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Chapter 5 The Three-dimensional
Structure of Proteins
1. Early studies on the peptide (protein)
structure
1.1 The peptide (O=C-N-H) bond was found to
be shorter than the C-N bond in a simple
amine and atoms attached are coplanar.
1.1.1 This was revealed by X-ray
diffraction studies of amino acids and of simple
dipeptides and tripeptides.
1.1.2 The peptide (amide) bond was
found to be about 1.32 Å (C-N single bond, 1.49;
C=N double bond, 1.27), thus having partial
double bond feature (should be rigid and
unable to rotate freely).
1.1.3 The partial double bond feature is
a result of partial sharing (resonance) of
electrons between the carbonyl oxygen and
amide nitrogen.
1.1.4 The atoms attached to the peptide
bond are coplanar with the oxygen and
hydrogen atom in trans positions.
1.2 X-ray studies of a-keratin (the fibrous
protein making up hair and wool) revealed a
repeating unit of 5.4 Å (Astury in the 1930s).
2. The likely regular conformations of protein
molecules were proposed before they were
actually observed!
2.1 This was accomplished by building precise
molecular models.
2.1.1 Experimental data (from X-ray
studies) were closely adhered, interpreted.
2.1.2 Single bonds other than the peptide
bond in the backbone chain are free to rotate.
2.2 The simplest arrangement of the polypeptide chain
was proposed to be a helical structure called a-helix
(Pauling and Corey, 1951)
2.2.1 The polypeptide backbone is tightly wound
around the long axis (rodlike).
2.2.2 R groups protrude outward from the
helical backbone.
2.2.3 A single turn of the helix (corresponding to
the repeating unit in a-keratin) extends about 5.6
Angstroms, including 3.6 residues (each residue arises
1.5 Å and rotate 100 degrees about the helix axis).
2.2.4 The model made optimal use of internal
hydrogen bonding for structure stabilization.
2.2.5 Each carbonyl oxygen of the residue n is
hydrogen bonded to the NH group of residue (n+4).
2.2.6 The residues forming one a-helix must all
be one type of stereoisomers (either L- or D-).
2.2.7 L amino acids can be used to build either
right- or left-handed a-helices (the helix spiraling
away clockwise or counterclockwise respectively).
2.3 b-pleated sheet was proposed to be the more
extended conformation of the polypeptide chain.
2.3.1 The conformation is formed when
two or more almost fully extended polypeptide
chains are brought together side by side.
2.3.2 Regular hydrogen bonds are formed
between the carbonyl oxygen and amide
hydrogen between adjacent chains (look like a
zipper).
2.3.3 The axial distance between the
adjacent amino acid residues is ~3.5 Angstroms.
2.3.4 The planes of the peptide bonds
arrange as pleated sheets.
2.3.5 The R groups of adjacent residues
protrude in opposite directions.
2.3.6 The adjacent polypeptide chains can
be either parallel (the same direction) or
antiparallel (the opposite direction).
3. Protein architecture can be understood at
different levels.
3.1 Each protein usually has one native conformation
3.1.1 Under physiological conditions of solvent
and temperature, each protein folds spontaneously into
one three-dimensional conformation, called the native
conformation.
3.1.2 This conformation is usually
thermodynamically the most stable (having the lowest
Gibb’s free energy), and predominates among the
innumerable theoretically possible ones.
3.1.3 Usually only the native conformation is
functional.
3.2 Protein structures have conventionally been
considered at four different levels.
3.2.1 The primary structure is the amino acid
sequence (including the locations of disulfide bonds).
3.2.2 The secondary structure refers to the regular,
recurring arrangements of adjacent residues resulting
mainly from hydrogen bonding between backbone
groups, with a-helices and b-pleated sheets being the two
most common ones.
3.2.3 The tertiary structure refers to the spatial
relationship among all amino acid residues in a
polypeptide chain, that is, the complete threedimensional structure.
3.2.4 The quaternary structure refers to the spatial
arrangements of each subunit in a multisubunit protein,
including nature of their contact.
3.3 Studies of protein conformation, function, and
evolution have revealed importance of two other levels
of organization.
3.3.1 The supersecondary structure refers to
clusters of secondary structures that repeatedly
appear in different proteins.
3.3.2 The already identified supersecondary
structures include mainly bab motif, Greek key motif,
b-hairpin loop, four-helix-bundle, …etc.
3.3.3 Supersecondary structure motifs are
usually also folding motifs of proteins. (a conjecture,
Not completely established experimentally).
3.3.4 A compact region (usually include
less than 200~400 residues) that is a distinct
structural unit within a larger polypeptide chain
is called a domain.
3.3.5 Many domains fold independently
into thermodynamically stable structures, and
sometimes, have separate functions.
Structural domains in the polypeptide troponin C,
two separate calcium-binding domains
Repeated usage of a pattern
Quanternary structure: macromolecular assembly
deoxyhemoglobin
4. The theoretically allowed conformations of peptide
main chains can be predicted by the Ramachandran plot
4.1 The backbone conformation of a peptide bond can
be defined by two sets of rotation angles.
4.1.1 The rotation angles around the N-Ca bonds
are labeled as phi, and around Ca-C bonds are psi.
4.1.2 By convention, both phi and psi are defined
as 0 degree in the conformation when the two peptide
planes connected to the same a carbon are in the same
plane.
4.1.3 In principle, phi and psi can have any value
between -180 and +180 degrees.
4.1.4 The conformation of the main chain is
completely defined when phi and psi are specified for
each residue in the chain.
4.2 The allowed combination values for phi and psi can
be graphically shown by plotting one against the other.
4.2.1 The Ramachandran plot is based on the
principle that no two atoms can come together closer
than the sum of their van der Waals radii (hard sphere
model).
4.2.2 Many combinations of rotation angles are not
allowed in peptide backbone conformation due to steric
hindrance.
4.2.3 Gly residues can take up many
conformations that are sterically forbidden for other
residues.
4.2.4 Every possible secondary structure is
described completely by the two bond rotation angles
that are the same for each consecutive residue. For righthanded a-helix, phi=-60 and psi=-45 to -50; for b-pleated
sheets, phi is around -120 and psi 120.
Phi=0, psi=0
5. a-helices and b-pleated sheets were confirmed
to be common secondary structures in proteins
5.1 Both a-helix and b-pleated sheet were later found to
be existing in proteins.
5.1.1 The existence of a-helices in protein was
confirmed (6 years after its prediction) by the
determination of the X-ray structure of myoglobin.
5.1.2 Right-handed a-helices widely exist in
proteins.
5.1.3 b-pleated sheets are also frequently found in
globular proteins (b-keratin, immunoglobular domain,
(b-barrel).)
5.2 The amino acid sequence affects the stability of ahelical structures.
5.2.1 Additional interactions (e.g., electrostatic
interactions, hydrophobic interactions, steric
hindrance) between amino acid side chains can
stabilize or destabilize the regular secondary structure.
5.2.2 Critical interactions occur between side
chains several residues (2-4) away on a helix structure.
5.2.3 Pro is rarely found in a-helices due to its
lack of rotation around the N-Ca bond and inability
to form hydrogen bond from its amide nitrogen.
5.2.4 Negatively charged residues at the
N-terminal end of a helical segment will have a
stabilizing effect on the positive charge of the
helix electric dipole. (Asp, Glu)
5.2.5 Positively charged residues will
have a similar stabilizing effect on the Cterminal end of the helical segment (negatively
charged). (Arg, Lys)
5.3 b turn (hairpin turn) is also a common secondary
structure found where a polypeptide chain abruptly
reverses its direction.
5.3.1 It often connects the ends of two adjacent
segments of an antiparallel b-pleated sheet.
5.3.2 It is a tight turn of ~180 degrees involving
four amino acid residues.
5.3.3 The essence of the structure is the hydrogen
bonding between the C=O group of residue n and the NH
group of the residue n+3.
5.3.4 Gly and Pro are often found in b turns. Gly is
there because it is small and flexible; Pro because the
peptide bond involving Pro can assume the cis
configuration, which in turn generates a tight turn on the
polypeptide chain.
5.3.5 b turns are often found near the surface of a
protein.
5.4 Some amino acid residues are accommodated
in the different types of secondary structure
better than others.
5.4.1 The probability is calculated from
known protein structures. It is used in predicting
secondary structures.
5.4.2 Some bias or propensities can be
explained easily.
5.4.3 Others are not yet understood.
With different phi, psi angles.
The crystal structure of pyruvate kinase
The two residues
that are 2-4 residues
away are close
in space.
Helical dipole
moment
Five different kinds of contraints affectting the
stability of the a helix
1) The electrostatic repulsion (or attraction) between
the neighboring amino acid residues with the charged
R groups.
2) The bulkiness of adjacent R groups.
3) The interactions between R groups spaced 2 to 4
residues apart.
4) The occurrence of Pro and Gly residues.
5) The interaction between amino acid residues at the
ends of a helix segment with the electric dipole
inherent to the helix.
6. The left-handed polypeptide chains wrap together
to form a right-handed triple helix in collagen
protein
6.1 The amino acid sequence of collagen is revealed to
be remarkable regular.
6.1.1 Nearly every third residue is Gly (X-X-G).
6.1.2 It is abundant in Pro and Hyp
(hydroxylproline).
6.1.3 The sequence Gly-Pro-Hyp recurs
frequently.
6.2 The helical motif of its three chains is entirely
different from that of the a-helix.
6.2.1 Intrachain hydrogen bonds are absent.
6.2.2 Each of the three helices is stabilized by
steric repulsion of the pyrrolidone rings of the Pro and
Hyp residues.
6.2.3 The rise per residue is 2.9 Å and there are
nearly 3 residues per turn.
6.2.4 Interchain hydrogen bonds are formed
(main chains ?).
6.2.5 Hyp also participate interchain hydrogen
bonding.
6.2.6 Collagens also contain hydroxylysine
that is believed to participate in hydrogen bonding.
6.2.7 The collagen polypeptide chains within
or between the triple helices are covalently cross
linked through Lys or Hylys side chains.
6.2.8 Only the small Gly can fit into the
crowded interior of the triple helix.
6.2.9 The superhelix provides great tensile
strength with no capacity to stretch.
6.2.10 Collagen fibers have similar tensile
strength as a steel wire of equal cross section.
6.3 Collagen is the most abundant protein in
mammals.
6.3.1 About 25% of the total protein mass in
mammals is collagen.
6.3.2 It is a major component of tendons, the
extracellular matrix of the connective tissues (skin,
bone matrix), and the cornea of the eye.
6.3.3 Collagen triple helices (also called
tropocollagen) self-assemble in the extracellular
space to form much larger collagen fibrils that
further aggregate into collagen fibers.
6.3.4 The collagen triple helices are
regularly staggered in fibril to give rise to the
striated appearance in negatively stained
electron micrograph.
6.3.5 The fibril formation involves many
enzymatic steps. Deficiency of these steps
generate many genetic diseases (e.g.,
osteogenesis imperfecta, Ehlers-Danlos
syndrome, both resulted from single amino acid
replacements of a Gly).
7. a-keratins contain a-coiled coils
7.1 a-keratins are rich in hydrophobic residues.
7.1.1 Phe, Ile, Val, Met, and Ala residues are rich.
7.1.2 This makes the protein insoluble in water.
7.2 Three helical strands wrap together to form a
superhelix (protofibril) in a-keratin.
7.2.1 Each strand is a a-helix.
7.2.2 The superhelical twisting is left-handed in
a-keratins (opposite to the individual strand).
7.2.3 The individual a-helices are cross linked by
interchain disulfide bonds.
7.3 a-keratins are the main components of skin and
many skin derivatives in vertebrate animals.
7.3.1 Including, e.g., hair, wool, feathers, nails,
claws, quills, scales, horns, hooves, tortoise shell, and
much of the outer layer of skin.
7.3.2 Usually harder a-keratins contain higher
number of Cys (18% of the residues are Cys in tortoise
shells and rhinoceros horns).
7.3.3 a-keratins can be stretched (to twice as its
original length) due to its structure springiness.
7.3.4 Permanent waving of hair is biochemical
engineering, where disulfide bonds between individual
chains are reduced (while the hair being heated), curled,
and reoxidized (cooled at the same time).
8. Elastin molecules contain random coils and
covalently cross-linked Lys residues.
8.1 The monomer elastin (called tropoelastin) is
rich in Gly, Ala, and Lys residues.
8.1.1 It contains alternating Gly-rich coiled
domains and Lys-rich domains.
8.1.2 Lys side chains are covalently linked
by enzymes.
8.2 Elastin is important component of elastic
connective tissues (lung, large blood vessels).
The combination of random coils and
covalent cross linking provide the elasticity of the
tissues.
9. Sperm whale myoglobin, the oxygen carrier in
muscle, was the first protein to be seen in atomic
detail by X-ray analysis (John Kendrew, 1950s)
9.1 The existence of a-helices were for the first time
directly observed in a protein.
9.1.1 The myoglobin molecule contains eight ahelices.
9.1.2 All the a-helices are right-handed.
9.1.3 All the peptide bonds are in the planar
trans configuration.
9.1.4 There is no b-pleated sheets observed in
the molecule.
9.2 The myoglobin molecule has a dense hydrophobic
core.
9.2.1 Many hydrophobic R groups (e.g., Leu, Val,
Met, Phe) were found to be in the interior of the
myoglobin molecule.
9.2.2 Hydrophobic interaction is important for the
stability of the protein structure.
9.2.3 Only two hydrophilic histidine residues were
found in the interior of the protein.
9.2.4 The compactness of the molecule is similar to
a solid.
9.2.5 Short-range van der Waals interactions make
a significant contribution to the stabilizing hydrophobic
interactions due to the solid-like compactness of the
molecule (residues of subtle difference were used to fill
the interior of a protein neatly and thus maximize van
der Waals interactions.
9.3 All but two of the polar R groups are located on
the outer surface and hydrated.
9.3.1 Nonpolar residues also present on the
outer surface!
9.4 Bending are made of residues or sequences that
are incompatible with a-helical structure.
9.4.1 All Pro residues are found at bends.
9.5 The flat heme group (the prosthetic group) was
revealed to rest in a crevice (pocket).
9.5.1 The heme group consists of a complex organic
ring structure, protoporphyrin, to which is bound an iron
atom in its ferrous (Fe2+) state.
9.5.2 The iron atom has six coordination bonds,
with four in the plane of and bonded to the flat porphyrin
molecule and two perpendicular to it.
9.5.3 One of the perpendicular coordination bonds
is bound to a nitrogen atom of an interior His residue.
9.5.4 The sixth coordination serves as the binding
site for O2.
9.5.5 The accessibility of the heme group to solvent
is highly restricted, thus preventing the oxidation of the
Fe2+ to the ferric ion (Fe3+), which is unable to bind O2.
Sir John Kendrew and Max Perutz won the
Nobel Prize in Chemistry in 1962 for
determining the complete atomic structure of
myoglobin and hemeglobin.
10. Atomic structures of many proteins have since
been determined.
10.1 Proteins differ in three dimensional structures.
10.1.1 Myoglobin represents only one of many
ways that a polypeptide chain can be folded.
10.1.2 Another small heme-containing protein,
cytochrome c, was found to contain only about 40% ahelices, but many irregularly coiled and extended
segments.
10.1.3 The heme group in cytochrome c is
covalently attached to a Met sulfur and a His nitrogen
atom (different from myoglobin).
10.1.4 Lysozyme, an enzyme that catalyzes the
hydrolytic cleavage of polysaccharides in some
bacterial cell walls, contains about 40% a-helices and
about 12% b-sheets.
10.1.5 Ribonuclease A contains more b-sheet
structures.
10.1.6 Disulfide bonds were observed in lysozyme
and ribonuclease A.
10.2 All the water soluble globular proteins have a
hydrophobic core and a mainly hydrophilic outer
surface.
10.2.1 Proteins are stabilized mainly by
noncovalent interactions (sometimes by disulfide
bonds).
10.2.2 Each protein has a unique structure to
perform its unique function.
11. Tertiary structure is determined by amino
acid sequence
11.1 Loss of tertiary structure (native conformation) is
accompanied by loss of function.
11.1.1 Proteins are relatively easy to lose their
tertiary structures due to their marginal stability
maintained by noncovalent interactions.
11.1.2 The process of total loss or randomization
of three-dimensional structure of proteins is called
denaturation.
11.1.3 Protein denaturation results from a
change in the solvent environment that is sufficiently
large to upset the forces that keep the protein structure
intact.
11.1.4 Many means can cause protein to denature:
Heating: unbalance the compensating
enthalpic and entropic contributions, thermal motion
causes melting.
Extreme pH: upset the balance of a
protein’s charge interactions.
Miscible organic solvents (alcohol and
acetone): solvent polarity and hydrogen bonding.
Solutes (urea, guanidine): provide
alternative hydrogen bonding.
Detergents (SDS): introducing their
hydrophobic tails into the protein’s interior.
11.1.5 The mechanism of many of these
denaturing processes are fully understood.
11.2 Denaturation of some proteins is reversible.
11.2.1 Some denatured globular proteins will
regain their native structure and their biological activity
once returned to conditions in which the native
conformation is stable. This process is called
renaturation.
11.2.2 The denaturation and renaturation
phenomena was originally observed on ribonuclease A
by chance by Christian Anfinsen (1950s).
11.2.3 Ribonuclease A became reduced and
randomly coiled (denatured) in 8 M urea plus bmercaptoethanol, with a loss of the enzymatic activity.
11.2.4 When urea and b-mercaptoethanol were
removed, the enzymatic activity was slowly regained
(enzymatic activity was fully regained under stable
conditions, with existence of trace amount of bmercaptoethanol.
11.2.5 All the physical and chemical properties of
the refolded enzyme were virtually identical with those
of the native enzyme.
11.2.6 Conclusion: the information needed to
specify the complex tertiary structure of ribonuclease A
is all contained in its amino acid sequence.
11.2.7 Subsequent studies have established the
generality of this central principle of molecular biology:
sequence specifies conformation.
Nobel Prize in Chemistry in 1972 for Anfinsen.
11.3 The tertiary structures of proteins are not rigid.
11.3.1 Many studies have found that globular
proteins have certain amount of flexibility in their
backbones and undergo short-range internal
fluctuations.
11.3.2 Many proteins undergo small
conformational changes in the course of their
biological function (e.g., O2-bound hemoglobin
differs from O2-free hemoglobin, substrate binding to
enzymes often cause conformational changes).
12. The polypeptide chain of a protein folds rapidly in
vivo
12.1 The protein folding problem is one of the
most challenging and important areas of inquiry
in biochemistry.
12.1 How does the amino acid sequence of
a protein specify its three-dimensional structure?
12.2 How does an unfolded polypeptide
chain acquire the form of its native
conformation?
12.2 Are all possible conformations searched to
find the energetically most favorable one?
12.2.1 The Levinthal’s paradox: the huge
difference between the calculated (theoretical) time
it may take for a polypeptide to fold by random
searching and the actual time it takes.
12.2.2 The cumulative selection, that is,
partially correct intermediates are (recognized by
nature and) retained due to sub-stability, makes
the searching process much more efficient.
12.3 Protein folding is an intriguing problem for both
theoreticians and experimentalists.
12.3.1 Proteins are only marginally stable. The
free energy difference between the folded and unfolded
states of a typical 100-residue protein is only 10
kcal/mol, meaning that correct intermediates can be
easily lost.
12.3.2 The criterion of correctness is the total
free energy of the transient species, not a residue-byresidue scrutiny of conformation (?).
12.3.3 Some intermediates, called kinetic traps,
have a favorable free energy but are not on the path to
the final folded form.
12.4 Molten globules are formed early in folding.
12.4.1 Molten gobule state contains native
secondary but not tertiary structure (an
experimental observation).
12.4.2 Hydrophobic collapse and
acquisition of stable secondary structure are
mutually reinforcing events in the formation of
molten globules (synergistic, helping each other).
12.5 Partially folded intermediates can be detected,
trapped, and characterized.
12.5.1 Rapid-kinetics studies, where protein
secondary structures are monitored by
spectroscopic methods (e.g., fluorescence, circular
dichroism), can reveal the progression of distinctive
intermediates during refolding processes.
12.5.2 Disulfide-bonded intermediates can be
trapped covalently by blocking uncombined
cysteines with iodoacetate.
12.5.3 Pulsed hydrogen-deuterium exchange
can be used to monitor the acquisition of secondary
structures in protein folding.
12.5.4 Our understanding of protein folding can
be stringently tested by designing novel proteins with
distinctive functions. For example, encouraging starts
have been made in synthesizing new scaffolds, metalbinding proteins, channels, and catalysts.
12.6 Protein folding in vivo is sometimes catalyzed by
isomerases and chaperone proteins.
12.6.1 The formation of correct disulfide pairing
in nascent proteins is catalyzed by protein disulfide
isomerase (PDI), which is especially important I
accelerating disulfide interchange in kinetically trapped
folding intermediates.
12.6.2 Peptidyl prolyl isomerases (PPIases)
accelerate cis-trans isomerization of Pro residues
during protein folding.
12.6.3 Molecular chaperones in cells
facilitate the correct assembly (including folding,
refolding, formation of oligomeric complexes, …
etc) of other polypeptides but are not themselves
part of the assembled, functional structure. Are
they enzymes? Facilitated search (e.g., avoiding
aggregates)? How? Certainly, they do not specify
the final structure? But do they specify the path?
May specify certain properties of the paths and/or
intermediates. May provide an appropriate
environment for folding. Many questions are yet
to be answered.