only a single type of attractive
force.
APOLARoR HYDROPHOBICBOND
Many of the amino acids found in proteins have
non-polar side chains such as the methyl group of
alanine, the isopropyl group of valine, the secondary
butyl group of lencine, the mercaptomethyl group
of eysteine, and the benzyl group of phenylalanine.
Since about 40~ of the total amino acids in most
proteins have non-polar side chains, it is to be
expected that the interactions and physical chemical properties exhibited by such groups would affect
the behavior of polypeptide chains and influence
their folding to produce characteristic secondary
and tertiary structures.
This was first pointed out by Kauzmann (1954)
who introduced the term hydrophobic bond to
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414
SCHACHMAN
represent the attractive forces responsible for the
tendency of the non-polar residues to avoid contact
with the aqueous phase and to adhere to one
another in the form of an intramolecular micelle.
Reasoning from thermodynamic data for solutions
of hydrocarbons in non-polar and polar solvents,
Kauzmann (1959a) made estimates of the strength
of this hydrophobic bonding and concluded that
the most stable conformations ofpolypeptide chains
would be those in which the non-polar side chains
are in contact with each other in the interior of
protein molecules where there is relatively little
water.
An alternative view has been formulated by
Klotz (1958, 1960) who very early stressed the
effect of the interactions between the non-polar
side chains and the water in determining the conformation of polypeptide chains in proteins. But
this view, though based on the same thermodynamic
studies of hydrocarbons in water, is markedly
different from that proposed by Kauzmann since
an alternative model system was employed in its
development. According to Klotz (1960), non-polar
side chains form crystalline hydrates with water
(just as do many hydrocarbons) and the coalescence
of these hydration "icebergs" produces stable icelattices in which the non-polar residues, rather than
being on the interior of the protein molecules, are
at the exterior in contact with the solvent.
Despite the divergence of the views of Kauzmann
and Klotz, both stress the importance of the nonpolar side chains in stabilizing tertiary structures.
Both attribute the effect of these groups to the
unique properties of water and to the unusual
interaction of the non-polar groups with water. The
disagreement is over the nature of the apolar or
hydrophobic bond, which is a theoretical problem.
Their differences stem from the alternative models
which were employed in the attempts to extrapolate
from the observed properties of the small molecules
to the expected behavior of macromolecules which
contain the non-polar groups as side chains on the
polypeptide backbone.
As evidence for the view that crystalline ice-like
lattices can form around non-polar side chains in
proteins, Klotz (1960) cites the isolation of crystalline hydrates of a variety of hydrocarbons and other
apolar molecules (Stackelberg and Miiller, 1954).
These structures, such as the crystalline hydrate
of tetra (n-butyl) ammonium bromide, contain as
many as 32 water molecules per tetrabutylammonium bromide molecule and have melting points
much higher than that of ordinary ice. Figure 2A
shows a model of a pentagonal dodecahedron
representing the crystalline hydrate of an apolar
molecule. According to this model, based on X-ray
diffraction studies the apolar molecule fits into the
FIGURE 2A. Model of pentagonal dodecahedron formed
in crystal hydrates of non-polar molecules. Each ball
represents a water molecule and the non-polar molecule
(not shown) would be in the center of cage-like structure.
B. Multiple polyhedra cage structure formed from the
dodecahedra shown in A. This represents the large ice-like
crystalline lattice of crystalline hydrates of non-polar
molecules. (From Klotz, 1960.)
hole in the sponge-like structure. In addition,
larger lattices of these apolar hydrates can be
formed by cooperative effects leading to stable
multiple polyhedra as shown in Fig. 2B. The
stability of these crystalline hydrates depends on
interactions among the water molecules themselves
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CONSIDERATIONS ON T H E T E R T I A R Y STRUCTURE OF P R O T E I N S
TABLE 2. HEATS OF HYDRATION OF
APOLAR HYDRATES*
Apolar molecule
--AH
16.6
13.9 -- 16.5
16.0 -- 17.7
16.5
15.0
16.6--19.0
CH4
14.5 -- 17
15
15
15
15--18.1
16.6
CsH 2
C2H4
C~H 6
CHaC1
CH3SH
water as illustrated b y the positive free energy
change for the process. Other things being equal,
the hydrocarbons would leave the aqueous phase
for the non-polar environment. I t should be noted
that the transfer of these apolar molecules to the
aqueous phase is favored energetically (as shown by
the negative value for the enthalpy change). However, there is invariably a large decrease in the
entropy resulting, no doubt, from the organization
of water molecules about the apolar solutes.
According to this model system the driving force
for the transfer of non-polar side chains to the
interior of the protein molecules arises from th.e
gain in entropy resulting when these side chains
leave the aqueous phase and the water becomes
more disordered.
This approach has now been the subject of more
detailed theoretical investigations by Ndmethy and
Scheraga (1962c), who extended the treatment to
account for the interactions not only between
freely moving small non-polar molecules but also
among analogous residues attached as side chains
to a polypeptide backbone. Their approach, illustrated in Fig. 3 for the interaction between two
side chains, is based on a statistical thermodynamic
treatment of pure liquid water (Ndmethy and
Scheraga, 1962a) and aqueous hydrocarbon solutions (N~methy and Scheraga, 1962b). In estimations of the free energy change accompanying the
formation of an apolar (or hydrophobic) bond,
allowances were made for the changes in the water
structure, the intermolecular forces between the
groups, and the restrictions in rotation about single
bonds resulting from the interaction. These calculations showed, in agreement with the position stated
earlier by Kauzmann (1954, 1959a), that the crucial
aspect of the bond in terms of its strength stems
from the change in the structure of water. The socalled van der Waals (London dispersion) forces
~ keal/mole
Ar
Kr
Cl~
H~S
PH 8
SO2
415
* From Klotz (1960).
and not on specific interactions between the apolar
molecule and water. Evidence for this conclusion is
summarized in Table 2, which shows that the heat
of hydration of apolar hydrates is remarkably constant despite the wide variation in the chemical
properties of the substances included in the list.
This is to be contrasted with the hydration energy
of ions which vary markedly with both the charge
and the size of the ions. Although there is a large
(unfavorable) entropy decrease (AS ~ 0) in the
formation of crystalline inclusion compounds of
non-polar molecules, the decrease in the enthalpy
(AH ~ 0) is sufficiently great to compensate for
the entropy change and to cause the (favorable)
decrease in free energy (AF ~ 0) required for
stabilization of the hydrates; i.e., A F - ~ A H TAS is negative for the reaction.
In formulating his view of the hydrophobie bond,
Kauzmann (1959a) considered the thermodynamic
data for the transfer of hydrocarbons from an
apolar solvent to water (Frank and Evans, 1945).
These data, summarized in Table 3, show clearly
that these hydrocarbons have a low affinity for
TABLE 3. THERMODYNAMIC CHANGES IN THE TRANSFER OF HYDROCARBONS FROM A I~ON-POLAR
SOLVENT TO WATER S
Temp.
(~
AS
(e.u.)
AH
(cal/mole)
AF
(cal/mole)
CHa in benzene -~ CH4 in H~O
CH4 in ether -~ CH4 in H~O
CH4 in CC14-~ CH4 in H~O
C2He in benzene -~ C2Hs in H~O
C~H6 in CCI~--~ C~H6 in H~O
298
298
298
298
298
--18
--19
--18
--20
--18
--2800
--2400
--2500
--2200
--1700
+2600
+ 3300
+2900
~-3800
T 3700
C2H4 in benzene --~ C2Ha in H20
C2H~ in benzene -* C~H~ in H~O
Liquid Propane -~ C3H8 in H20
Liquid n-butane -~ C~Hlo in H~O
298
298
298
298
-- 15
--7
--23
--23
-- 1610
--190
--1800
--1000
+2920
+ 1870
+ 5050
+ 5850
Liquid
Liquid
Liquid
Liquid
291
291
291
291
--14
--16
--19
--20
0
0
0
0
+4070
+4650
+ 5500
+5800
Process
benzene --* Cell s in H20
toluene --~ CTHs in H20
ethyl benzene -* CsH10 in H20
m- or p-xylene --~ CsHlo in H20
* From Kauzmaml (1959a).
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416
SCHACHMAN
I
!
CH3
CVl~, CH3
FIOURE 3. Schematic representation of the formation of a
hydrophobic bond between two isolated side chains
(alanine and leucine). The bond is formed through the
approach of the two side chains until they touch, with a
consequent reduction in the number of nearest water
neighbors. The extent of contact shown here is less than
the maximum obtainable for the two side chains. Water
molecules are shown only schematically without indicating
particular orientation or hydrogen bonded networks.
(From N6methy and Scheraga, 1962c).
c o n t r i b u t e o n l y a v e r y small a m o u n t to t h e overall
e n e r g y o f the h y d r o p h o b i c bond. W e r e it n o t for the
unique p h y s i c a l p r o p e r t i e s o f liquid w a t e r a n d the
t e n d e n c y for w a t e r molecules to exist as h y d r o g e n b o n d e d clusters, the i n t e r a c t i o n o f n o n - p o l a r groups
w o u l d be so w e a k as to be insignificant as a
stabilizing force for the folded p o l y p e p t i d e chains.
T A B L E 4. F R E E E N E R G Y C H A N G E FOR T R A N S F E R
FROM E T H A N O L TO W A T E R AT 2 5 ~
AF
Whole molecule
cal/mole
Af
Side chain
contribution
cal/mole
Glycine
Alanine
Valine
Leucine
Isoleucine
1)henylalanine
Proline
Non-polar side chains
-- 4630
-- 3900
-- 2940
-- 2210
-- 1690
-- 1980
-- 2060
0
+ 730
+ 1690
+ 2420
+ 2970
+2650
+ 2600
Methionine
Tyrosine
Threonine
Serine
Asparagine
Glutaminc
Aspartic acid
Glutamic acid
Other side chains
-- 3330
-- 930
-- 4190
-- 4590
-- 4640
-- 4730
-- 4090
--4080
+ 1300
+ 2870
+ 440
+ 40
-- 10
-- 100
+ 540
+550
Contribution of a CH 2 group
Ethane
+ 3020
Methane
+ 2260
Ethane-methane
-Alanine-glycine
-Leucine-valine
-* From Tanford (1962b).
--+ 760
+ 730
+ 730
I n t h e i r t r e a t m e n t , N 6 m e t h y a n d Scheraga
(1962e) included t h e results of calculations o f t h e
t h e r m o d y n a m i c p a r a m e t e r s for t r a n s f e r r i n g t h e
v a r i o u s t y p e s o f a m i n o acid side chains from w a t e r
to a n o n - p o l a r e n v i r o n m e n t . F o r leueine, isoleucine,
a n d valine the free e n e r g y change was - - 2 kcal/mole
a n d the e n t h a l p y a n d e n t r o p y changes were a b o u t
+ 2 kcal/mole a n d 14 e n t r o p y units, respectively.
These changes in free e n e r g y a n d e n t r o p y are
s l i g h t l y less t h a n those e s t i m a t e d b y K a u z m a n n
(1959a) from t h e r m o d y n a m i c d a t a for h y d r o carbons. The slight differences m i g h t be a t t r i b u t e d
t o t h e limitations in t h e clustering o f w a t e r molecules a r o u n d a non-polar group, when the group is
p a r t o f a macromolecule, as c o n t r a s t e d to the same
group in a freely moving small molecule. This
restriction was t a k e n into account b y N 6 m e t h y a n d
Scheraga (1962c), whereas no allowance for it was
m a d e b y K a u z m a n n (1959a).
A n o t h e r e s t i m a t e o f the s t r e n g t h of h y d r o p h o b i c
b o n d s comes from t h e work o f T a n f o r d (1962b) who
used the procedures described b y Cohn a n d E d s a l l
(1943) for calculating values for the free e n e r g y
change i n v o l v e d in the t r a n s f e r o f various amino
acid side chains from e t h a n o l to water. The results,
shown in Table 4, were b a s e d on the suggestion o f
Cohn a n d E d s a l l t h a t t h e t o t a l free e n e r g y change
can be a c c o u n t e d for as the sum o f constant cont r i b u t i o n s from various p a r t s o f the a m i n o acid
molecules. The r e a r r a n g e m e n t o f the molecule
H2N--CO--CHR--0H
to + H a N - - C H R - - C O O - ,
for e x a m p l e , decreases t h e free e n e r g y o f t h e transfer b y a b o u t 3,700 cal. As seen in the lower p a r t o f
t h e t a b l e which r e p r e s e n t s d a t a on hydrocarbons,
a CH 2 group increases the free energy o f transfer b y
a b o u t 730 cal. B y c o m p a r i n g the values for the
v a r i o u s a m i n o acids with t h a t for glycine, t h e free
e n e r g y changes in t r a n s f e r r i n g the side chains are
calculated directly. The a g r e e m e n t f o u n d for the
h y d r o c a r b o n side chain in Manine w i t h t h a t for the
CH 2 group in the h y d r o c a r b o n s is striking. Also, as
T a n f o r d p o i n t e d out, similar free energies were
o b t a i n e d for the t r a n s f e r o f t h e side chain o f norleucine from a v a r i e t y o f organic solvents to water.
This s u p p o r t s the view t h a t t h e calculated values
for the n o n - p o l a r side chains are r e p r e s e n t a t i v e o f
the h y d r o p h o b i c i n t e r a c t i o n s when w a t e r is one o f
the solvents.
N 6 m e t h y a n d Scheraga (1962c) also calculated
the t h e r m o d y n a m i c change in pair-wise h y d r o p h o b i c
b o n d f o r m a t i o n b e t w e e n two isolated side chains
such as t h a t i l l u s t r a t e d in Fig. 3. The results o f such
calculations for t h e f o r m a t i o n o f a h y d r o p h o b i c
b o n d b e t w e e n leucine a n d isoleueine are shown in
Table 5. I t is a p p a r e n t t h a t t h e results are qualit a t i v e l y similar to those given a b o v e for transferring
aliphatic side chains from an aqueous e n v i r o n m e n t
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CONSIDERATIONS ON T H E T E R T I A R Y STRUCTURE OF PROTEINS
TABLE 5. THERMODYNAMIC PARAMETERS FOR FORMATION
OF A LEUCINE-ISOLEUCINE HYDROPttOBIC BOND*
t
AFo
AHo
ASo
o
kcal/mole
kcal/mole
e.u.
0
25
50
70
--0.8
--1.1
--1.28
--1.33
2.3
1.5
0.4
--0.6
11.5
8.6
5.1
2.1
* F r o m N ~ m e t h y a n d Scheraga (1962c).
to a non-polar medium. The decrease in free energy
due to the interaction is about 1 kcal/mole despite
the unfavorable (positive) enthalpy change. As in
the other types of processes, the decrease in the
ice-like clusters accompanying the interaction produces a large positive entropy change which more
than offsets the positive enthalpy change.
It should be noted that these thermodynamic
considerations lead to conclusions about the thermal
stability of hydrophobic bonds (Kauzmann, 1959a;
Scheraga et al., 1962). Since the transfer of hydrocarbons from water to a non-polar environment
(hydrophobic bond formation) is an endothermic
process (the enthalpy change is positive) such processes are favored by an increase in temperature.
Thus, if protein conformations were stabilized
principally by hydrophobic bonds of the type
described by Kauzmann (1959a), raising the temperature would lead to enhanced stability until
about 60~ where the enthalpy change for this
transfer is about zero (at even higher temperatures
it becomes negative). Most proteins become denatured as the temperature is raised (even before a
temperature of 500C is reached), indicating that
hydrophobic bonds of the type described here
cannot by themselves be responsible for stabilizing
the secondary and tertiary structures. If, however,
the overall stability of folded polypcptide chains
were due to the cooperative action of hydrophobic
bonds and interpeptide hydrogen bonds, then the
temperature of maximum stability would be decreased to lower temperatures and the macromolcculcs would be more readily denatured as the
temperature is raised above that point.
Ndmethy and Scheraga (1962c), like Kauzmann
(1959a), concluded that ice-like lattices around
non-polar groups (Klotz, 1960) cannot serve as
stabilizing structures in proteins. It is generally
agreed that there is a large negative (unfavorable)
entropy change when non-polar groups come in
contact with an aqueous medium due to the
ordering of the water molecules. But there is
disagreement about the magnitude of the enthalpy
term to be used in estimations of the free energy
change. Hence, opposing conclusions have been
reached as to which state is more stable thermodynamically. Klotz (1960), by using a value of
417
--15 kcal/mole for the enthalpy of formation of
crystalline gas hydrates (see Table 2), concludes
that the free energy change would indeed be negative for the non-polar side chains interacting with
water, since the favorable enthalpy change would
more than compensate for the unfavorable entropy
change. On the basis of Kauzmann's model (1959a)
of small hydrocarbon molecules in solution (or the
extension of the treatment of Ndmethy and Schcraga
[1962c] described above) the enthalpy change for
these groups coming in contact with water would
be only about --2 kcal/mole. This would be insufficient to counteract the large unfavorable
(negative) entropy change and the free energy
change (AH-TAS) as a consequence would be
positive (unfavorable).
The conflict then is not on grounds of thermodynamic considerations but rather in terms of the
models assumed to be appropriate for treatment of
the very complicated problem of the thermodynamic
behavior of the folded polypeptide chains. Plausible
arguments have been raised by Kauzmann (1959a)
and Ndmethy and Scheraga (1962c) indicating that
the crystalline ice-like lattices (which can form
under certain conditions around hydrocarbons)
would not be stable when the non-polar groups are
attached to a polypeptide chain. But the crucial
test of the validity or the inadequacy of one of the
model systems nmst await the results of other types
of investigations. According to Klotz (1960), in the
molecular domain of proteins, as in ice, a substantial
amount of water is immobilized in an ordered form
with a long lifetime. Also, the non-polar groups
would be concentrated at the exterior of the protein
molecules in order to cause the formation of the
cooperative ice-like lattices. On the other hand,
according to Kauzmann (1959a) and Ndmethy and
Scheraga (1962c) there would be little bound,
ordered water, and the non-polar side chains would
avoid contact with the aqueous medium by being
buried in the interior of the protein molecules. At
present there is information pertaining to these
alternatives for only one protein-myoglobin (Kendrew, 1962). The results of the X-ray diffraction
studies indicate that there is virtually no water
inside the compact folded structure, most of the
polar groups are on the surface, and the non-polar
residues are in the interior in close contact with one
another. Also, with the exception of bound water
molecules attached to polar groups at the surface,
no regions of ordered liquid have been detected.
Some regions of the patterns have not been investigated in detail, however. Until they are explored
thoroughly in myoglobin, as well as in other
proteins (e.g., Avey et al., 1962; Filmer and
Kaesberg; 1962) neither of the models used in the
formulation of the apolar or hydrophobic bond can
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418
SCHACHMAN
be dismissed or adopted unequivocally. Studies with
other proteins are particularly important since
myoglobin and hemoglobin seem to be unusual
when compared to the few other proteins whose
structures now are being deduced by X-ray
diffraction measurements.
THERMODYNAMICS OF FOLDING OF
P O L Y P E P T I D E CHAINS
Theoretical demonstrations that the native conformation of folded polypeptide chains is the
thermodynamically stable form of lowest free energy
must await much more detailed understanding of
the strength of the various forces discussed above
as well as an evaluation of other types of interactions such as ionic bonds and side chain hydrogen
bonds. Nonetheless, it seems profitable to examine
this question by way of summarizing existing concepts and illustrating the complexity of the problem
(For a somewhat analogous treatment see Tanford,
1962.)
Figure 4 is a schematic diagram showing the
forces involved in stabilizing the secondary and
tertiary structures of proteins and the thermoIntramolecular Non-covalent Bonds
Responsible For
Secondary and Tertiary Structures
COO.H
...R..
0
:
'.
9
i
C
I
INTERPEPTIDE
HYDROGEN
BOND
NH +
,
oc--o
"
I
SIDE CHAIN
HYDROGEN
BOND
IONIC
BOND
UNFOLDED C H A I N S .
~ NATIVE
APOLAR OR
HYDROPHOBIC
BOND
STRUCTURE
A F (col)
CONFORMATIONAL ENTROPY
,~,
INTERPEPTIDE HYDROGEN BONDS
"
APOLAR OR HYDROPHOBIC BONDS '~'
+ 15 x I 0 5
-05
x 105
- 1 . 0 x 105
SIDE CHAIN HYDROGEN BONDS
"-'
IONIC BONDS
~
-0.1
x 10 `5
,--,
-01
x 10 `5
TOTAL
-003
x 105
FIOU~E 4. Schematic diagram illustrating the types of
non-covalent bonds responsible for the maintenance of the
secondary and tertiary structures of proteins. Below are
estimates of the free energy terms involved in consideration
of the stability of the folded, native structure. A polypeptide chain of 100 residues was considered in the
calculations. Other details are in the text.
dynamic parameters that must be evaluated. In
estimating the magnitude of the various terms it
was assumed that the polypeptide chain was composed of 100 amino acid residues. No allowances
were made for the presence of crosslinking covalent
bonds such as disulfide bonds.
The unfolded structure is considered to be a
randomly coiled polypeptide chain possessing considerable flexibility and with the various side chains
in contact with the medium. Presumably it exists
in this form just at the completion of the biosynthesis of the polypeptide chains. Different
unfolded conformations have equivalent stabilities;
as a consequence, a given polypeptide chain will
experience different conformations as a function of
time and at any instant there will be a distribution
of different conformations among the population
of chains. In the folding process many fluctuations
would occur until a unique, rigid conformation is
attained which represents the native structure.
Clearly, the unfolded structures by reason of
their flexibility and equivalence of energy for the
many different conformations possess a high
entropy. In the folding process there is a great
decrease in conformational entropy which contributes an unfavorable (positive) change in free
energy (--TAS). Schellman (1955b) has dealt with
this problem in detail pointing out that an effective
number of conformations must be counted and this
number is always less than the total possible
number of conformations. This is done in recognition of the fact that not all of the possible forms
are of equivalent energy. In this way Schellman
made both high and low estimates of the change in
conformational entropy. For the former he used
the conclusions of Pauling and Corey (1952) that
there are six possible orientations about each of the
two single bonds in a peptide unit and two (cis and
trans) about the partial double bonded peptide
group (--CONH--). Of these 72 configurations onehalf were considered to be sterically improbable.
Thus Schellman counted only 36 stable conformations and calculated for the change in eonformational entropy per residue (RlnP where P is the
number of possible conformations of equal energy)
a value of about --7 entropy units. To provide a
minimum value Schellman (1955b) used data for
the configurational entropy of each single bond in
long chain paraffins. With the assumption that this
applies to the two single bonds in an amino acid
residue and no eis-trans isomers need be considered,
he arrived at --3 entropy units per residue for the
change in conformational entropy.
I f an average of --5 entropy units per residue is
used for the change in conformational entropy
when the random chain becomes folded to form the
native protein, there is an increase of free energy
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CONSIDERATIONS ON T H E T E R T I A R Y STRUCTURE OF PROTEINS
(--TAS) of 1.5 • 105 ca] (for a protein composed
of 100 amino acid residues). I f the folded polypeptide chains are to be stable despite this unfavorable
entropic contribution, there must be large negative
contributions to the free energy from the formation
of hydrogen bonds, hydrophobic bonds, and ionic
bonds.
On the basis of the discussion in one of the preceding sections, a value of --500 cal/mole was used
for the enthalpy of formation of a hydrogen bond,
thereby giving --0.5 • 105 cal as the free energy
of stabilization from the enthalpy contribution of
the interpeptide hydrogen bonds. This assumes that
about 50% of the amino acid residues were in the
form of an ~-helix involving two hydrogen bonds
per residue.
In order to provide some estimate of the free
energy of stabilization from the hydrophobic bonds,
a value of --2,000 cal/mole was used as the free
energy of formation of such bonds. It is obvious
that higher or lower estimates could be used with
equal validity. Also, it is well to bear in mind that
hydrophobic bond formation cannot be taken as
"all or none" in the native and random-coil states.
There are risks, obviously, in considering the free
energy change in transferring a single non-polar
side chain from the aqueous phase to the interior
of the molecule and then multiplying by the number
of such residues to give the total contribution of the
hydrophobic bonds. Some residues can participate
in hydrophobic bonds even in randomly coiled
chains, and not all groups can be packed perfectly
in the native structure. For the purposes of the
present approximate treatment, however, this
procedure was employed assuming 50% of the
residues had non-polar side chains. This gives
--1 • 105 cal as the free energy of stabilization
from hydrophobic bonds.
In a small protein molecule (about 100 amino
acid residues) there cannot be very many side
chain hydrogen bonds such as that formed between
tyrosine and a carboxylate ion. As a result, the
free energy of stabilization from such interactions
must be very small. For the value shown in Fig. 4,
it was assumed that there were six such bonds with
an enthalpy of formation of --500 calories.
Similarly the free energy of stabilization due to
ionic bonds is likely to be small both because the
number of such interactions is limited (see the
relative content of the charged amino acids in
proteins) and because the interaction energy itself
is not very large. The value --0.1 • 105 cal was
based on calculations of Tanford (1957) for a
variety of models containing different distributions
of charged groups.
It is clear that no unequivocal conclusions can
be drawn from the data summarized in Fig. 4, in
419
view of the crudity of some of the estimates.
Despite the uncertainties in the various values it
seems likely that the total change in free energy
for the folding of disorganized, random chains to
produce a compact structure with a unique conformation is close to zero or slightly negative.
Slight modifications in any of the estimates can
make the free energy change more favorable
(negative), but they could as well have the effect
of increasing the relative stability of the unfolded
chains9 One or more disulfide cross links would
reduce considerably the conformational entropy
of the unfolded chains (Sehellman, 1955b; Kauzmann, 1959a, 1959b) thereby enhancing the stability
of the native structures. I f it is assumed that the
above values are valid and hence that the native
structure is marginally stable from the viewpoint
of thermodynamics, then it is obvious that a change
in the environment (such as the addition ot organic
solvents, acid or alkali, or denaturants like urea or
sodium dodecyl sulfate) which alters one or more
of the different quantities could have a large effect
in making the unfolded chains relatively more
stable than the native structure.
Another approach to the evaluation of the
stability of folded structures was used by Tanford
(1962b), who considered the stabilization resulting
from hydrophobic bonds. The results summarized
in Table 6 are similar to those illustrated in Fig. 4.
The decrease in free energy in the unfolding process
due to the gain in conformational entropy was
virtually counterbalanced by the positive free
energy change required for the transfer of the side
chains from the interior of the molecule to the
aqueous medium. Once again, slight changes in
some of the estimates (Tanford used 4 entropy
units per residue for the change in conformational
entropy) could alter the balance so that the total
free energy change in the unfolding process would
become positive, thereby favoring the native
conformations.
The above discussion shows clearly that knowledge concerning the thermodynamic stability of
proteins is only superficial. Large uncertainties
exist and there is a need for a refined quantitative
treatment of the type sketched above. But the
development of such a theory is bound to prove
awesome in its complexity. It seems unlikely that
meaningful computations can be made until more
is known about the conformation of the polypeptide
chains in native proteins. Surprises can be expected
when such information becomes available, and
interactions which haven't been considered heretofore will require attention. Already in the preliminary surve.y of the polar interactions in myoglobin
(Kendrew, 1962), there have been unanticipated
findings. For example, the hydroxyl groups of serine
9
/.
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420
SCHACHMAN
TABLE 6. CONTRIBUTION OF THE MOST IMPORTANT HYDROPHOBIC INTERACTIONS TO THE
FREE
ENERGY OF UNFOLDING
AT 250$
Side chain
Afu per side chain,
cal/mole
Number present in
fl-lactoglobulin
Myoglobin
Ribonuclease
Tryptophan
Isoleucine
Tyroslne
Phenylalanine
3000
2970
2870
2650
2
9
3
6
2
10
4
4
0
3
6
3
Proline
Leucine
Valino
Lysine
2600
2420
1690
1500
4
18
8
19
8
22
10
15
4
2
9
10
Methionine
Alanine
Arginine
Threonine
1300
730
730
440
2
17
4
5
4
14
3
8
4
11
4
10
153
-- 184
+ 173
162
-- 194
+ 192
124
-- 149
+ 100
Total n u m b e r of residues
--TASconr, kcal/mole
]gAfu, kcal/mole
* From Tanford (1962b).
and threonine have been found to be hydrogen
bonded with backbone carbonyl groups a few
residues further along the chain. In some cases,
forked hydrogen bonds were formed without interfering with the regularity of the helix; but in one
case the side chain interaction with the backbone
caused a distortion of the helix (Kendrew, 1962).
Additional observations of this type are bound to
have a sobering influence on those attempting to
develop a quantitative general theory.
because of limitations in knowledge of basic para m e t e r s s u c h as t h e t o t a l w e i g h t a n d n u m b e r o f
polypeptide chains in the biologically active molecules. I n a d d i t i o n , t h e p r o t e i n s w h i c h h a v e b e e n
studied exhibit such diversity in their structural
patterns that unified concepts are not readily
apparent. Some generalizations are evolving, however, and they are presented here along with
specific e x a m p l e s ( T a b l e 7), t o i l l u s t r a t e t h e g r e a t
variations in protein structures.
T H E D I V E R S I T Y OF P R O T E I N
STRUCTURES
The formulation of generalizations about the
structure of protein molecules has been impeded
ASSOCIATION-DIssOCIATION EQUILIBRIA AND
MOLECULAR WEIGHTS
Many proteins undergo reversible association
and dissociation reactions to form equilibrium
TABLE 7. PROTEIN STRUCTURE
Protein
Molecular
weight
No. of
chains
No, of
- - S - - S - - Bonds
No. of
binding sites
Insulin
Ribonuclease
Lysozyme
Myoglobin
Papain
Trypsin
5,800
13,700
14,400
17,000
20,900
23,800
2
1
1
1
1
1
3
4
5
0
3
6
-1
-1
1
1
Chymotrypsin
Carboxypeptidase
Hexokinase
Taka-amylase
Bovine Serum Albumin
Yeast Enolase
24,500
34,300
45,000
52,000
66,500
67,000
3
1
2
1
1
1
5
0
0
4
17
0
1
1
--24
--
Hemoglobin
Alkaline Phosphatase
Liver Alcohol Dehydrogenase
Hemerythrin
Glyceraldehyde-3P-Dehydrogenase
Lactic Dehydrogenase
68,000
80,000
83,000
107,000
140,000
140,000
4
2
2
8
4
4
0
4
0
0
0
0
4
2
2
8
4
4
Aldolase
Yeast Alcohol Dehydrogenase
y- Globulin
Glutamic Dehydrogenase
Myosin
142,000
150,000
160,000
250,000
620,000
3
4
4
4
3
0
0
25
0
0
1
4
2
4
3
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CONSIDERATIONS ON T H E T E R T I A R Y STRUCTURE OF PROTEINS
,'2[ ..............
r
4
-
7o f -
~0
-~ I10
9o
"~
80
6o,
,
421
tetramer, is usually cited in discussions of the
molecular weight of glutamic dehydrogenase, but
it now seems clear that the value of 2.5 • 105 is
more appropriate. Hexokinase can be listed as
another example of this type of system. Under
many experimental conditions this enzyme exhibits
hydrodynamic and thermodynamic properties corresponding to molecules with a weight of 90 • 10a,
but in the presence of the substrate, glucose, there
is dissociation to the monomer of molecular weight
45 • 10a (Ramel and Schachman, unpublished;
Schachman, 1960).
Concentration Grams/Liter
FmURE 5. The association-dissociation properties of beef
heart lactic dehydrogenase as a function of enzyme
concentration and temperature. Filled triangles represent
results obtained at 5~, filled circles show results at 20~, and
open circles the results of the equilibrium test experiment
conducted at 20~ The lines were eMeulated for an equilibrium mixture of monomers, dimers, and trimers. (From
Millar, 1962.)
mixtures containing molecules in varying states of
aggregation. As a consequence, difficulties are
encountered in molecular weight determinations.
The values for insulin, hexokinase, lactic dehydrogenase, and glutamic dehydrogenase, for example,
have fluctuated widely not because of experimental
errors but because of changes in the proportions
of the different aggregates under the various experimental conditions. The degree of association is
influenced frequently by temperature, p H and concentration as well as by the presence or absence of
coenzymes, substrates, or specific ions. This is illustrated in Fig. 5 by the data of Millar {1962) from
studies on beef heart lactic dehydrogenase. These
experiments show that the apparent molecular
weight varies markedly with protein concentration.
At low concentrations dissociation is favored and
the molecular weight is 70 • 10a. When the concentration is increased, association occurs and molecular
weights of about 140 • 10a are observed. Markert
and Appella (1961), in other studies with solutions
of higher concentrations, obtained 135 • ]03for the
molecular weight of this enzyme (see also Appella
and Markert, 1961).
For those proteins which exhibit associationdissociation equilibria, the molecular weight is
taken to represent the smallest molecule which
possesses the characteristic biological activity.
Higher values correspond, therefore, to dimers,
trimers, or even higher states of aggregation.
Glutamie dehydrogenase, under conditions resembling those employed for assay of enzymic activity,
has a molecular weight of 2.5 • 105 (Frieden, 1963),
whereas earlier physical chemical studies on this
enzyme yielded a value of 1 • 106 (Olson and
Anfinsen, 1952). The latter value, representing the
SUBUNITS
In the presence of denaturing agents, both of the
enzymes cited above, as well as many other
proteins, dissociate still further to give subunits
which are smaller in molecular weight than the
monomers. These subunits which are devoid of
biological activity represent the individual polypeptide chains. In some proteins such as insulin
(Sanger, 1956) and y-globulin (Porter, 1962) the
individual chains are crosslinked by disulfide bonds.
More frequently, the association of the inactive
subunits to produce active monomers does not
involve covalent bonds; instead secondary (noncovalent) forces alone cause the necessary folding
and association. Such is the case with aldolase
which has been shown to be composed of three
polypeptide chains (Stellwagen and Sehachman,
1962; Deal et al., 1963). Lactic dehydrogenase from
chicken heart or chicken muscle also seems to be
composed ofsubunits, since the enzyme of molecular
weight 1.4 • 105 dissociates upon the addition of
guanidine hydrochloride to give chains of 35 • 103
molecular weight (Pesce and Kaplan cited by Cahn
et al., 1962). This is also true of beef heart lactic
dehydrogenase (Appella and Markert, 1961). Some
of the relatively small protein molecules contain
even more than four subunits. This has been shown
recently with hemerythrin (Klotz and KeresztesNagy, 1963) which is composed of eight polypeptide
chains having molecular weights about 13 • 10a.
This association of individual polypeptide chains
to produce a biologically active protein is not
restricted to the globular proteins for the fibrous
protein, myosin, also has been shown to be composed of subunits (Kielley and Harrington, 1960;
Woods et al., 1963).
BINNING (OR ACTIVE) SITES
For many of the enzymes there seems to be an
equivalence between the number of binding (or
active) sites and the number of polypeptide chains.
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422
SCHACHMAN
NUMBER OF BINDING SITES tN LACTIC D~EHYOROGENASE
I
0
0
~
80
z I.Oi
i
i
i
r
J
i
i
~
6
50
I
i
I
i
40~
0.
8.
~
~
0
~
02
0.4
06
moles ENZYME
mole DPNH
o
I
0
~ , I , I , I
2
6
I0
12
( I / F R E E DPNH) xlO "4
I I , I'~
0 I
3
moles BOUND DPNH
mole ENZYME
FmVRE 6. Determination of the number of binding sites
in chicken heart lactic dehydrogenase. Data were obtained
from sedimentation velocity experiments with an automatic photoelectric scanning absorption optical system
(Schachman, 1963). On the left is the plot of the fraction
of D P N H bound as a function of the ratio of the total
moles of enzyme to moles of DPNH. In the center and
right are conventional plots (according to theories for
multiple equilibria) for evaluating the number of binding
sites and the binding constants. The intercept on the
ordinate in the center graph gives the reciprocal of the
number of binding sites and the intercept on the abscissa
in the right hand graph gives directly the number of binding
sites.
A typical example is illustrated by the data in Fig.
6 showing the binding of DPNH to chicken heart
lactic dehydrogenase. Under the conditions used
for these experiments, the enzyme has a molecular
weight of 1.4 • 105 and there are four binding
sites corresponding to the four subunits in the
enzyme molecule. Hemerythrin also shows a similar
correlation in the number of binding sites and the
number of polypeptide chains; Klotz and KeresztesNagy (1963) found eight oxygen binding sites per
monomer of molecular weight, 107 • 103. The
recent studies (Harris et al., 1963) with glyceraldehyde-3-phosphate dehydrogenase are particularly
noteworthy in this regard. Although the molecular
weight of this enzyme has not as yet been established unequivocally (values from 120 • 103 to
140 • 103 have been reported) there seems to be
an excellent correlation between the number of
polypeptide chains and the number of active sites.
The latter has been established both by quantitative
studies of the binding of iodoacetate which causes
inactivation and by reaction with p-nitrophenylacetate which is hydrolyzed to give the acetylenzyme. Harris et al. (1963), by using either the
radioactive inhibitor or the radioactive substrate
to label the cysteine residues followed by tryptic
digestion and analysis of the resulting radioactive
peptides, were able to demonstrate that each
active enzyme molecule contains at least three
structurally equivalent catalytic centers. Earlier
experiments (Racker et al., 1959) showed that the
crystalline enzyme contained about 3.5 moles of
bound DPN per mole of enzyme (assuming a
molecular weight of 120 • 103). If a higher value
(140 • 103) is used for the molecular weight, the
number of active sites and subunits would be four
instead of three.
Not all proteins exhibit this equivalence between
the number of binding sites and the number of
subunits. Equilibrium dialysis studies on the interaction of antibodies with specific haptens showed
that the antibody molecules possess two binding
sites (Karush, 1956). According to present views
(Fleischman et al., 1962; Porter, 1962) the antibody
molecules are composed of four polypeptide chains
held together both by disulfide bonds and secondary forces. It should be noted, however, that there
appear to be two types of polypeptide chains
(Porter, 1962) and the two combining sites may
indeed be confined to only one of the two pairs of
chains. In this way the various experimental data
could be rationalized with the view that an active
site is formed by the folding of a single polypeptide
chain and that the participation of a second chain
is not required to produce a single specific site.
Although this view may be tenable for antibodies,
it appears to be inconsistent with the experimental
observations on aldolase. As indicated above,
aldolase of molecular weight about 1.4 x 105 can
be dissociated by urea, acid, or detergents to give
material of about 50 x 103 in molecular weight
(Stellwagen and Schachman, 1962; Deal et al.,
1963). These data taken together with end group
analyses (Udenfriend and Velick, 1951; Kowalsky
and Boyer, 1960) indicate that the native enzyme
molecules contain three polypeptide chains which
are held together in a compact form by noncovalent bonds. Grazi et al. (1962) determined the
number of binding sites per enzyme molecule by
reacting the substrate, dihydroxyacetone phosphate, with the enzyme and then reducing the
complex with sodium borohydride. From measurements of the covalently bound isotope (in the
labeled substrate) they showed that the three chain
enzyme molecule possessed only one binding site.
Independent studies of the equilibrium binding of
fructose 1,6-diphosphate or dihydroxyacetone phosphate to aldolase also showed only a single binding
site per enzyme molecule (Westhead et al., 1963;
Stellwagen and Schachman, 1962; Putney, Stellwagen and Schachman, unpublished). In this
protein, therefore, it appears that the cooperative
interaction and folding of three polypeptide chains
is required for the formation of a single active site.
It should be noted that aldolase appears to be
exceptional with regard to the relationship between
the number of binding sites and the number of
subunits. Obviously, additional experimental evidence both on aldolase and other enzymes would be
of value if generalizations are to be drawn from
these limited experimental results. For purposes of
comparison, the data for a large number of proteins
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CONSIDERATIONS ON T H E T E R T I A R Y STRUCTURE OF PROTEINS
are summarized in Table 7. Many proteins such as
fl-amylase, muscle enolase, phosphorylase, flgalactosidase, and apoferritin have been omitted
because of insufficient documentation in the published literature. In some instances the complications resulting from association-dissociation
equilibria are not completely unravelled, so that
the molecular weights are still in doubt. For other
proteins research on the number of subnnits or the
number of binding sites is still incomplete. Even
for the proteins listed in Table 7 some of the
parameters may be changed as a consequence of
further research. For example, the molecular weight
of glyceraldehyde-3-phosphate dehydrogenase is
given as 140 • ] 0 a although it may be as low as
120 • 10a. Similarly, the listed molecular weight
of myosin may have to be changed when the existing discrepancies are resolved after further experimental work (Kielley and Harrington, 1960; Lowey
and Cohen, 1962; Gellert and Englander, 1963;
Woods et al., 1963). Changing these values of the
molecular weight may necessitate corresponding
modifications in some of the other parameters
listed in Table 7.
DISULFIDE BONDS
As seen in Table 7, most of the larger, multichain proteins do not contain disulfide bonds;
therefore the maintenance of their inter-chain
tertiary structures can be attributed solely to noncovalent bonds. Even for those few proteins which
do possess disulfide bonds, the bonds are generally
intra-chain and only rarely inter-chain. All four disulfide bonds in alkaline phosphatase, for example,
are intra-chain bonds and the two subunits are
held together by non-covalent bonds (Schlesinger
et al., 1963). In y-globulin the large majority
of the disulfide bonds are intra-chain and only a
few are involved in crosslinking the various chains
(Porter, 1962). Although chymotrypsin is listed as
a three chain protein, it should be recalled that the
enzyme is derived from a single chain protein which
is crosslinked by five disulfide bonds. When the
zymogen, chymotrypsinogen, is converted into the
active enzyme by proteolytic action, several small
peptides are released and the resulting protein
molecules have three chain structures in which
some of the disulfide bonds participate as crosslinks (Hartley, 1962). As indicated earlier, one of
the disulfide bonds in insulin is involved in intrachain bonding and the other two contribute to the
inter-chain structure (Sanger, 1956).
It is of interest that many of the single chain
proteins, both small and large, contain an appreciable number of disulfide bonds. Although three
disulfide bonds are listed for papain, it should be
423
noted that there is conclusive evidence for only
two such bonds and the nature of the other two
half-cystine residues has not as yet been elucidated
(Smith et al., 1962). Both ribonuclease and lysozyme are noteworthy examples of disulfide containing small proteins. These enzymes exhibit unusual resistance to denaturing agents as compared
to other proteins, and their stability as well as
their striking capacity for reversibility of denaturation can be attributed to the high concentration of
disulfide crosslinks.
The behavior of serum albumin provides another
notable illustration of the role of disulfide bonds in
a single chain protein. Various types of experiments
(Karush, 1950; Yang and Foster, 1954; Tanford et
al., 1955; Kauzmann, 1956) indicate that the
albumin molecules are unusually flexible and
deformable. Whereas many enzymes appear as
rigid particles in terms of their physical chemical
behavior and they interact specifically with only
certain small molecules such as substrates or
coenzymes, serum albumin expands readily under
a variety of experimental conditions and possesses
a great affinity for many small molecules of widely
diverse chemical properties (Klotz, 1950). This
unique behavior indicates that the secondary and
tertiary structures of serum albumin are extremely
labile and easily modified. Not only is there binding
of many small ions and molecules, but an ion of
one type can cause the displacement of another
which is already bound, i t thus appears that the
same sites are involved in the binding of diverse
substances. These competitive binding experiments, along with other detailed investigations of
the nature of the interacting groups, indicate that
the coiled polypeptide chain can assume a great
variety of stable conformations. One or another is
favored relative to the others depending on the
local ionic environment. Since the albumin molecules are capable of binding a large number of
different substances, Karush (1950) concluded that
the binding sites (as many as 24) could assume
different conformations of approximately equal
energy. This unusual property was termed "configurational adaptability." According to this
concept, in the presence of any specific ion or
molecule a particular conformation of the protein
is stabilized which, by virtue of its structural
relation to the small ion or molecule, permits the
various portions of the small molecule to interact
with the appropriate groups in the protein. From
studies with optically active dyes (Karush, 1952)
it appeared that structural alterations of the protein as a consequence of the binding of the first dye
anions facilitated the binding of additional ions.
The appearance of these additional combining
sites is a manifestation of the adaptability which
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424
SCHACHMAN
is so striking in the serum albumin molecules. I t
would seem that the 17 disulfide crosslinks,
strategically placed along the polypeptide chain of
about 600 amino acid residues, play a crucial role
in conferring over all stability while still permitting
enormous flexibility and adaptability within many
regions of the folded structure.
Only one other group of proteins, the y-globulins,
possesses more disulfide bonds than do the serum
albumins. Here, too, the disulfide bonds occupy a
crucial role not only in crosslinking the various
chains (Porter, 1962) but also in directing the
refolding of the polypeptide chains after denaturation of the antibody. In studies of hapten-antibody
interactions, Karush (1962) found that there was a
loss of specific reactivity when urea was added
and that the specific bonding capacity was regenerated upon removalof the urea. The loss in combining
power was attributed to the disruption of the
architecture of the antibody sites, and the restoration was ascribed to the reappearance of the
original structure under the influence of the many
restraining disulfide bonds. Extensive reduction of
these disulfide bonds also led to a loss of the
combining power of the antibody (Karush, 1962).
Most proteins containing disulfide bonds as part
of the tertiary structure do not also possess free
sulfhydryl groups. But it would be inappropriate
to conclude that there is an absolute exclusion
principle that all proteins cannot possess both
sulfhydryl groups and disulfide bonds. Serum
albumin and ovalbumin, in particular, are examples
of proteins having both cysteine and cystine
residues. The presence of both groups in the same
molecule creates a potentially unstable situation
since exchange reactions occur readily between
SH and SS groups, thereby leading to possible
scrambling of the disulfide bonds. It is not surprising therefore that this combination is not
prevalent in most proteins. There is no apparent
reason for their combined presence in the two
proteins cited. It may be that certain side chains
near the various cystine residues favor the disulfide
bond over the sulfhydryl group and that all molecules are identical in their distribution of disulfide
bonds, but it also may be that different albumin
molecules do not contain identical disulfide bonds
and that exchange reactions help to provide the
flexibility and adaptability required of serum
albumin in its physiological role as a transport
protein.
SIZE OF POLYPEPTIDE CHAI:NS
In view of the current research on the biosyntheses of proteins and the role of messenger RNA,
it is of interest to examine the available data per-
raining to the size of polypeptide chains in proteins.
Some of this is summarized in Table 7. Although
many polypeptide chains (in both single chain and
multi-chain proteins) have molecular weights of
about 10,000 to 20,000, there are several examples
(serum albumin and yeast enolase) in which the
polypeptide chains are composed of about 600
amino acid residues (corresponding to molecular
weights of about 70,000). In myosin of molecular
weight 6 x 105, there appear to be three polypeptide chains of equal size (Kielley and Harrington, 1960; Kielley and Barnett, 1961). These chains
of 2 • 105 molecular weight appear to be the
largest naturally occurring polypeptide chains
observed thus far.
P R O T E I N DENATURATION
Experimental investigations aimed at demonstrating that biologically active proteins can be
reconstituted from the denatured molecules are
based on the tacit assumption that the disorganized
polypeptide chains resulting from exposure of
enzymes to powerful denaturing agents are totally
devoid of the secondary and tertiary structures
characteristic of the native molecules. Although
denaturation of a protein molecule may be "allor-none" in that the unfolding of the polypeptide
chain in one region is accompanied by the loss of
organized structure in all other regions, it is also
possible that local side-chain or backbone interactions may be sufficiently strong at a particular
site that some "ordered" structure persists even
though the bulk of the specific architecture is
destroyed by the denaturant. I f the latter were to
occur, success in restoring the denatured protein
to active molecules would be misleading. For such
systems it could be argued that the small regions
of the polypeptide chains in which the specific
folding survived, act as nuclei for the reconstitution
reactions. Because of limitations in the sensitivity
of the physical chemical techniques customarily
employed in the study of denatured proteins, the
existence of such ordered regions could easily
escape detection. Under the circumstances the
only recourse is the use of many different methods
to examine the denatured protein and the employment of denaturing agents which are effective in
disrupting the various bonds described earlier.
Frequently, dissociation of proteins into subunits
is achieved without the simultaneous disruption
of much of the secondary and tertiary structures.
This is observed, for example, with muscle
phosphorylase (Madsen and Cori, 1956) which is
dissociated into four inactive subunits upon the
addition of p-chloromercuribenzoate (PCMB).
Similar results were obtained when sulfhydryl
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CONSIDERATIONS ON THE TERTIARY STRUCTURE OF PROTEINS
reagents were added to hemerythrin (KeresztesNagy and Klotz, 1963). In both cases the reaction
appeared to be "all-or-none" in that the fraction
of the molecules which was inactivated and dissociated was equal to the fraction of the sutfhydryl
groups which had reacted with PCMB. Moreover,
both proteins were reconstituted when excess
eysteine was added. Detailed physical chemical
studies have not as yet been conducted on these
subunits, but it is doubtful that much of the
secondary and tertiary structures of the individual
polypeptide chains could have been disrupted by
reagents like PCMB. This compound is known to
have great affinity for sulfhydryl groups and its
effectiveness in causing dissociation must be
attributed to this highly specific reactivity and to
indirect steric effects caused by the bulky group
interfering with other side-chain interactions.
Indications of a secondary, indirect effect of
PCMB come from the kinetic studies of the inactivation and dissociation of phosphorylase (Madsen
and Cori, 1956; Madsen, 1956). The rate of reaction
of the sulfhydryl groups with PCMB was greater
than the rate of inactivation. Moreover, the dissociation into subunits proceeded even more
slowly. It appears as if the reaction of PCMB with
the first sulfhydryI group of a particular molecule
caused a structural change which facilitated reaction with the remaining 17 sulfhydryl groups in
the same protein molecule. After all the sulfhydryl
groups had reacted, further conformationa] changes
led to inactivation of the enzyme. Dissociation into
the four subunits occurred apparently after there
had been additional alterations in the tertiary
structure.
Yeast alcohol dehydrogenase also is inactivated
and dissociated into subunits upon the addition of
PCMB; similarly, silver ions which likewise have a
high affinity for sulfhydryl groups caused inactivation and dissociation. Snodgrass et al. (]960)
showed that the loss of activity was due to the
formation of mercaptides, and to consequent conformational changes (see also Li et al., 1962), rather
than to the displacement of zinc which is known
to be essential for enzymic activity. After prolonged
exposure of the enzyme to Ag+ or PCMB, zinc is
released from the enzyme which then dissociates
into subunits (Vallee, 1961). The importance of
zinc in the maintenance of the tertiary structure
of this enzyme was demonstrated even more directly by K~tgiand Vallee (1960). Upon the addition of
different chelating agents, such as 1,10-phenanthroline or 8-hydroxyquinoline-5-sulfonic acid,
the enzyme is first inhibited, and then, if the chelating agents are present in excess, the molecules
dissociate into four subunits which are free of zinc
atoms. These experiments show that in yeast
425
alcohol dehydrogenase (and probably in other
metal-containing enzymes as well) the zinc atoms,
in the form of metal-protein chelates, contribute
to the stabilization of the tertiary structure
required for enzymic activity. The conformational
change caused by the inactivation of the enzyme
with 1,10-phenanthroline is much less than that
caused by denaturing agents such as urea or
alkali (Ulmer and Vallee, 1961).
The above examples illustrate the partial disruption of tertiary structures with specific reagents
which are effective against only certain types of
bonds. More extensive denaturation is generally
caused by reagents like hydrogen and hydroxyl
ions, urea, guanidine hydrochloride, detergents
like sodium dodecyl sulfate, and organic solvents
(Neurath et at., 1944). The disorganization caused
by acid or alkali can be attributed to the large
intra-molecular repulsive forces which develop as
the net charge is increased. Although urea and
guanidine hydrochloride have been used as denaturants for many years, it is still not known whether
the disruptive effect of these reagents is to be
attributed to the breakage of hydrogen or hydrophobic bonds (Kauzmann, 1954, 1959; Klotz and
Stryker, 1960; Bruning and Holtzer, 1961; Levy
and Magoulis, 1962; Whitney and Tanford, 1962;
Gordon and Jencks, 1963; Robinson and Jeneks,
1963). The effectiveness of sodium dodecyl sulfate
stems in part from the repulsive forces generated
as a result of binding of the anionic detergents and
also from the rupture of hydrophobic bonds in the
protein through interaction with the long hydrocarbon chain of the detergent. Organic solvents are
effective as denaturants because of their affinity
for non-polar side chains in the proteins, but it
shouid be noted that ionic bonds would be strengthened by the addition of non-polar organic solvents
like dioxane because of the consequent decrease
in the dielectric constant of the medium.
The effect of some of these denaturing agents on
aldolase is shown by the data summarized in Table
8. The large decrease in sedimentation coefficient
(compared to the native enzyme) indicates that
the molecular weight and/or the shape of the
molecules had changed drastically. Actually both
the weight and shape had changed. This is evident
from the direct measurement of the molecular
weight by sedimentation equilibrium experiments
and from the viscosity data. Not only was there
dissociation into three subunits but the polypeptide
chains were unfolded and had dimensions similar
to those of randomly coiled chains. Further evidence
for the marked conformationat change was obtained fr6m the optical rotation data. Detailed
spectral studies (Stellwagen and Sehachman, 1962;
Donovan, 1963) indicated that the environment
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426
SCHACHMAN
TABLE 8. PHYSICAL AND CHEMICAL PROPERTIES OF NATIVE AND DENATURED ALDOLASE $
$20 ' W
M.W. a
?]sp/C b
~raax c
[~]D
~e d
Denaturing agent
S
x 10-5
ml/g
m/~
deg
m/z
None
Sodium Dodecyl Sulfate--1%
Acetic Acid--5%
HC1--0.0I M
Urea--4 M
7.9
2.2
1.9
2.0
1.6
1.42
0.43
-0.46
0.42
4
15
23
23
18
279.7
--23
283
.
.
.
.
277.0
.
.
.
277.0
--62
238
277.0
--83
223
Spec. Act.
units/mg
SH
mole-1
11
.
.
-27
73e
.
.
-<1
13f
---
* From Stellwagen and Sehachman (1962).
a Apparent weight average molecular weights from sedimentation equilibrium experiments at 2.5 mg]ml.
b Experiments at 5 mg/ml.
c Maxima in the absorption spectra.
d Crude constants evaluated from plots of optical rotation data: [~]x ~t2 vs [u];t.
e Hydrazine assay.
t a-Glycerophosphate dehydrogenase assay.
of the tyrosyl, tryptophanyl, and phenylalanyl
residues had been altered. In the native enzyme
m a n y of these residues are" buried" in the interior
of the protein and upon denaturation they are no
longer shielded from contact with the aqueous environment. Similarly, 16 of the 27 sulfhydryl groups in
the native enzyme are "masked" and unavailable
for titration with PCMB, but upon denaturation
all of the sulfhydryl groups are readily titrated
(Swenson and Boyer, 1957). The results in Table 8
show clearly that the enzyme is not only inactivated
and dissociated into subunits but also that the
bulk of the organized structure is destroyed in the
various denaturing media. However, this transition
from the native structure to the three disorganized
polypeptide chains does not occur as a single
reaction (Deal et al., 1963). Various stages in the
process can be distinguished by analysis of the
sedimentation patterns (Stellwagen and Sehachman, 1962; Deal et al., 1963) and the kinetics of
the change in the spectral properties and the optical
rotation (Donovan and Schachman, unpublished).
Although no detailed interpretation of the various
time-dependent intra-molccnlar changes is yet
feasible, it appears that there is a swelling of the
molecule first to give a more slowly sedimenting
species (Deal et al., 1963) followed by a separation
of the subunits and their eventual unfolding (Donovan and Schachman, unpublished).
T H E ACQUISITION OF T E R T I A R Y
STRUCTURES
Early attempts to regenerate biologically active
molecules from denatured proteins were frequently
unsuccessful. There were, to be sure, notable
exceptions such as soy bean trypsin inhibitor
(Kunitz, 1948) and chymotrypsinogen (Eisenberg
and Schwert, 1951). Despite these two instances
(and others less thoroughly investigated) some
workers, because of the inability to reconstitute
biologically active proteins from the disorganized
polypeptide chains, adopted the view that protein
denaturation was an irreversible process (see
Neurath et al., 1944, for alternative views). In the
light of more recent knowledge, conclusions that
denaturation is irreversible appear unwarranted.
Frequently, the failure to restore the unfolded
chains to the native conformation was due to
unforseen, subsidiary chemical modifications which
occurred during the denaturation process and
subsequently prevented its reversal. The amino
groups in proteins, for example, are now known
to react with the cyanate which accumulates in
concentrated urea solutions. Such unsuspected
chemical modification of the polypeptide chains
could have precluded the regeneration of enzymic
activity. Other examples can be cited as well. The
sulfhydryl groups which are "unmasked" during
denaturation are very easily oxidized (especially
in the presence of metal ions and at high pH) to
give intra- and inter-molecular disulfide bonds
which then prevent the proper refolding of the
chains. In some instances insufficient attention was
given to possible kinetic barriers to the reversal
of protein denaturation. The solutions of denatured
protein in 8 M urea, for example, were generally
diluted rapidly with buffer when the denaturation
reactions were to be terminated and the urea
removed. This led generally to the coagulation of
the protein and not to the native, soluble form
even though on thermodynamic grounds the latter
m a y have been favored.
Now that precautions are being taken to protect
the exposed side chains from interfering chemical
reactions and the denaturing agents are removed
slowly so as to facilitate necessary annealing reactions, reconstitution of active molecules is becoming
commonplace. The regeneration of enzymic activity
in single chain molecules containing disulfide bonds
is discussed elsewhere in this volume (C. J. Epstein
et al., 1963); therefore only multichain proteins are
considere~l here with illustrations from studies with
aldolase (Stellwagen and Schachman, 1962; Deal
et al., 1963).
Initial attempts to regenerate enzymic activity
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CONSIDERATIONS ON THE TERTIARY STRUCTURE OF PROTEINS
427
TABLE 9. P H Y S I C A L A N D C H E M I C A L P R O P E R T I E S OF N A T I V E A N D R E C O N S T I T U T E D A L D O L A S E *
Sample a
Native
R e c o n s t i t u t e d f r o m acid b y dilution
R e c o n s t i t u t e d f r o m acid b y dialysis
R e c o n s t i t u t e d f r o m u r e a b y dialysis
s~0, wb
S
7.6
7.5
7.8
7.8
~sp/C
ml[g
M.W. b
• 10 -6
4.0 c
4.2 e
---
1.42
1.48
1.60
1.59
Spec. Act.
units/rag
73 d
73
---
SH
mole -1
13.0 e
13.2
13.5
11.5
10.6
11.5
11.4
10.4
~tmax~
m#
[r162 ~e~
deg
~tc
m#
279.7
279.7
279.7
279.7
--23
--22
--21
--20
283
290
292
287
* F r o m Stellwagen a n d S c h a c h m a n (1962).
a AU p h y s i c a l chemical m e a s u r e m e n t s were m a d e on solutions of aldolase in 0.2 M NaC1-0.01 M p h o s p h a t e buffer, p H 7.
b T h e c o n c e n t r a t i o n s were 5.0, 4.0, 2.5, a n d 2.5 m g / m l respectively,
c T h e c o n c e n t r a t i o n s were 4 m g / m l .
d H y d r a z i n e assay.
e ac-Glycerophosphate d e h y d r o g e n a s e assay. O n e u n i t is 1/~M fructose 1,6.diphosphate c l e a v e d / m i n u t e .
t These are t h e m a x i m a in t h e a b s o r p t i o n spectra.
g T h e s e were e v a l u a t e d f r o m plots o f [~])t )2 v s [~c]~.
from acid-dissociated (pH 2) aldolase solutions
involved direct neutralization of concentrated
solutions (5 mg/ml). This led invariably to aggregation of the polypeptide chains and the resulting
turbid solutions contained rapidly sedimenting
material with little or no enzymic activity. When,
however, the hydrogen ion concentration was
reduced by dialysis of the acidified solutions against
buffers of the desired pH (a pH of about 5.5 proved
optimal), considerable reactivation occurred. Also,
dilution of the acidified solutions with the appropriate buffer led to regeneration of the enzymic
activity. By means of these procedures, about
70% of the original activity was restored from
aldolase which had been denatured by hydrogen
ions, acetic acid, urea, and guanidine hydrochloride
(Stellwagen and Schachman, 1962; Putney and
Schachman, unpublished). The remaining inactive
material was separated from the reconstituted
enzyme by appropriate changes of the pH of the
solution (which caused the inactive material to
precipitate) or by fractionation on phosphocellulose.
In this way, purified reconstituted aldolase was
obtained for detailed characterization in terms
of physical, chemical, and enzymic properties.
The results summarized in Table 9 show that the
reconstituted enzyme was virtually indistinguishable from the native enzyme molecules (Stellwagen
and Schachman, 1962). The 16 sulfhydryl groups
which were "unmasked" upon dissociation and
denaturation of the enzyme (see Table 8) were
once again not titratable in the reconstituted
enzyme. Similarly, the side chains of the aromatic
amino acids were "buried" in the interior of the
reconstituted molecules as in the native enzyme.
As expected for a process involving the association of three polypeptide chains, the restoration of
activity was influenced by the concentration of
the denatured protein. At very low concentrations
(1 7/ml) there was little or no restoration of activity
and the yield of active material increased to a
maximum as the concentration was raised (Stellwagen and Schachman, 1962). These results a r e
similar to those of Levinthal et al. (1962) on the
reconstitution of alkaline phosphatase. The observations with these two multichain enzymes
should be contrasted with those for the restoration
of ribonuclease activity from the denatured,
reduced single polypeptide chain. For this enzyme
lower concentrations favor the reconstitution of
active enzyme molecules since intra-moleeular
disulfide bonds predominate. When the oxidation
is performed at higher concentrations inter-molecular disulfide bonds form, and there is a long lag
period before activity is regenerated by shuffling
of the disulfide bonds (Epstein et al., 1962).
Kinetic studies (Fig. 7) of the reconstitution of
active molecules from the acid dissociated aldolase
showed that there was a very rapid partial restoration of activity followed by a slower process
leading to 60% recovery of the total activity.
At 25 ~ the reaction was complete in about 30 min,
whereas the rate at 2~ was substantially less. The
initial activity formed in the first phase of the
process was lost rapidly if the reconstitution was
I00
/ 1
I
I
I
I/r---V
- ~
RECONSTITUTION OF ALDOLASE
% Activity
25 ~
.
~
c.
/A---o
50
I
I//j
90
120
i80 200
Minutes
FIGURE 7. K i n e t i c s o f t h e r e g e n e r a t i o n o f e n z y m i c
a c t i v i t y a t different t e m p e r a t u r e s . T h e o r d i n a t e gives t h e
t o t a l yield (in per c e n t o f t h e original aldolase activity)
a f t e r r e e o n s t i t u t l o n , a n d t h e abscissa gives t h e time.
Aldolase a t 5 m g / m l in 0.05 M NaC1 w a s dissociated b y t h e
a d d i t i o n of 1 M HC1 to p H 2 a n d r e c o n s t i t u t i o n w a s
effected b y diluting aliquots w i t h 0.05 M a c e t a t e buffer a t
p H 5.5. A s s a y s were t h e n p e r f o r m e d o n a l i q u o t s o f t h e
r e c o n s t i t u t e d s a m p l e s b y f u r t h e r dilution a t t h e t i m e s
indicated. ( F r o m Stellwagen a n d S c h a c h m a n , 1962.)
30
a
60
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Press
428
SCHACHMAN
performed at 37 ~. The native enzyme at this
temperature is very stable by comparison. It would
appear, therefore, that the initial rapid reaction
leads to an intermediate product which is enzymically active but very labile. At 37 ~ this material
becomes inactivated but at the lower temperatures
an annealing process occurs leading to active,
stable enzyme molecules. Analyses of the kinetic
data (Stellwagen and Schachman, 1962) showed
that both the rapid and slow phases are first order
with respect to concentration. The rate limiting
steps in the reconstitutiou process, therefore, are
not the association of the three polypeptide chains.
Deal et al. (1963), by studying the reconstitution
at intermediate pH values, detected an inactive
intermediate composed of the three polypeptide
chains and concluded also that the process occurred
in two stages. It appears from kinetic studies of
the spectral changes upon reconstitution (Donovan
and Schachman, unpublished) that marked changes
in conformation continue even after association of
the three subunits.
Although the studies on the denaturation and
reconstitution of aldolase (Stellwagen and Schachman, 1962) are consistent with the view that
multichain enzymes are formed in vivo by the
spontaneous folding and association of the newly
synthesized polypeptide chains, further support
for this hypothesis is required. Additional investigations on this particular system as well as comparable studies on the denaturation and reconstitution
of other enzymes would be of value. At this writing
only two other examples can be cited. Alkaline
phosphatase of E. cell has been dissociated and
reconstituted with most of the enzyme activity
being restored, but no evidence has yet been
presented to indicate the extent of the disorganization of the two separated polypeptide chains
(Levinthal et al., 1962). In the case of rabbit
myosin, the dissociated subunits behave as
randomly-coiled chains and the reconstituted
protein exhibits the unique physical properties of
the native fibrous protein (Young et al., 1962).
However, the proper conditions for preventing the
oxidation of sulfhydryl groups have not been found
nor has the ATPase activity been restored after
the inactivation and dissociation. Despite the
limited number of available examples, and the
inadequacy of some of the data, it seems likely
that for the multichain enzymes the amino acid
sequence in the polypeptide chains uniquely
determines the chain conformation as well as the
spatial relationships of the subunits in the native
enzyme molecules.
Much more information must be accumulated
before a definitive mechanism can be proposed for
the sequence of reactions leading from the indivi-
dual random polypeptide chains to the biologically
active multichain proteins. In such proteins it is
important to determine whether the chains are
identical or different in their primary structure.
For aldolase, end group analyses are consistent
with the view that the chains are similar and thus
far only one component has been detected by
electrophoresis of the subunits. But these experiments are hardly conclusive and data pointing to
differences in the chains can also be cited. Also,
it is uncertain whether the active site is formed
by the folding of only one chain which is stabilized
through interaction with the other two, or whether
residues from all three polypeptide chains participate directly in the catalytic function. The successful refolding and association of the three polypeptide
chains in aldolase shows clearly that non-covalent
interactions are sufficient to direct and maintain
the proper refolding. This indicates that the
disulfide bonds in proteins like ribonuclease and
taka-amylase form correctly because the noncovalent forces create the proper spatial orientation
of the participating sulfhydryl groups. No postulate
about shuffling of previously formed incorrect
disulfide pairing seems necessary.
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Considerations on the Tertiary Structure of Proteins
H. K. Schachman
Cold Spring Harb Symp Quant Biol 1963 28: 409-430
Access the most recent version at doi:10.1101/SQB.1963.028.01.057
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