Transcript Chapter 4B Lecture

Chap. 4B The Three-dimensional Structure
of Proteins
Topics
• Overview of Protein Structure
• Protein Secondary Structure
• Protein Tertiary and Quaternary Structure
• Protein Denaturation and Folding
Fig. 4-1. Structure of the enzyme
chymotrypsin, a globular protein
Fibrous Proteins: Silk Fibroin
Fibroins, the proteins of silks, are produced by insects and
spiders. Their polypeptide chains are predominantly in the ß
conformation. Fibroins are rich in Ala and Gly residues,
permitting close packing of ß sheets and an interlocking
arrangement of R groups (Fig. 4-14a). The overall structure is
stabilized by extensive hydrogen bonding between all peptide
linkages in the polypeptides of each ß sheet and by the
optimization of van der Waals interactions between sheets. Silk
does not stretch because the ß conformation is already highly
extended. However the structure is flexible because the sheets
are held together by weak interactions rather than by covalent
bonds as with the disulfide bonds in -keratins.
Globular Proteins: Myoglobin (I)
Myoglobin is a relatively small (Mr 16,700) oxygen-binding protein
of muscle cells. It functions to store oxygen and to facilitate
oxygen diffusion in contracting muscle tissue. It is particularly
abundant in the muscles of diving mammals such as whales, seals,
and porpoises. Myoglobin contains a single polypeptide chain of
153 amino acids and a single prosthetic group, iron protoporphyrin
(heme) (Fig. 4-16). The same heme group is found in erythrocyte
hemoglobin, and is responsible for the deep red-brown color of
both myoglobin and hemoglobin. Myoglobin was the first globular
protein whose structure was determined by x-ray diffraction. In
the diagram in Fig. 4-16, myoglobin structure is represented via
ribbon (a), surface contour (b), ribbon plus side-chain (c), and
space-filling (d) models.
Globular Proteins: Myoglobin (II)
The backbone of myglobin consists of eight relatively straight
right-handed  helices interrupted by bends, some of which are ß
turns (Fig. 4-16a). Seventy-eight per cent of the residues of
myoglobin occur in the  helical regions (Table 4-4, see below).
The heme group is mostly surrounded by the polypeptide chain and
sits in a crevice which is barely exposed at the surface of the
structure (Fig. 4-16 b & d). Most of the hydrophobic amino acid R
groups are in the interior of the molecule, hidden from exposure to
water. All but two polar R groups are located on the outer surface
of the molecule. The atoms inside myoglobin are densely packed,
and thus short-range van der Waals interactions make a significant
contribution to stability. All of the peptide bonds are in the planar
trans configuration. Three of the four Pro residues are found in
bends.
The Heme Group of Myoglobin
A heme group is present in myoglobin, hemoglobin, cytochromes,
and many other proteins. Heme consists of a complex organic ring
structure, protoporphyrin, which binds an iron atom in its ferrous
(Fe2+) state (Fig. 4-17a). The iron atom has six coordination
positions, four of which are in the plane of the protoporphyrin and
bonded to it, and two of which are perpendicular to the ring. In
myoglobin and hemoglobin one of the perpendicular coordination
positions is bound to a nitrogen atom of a His residue in the
polypeptide chain (Fig. 417b). The sixth coordination
position is open and serves as
the binding site for an O2
molecule. It is important that
the myoglobin heme group is
largely sequestered away from
the solvent. The iron of free
heme in solution is readily
oxidized to the ferric (Fe3+)
state, which does not bind O2.
Protein Folding Patterns: Motifs
To understand the complete three-dimensional structure of a
protein, it is important to analyze its folding pattern. Two terms
are used to describe a folding pattern--motif (a.k.a., fold) and
domain. A motif is a recognizable folding pattern involving two or
more elements of secondary structure and the connections
between them. Motifs can be simple, such as the ß--ß loop
motif; motifs also can be elaborate, such the ß barrel motif (Fig.
4-18). In some cases a single large motif may comprise the entire
protein. Motif and fold can be
used interchangeably, however
the term fold is more commonly
applied to describe more complex
folding patterns. For example,
the distinctive arrangement of
eight  helices in myoglobin is
found in all globins and is called
the globin fold.
Protein Folding Patterns: Domains
A domain is a part of a polypeptide chain that is independently
stable or could undergo movements as a single entity with
respect to the entire protein. Each domain can appear as a
distinct globular lobe region as is true for the calcium binding
protein of muscle, troponin C (Fig. 4-19). However, extensive
contacts can occur between domains and make individual domains
hard to discern. The two or more domains of a protein often
have different functions, which are coordinated in the overall
function of the protein. Small proteins such as myoglobin usually
only have one domain.
Protein Folding Patterns: Some Rules
Several rules have emerged from
studies of common protein folding
patterns. 1) Hydrophobic
interactions are important for
protein stability. At least two
layers of secondary structure are
required for shielding of
hydrophobic residues from water.
2)  helices and ß sheets usually
are found in different structural
layers of a protein. 3) Segments
adjacent to each other in the
primary structure are usually
stacked next to each other in the
folded structure. 4) The ß
conformation is most stable when
the individual strands are twisted
slightly in a right-handed sense
(Fig. 4-20c).
Large Motifs Can Be Constructed from
Smaller Ones
Complex motifs can be built up from simpler ones. For example,
a series of ß--ß loops arranged so the ß strands form a
barrel creates a particularly stable and common motif, the /ß
barrel (Fig. 4-21). In this structure each parallel ß segment is
connected to its neighbor by an -helical segment.
Structural Classification Via Motifs
Based on similarities in tertiary structure, different folding
motifs are now grouped into larger classes (e.g., the all  class,
Fig. 4-22 top). Other classes include the all ß, the /ß (with 
and ß segment interspersed or alternating), and the  + ß (with
 and ß regions somewhat segregated) (Fig. 4-22, not shown).
Protein Families and Superfamilies
Based on motif analysis of many proteins, it now is clear that
protein tertiary structure is more reliably conserved than is
amino acid sequence. Proteins with significant similarity in
primary structure and/or with similar tertiary structure and
function are said to be in the same protein family. Proteins
within a family are evolutionarily related. Two or more protein
families that have little similarity in primary structure but
display the same major structural motif are grouped together in
a superfamily. An evolutionary relationship among families in a
superfamily is considered probable even though time and
functional distinctions have erased many of the telltale sequence
relationships.
Multisubunit Proteins
Many proteins have multiple polypeptide subunits (from two to
hundreds). A multisubunit protein is referred to as a multimer. A
multimer with just a few subunits is often called an oligomer. The
repeating structural unit in such a multimeric protein, whether a
single subunit or a group of subunits, is called a protomer. Greek
letters are sometimes assigned to distinguish the individual
subunits that make up a protomer. Multisubunit proteins have
quaternary structure.
Quaternary Structure of Hemoglobin
Hemoglobin (Mr 64,500) contains four polypeptide chains with
one heme prosthetic group each, in which the iron atoms are in
the ferrous (Fe2+) state (Fig. 4-23). The protein portion, globin,
consists of two  chains (141 residues each) and two ß chains
(146 residues each). The subunits of hemoglobin are arranged in
symmetric pairs, each pair having one  and one ß subunit.
Hemoglobin can therefore be described either as a tetramer or
as a dimer of ß protomers.
Intrinsically Disordered Proteins
As many as a third of all human proteins may be unstructured or
have significant content of unstructured segments. These
intrinsically disordered proteins have properties that are distinct
from classical structured proteins. Namely, they can lack a
hydrophobic core, and instead may contain high densities of
charged amino acid residues such as Lys, Arg, and Glu. Pro
residues are also prominent as they tend to disrupt ordered
structures. The lack of an ordered structure can allow a protein
to interact with multiple partners. The mammalian protein p53
plays a crucial role in the
control of cell division. It
features both structured and
unstructured segments (Fig. 424). An unstructured region at
the carboxyl terminus interacts
with at least four different
binding partners and assumes a
different structure in each of
the complexes.
Proteostasis
The maintenance of an active set
of cellular proteins required under
a given set of conditions is called
proteostasis (Fig. 4-25). Three
kinds of processes contribute to
proteostasis. First, proteins are
synthesized on a ribosome.
Second, multiple pathways
contribute to protein folding, many
of which involve the activity of
complexes called chaperones.
Chaperones (including chaperonins)
also contribute to the refolding of
proteins that are partially and
transiently unfolded. Finally,
proteins that are irreversibly unfolded are subject to sequestration
and degradation by several additional pathways. Partially unfolded
proteins and protein-folding intermediates that escape the quality
control activities of the chaperones and degradative pathways may
aggregate, forming both disordered aggregates and ordered
amyloid-like aggregates that contribute to disease processes.
Protein Denaturation (I)
The conformation of a native protein is
only marginally stable. Conditions
different from those in a cell, etc.,
therefore can cause protein structural
changes resulting in the loss of threedimensional structure needed for function
(i.e., denaturation). In the denatured
state, the conformation of the protein
need not be completely randomized. A
number of physical and chemical agents
can cause protein denaturation. A classic
agent is heat, which has complex effects
on many weak interactions in a protein
(primarily on the hydrogen bonds). On
heating, a protein’s conformation generally
remains intact until an abrupt loss of
structure occurs over a relatively narrow
temperature range (the Tm) (Fig. 4-26a).
The abruptness of the loss of structure
suggests a cooperative process in which
loss of structure in one or more parts of
the protein rapidly destabilizes the
structure of other parts.
Protein Denaturation (II)
Proteins also can be denatured by
extremes in pH, by miscible organic
solvents such as alcohol and acetone, by
certain solutes such as urea and guanidine
hydrochloride, or by detergents (Fig. 46b). None of these agents breaks
covalent bonds. Organic solvents, urea
and detergents act primarily by disrupting
the hydrophobic interactions that produce
the stable core of globular proteins. Urea
and guanidine hydrochloride also disrupt
hydrogen bonds. Extremes of pH alter
the net charge on the protein causing
electrostatic repulsion and the disruption
of some hydrogen bonding. The denatured
structures resulting from these various
treatments are not necessarily the same.
Lastly, denaturation often results in
aggregation and precipitation of the
unfolded protein. The protein precipitate
that is seen after boiling an egg white is
a well known example.
Renaturation of Ribonuclease
Early studies examining the
renaturation (refolding) of the enzyme
ribonuclease provided experimental
proof that the tertiary structure of a
protein is determined by its primary
structure (Fig. 4-27). In this
experiment, purified ribonuclease was
denatured to its unfolded inactive state
by exposure to a concentrated solution
of urea and the reducing agent,
mercaptoethanol. This unfolded the
protein and reduced all disulfide crosslinks to cysteine residues. When urea
and mercaptoethanol were removed, the
randomly-coiled, denatured ribonuclease
spontaneously refolded to its
catalytically active state. Even the
protein’s four unique disulfide bonds
reformed correctly. This experiment
provided the first evidence that the
amino acid sequence of a polypeptide
chain contains all the information
required to fold the chain into its
native, three-dimensional structure.
Overview of Protein Folding
Protein folding is thought to occur
via a hierarchical pathway (Fig. 428). Certain amino acid sequences
fold first into local secondary
structures, guided by constraints
discussed earlier in Chap. 4.
Assembly of local structures is
followed by formation of longerrange interactions between these
secondary structure elements to
obtain folding motifs described
above. Throughout the process
hydrophobic interactions play a
significant role in aggregating
nonpolar amino acid side chains in the
core of folding intermediates and the
final tertiary structure. Computer
programs have now been developed
that can often predict the structures
of small proteins on the basis of
their amino acid sequences.
The Thermodynamics of Protein Folding
Thermodynamically, the protein folding process can be viewed as
a kind of free energy funnel (Fig. 4-29). The unfolded states
(top of funnel) are characterized by a high degree of
conformational entropy and relatively high free energy. As folding
proceeds, the narrowing of the funnel reflects the decrease in
the conformational space that must be searched as the protein
approaches its native state (N, at the bottom of the funnel).
Small depressions along the sides of the free energy funnel
represent semistable intermediates that can slow the folding
process. The funnels can have a variety of shapes depending on
the complexity of the folding pathway, the existence of
semistable intermediates, and the potential for particular
intermediates to assemble into aggregates of misfolded proteins.
Chaperones
Many proteins do not fold spontaneously as they are synthesized
by a cell. The folding of these proteins requires the general class
of proteins called chaperones. Chaperones interact with partially
folded or improperly folded polypeptides, facilitating correct
folding pathways or providing microenvironments in which folding
can occur. Several types of chaperones are found in organisms
ranging from bacteria to humans. The two major classes of
chaperones are the molecular chaperones (e.g., the Hsp70 family
of proteins) and chaperonins (e.g., the GroEL/Hsp60 family of
proteins). (Hsp stands for heat shock protein. These proteins are
induced in bacterial cells by exposure to high temperatures.)
Molecular chaperones act as monomers, whereas chaperonins are
large multisubunit complexes.
Chaperone-assisted Protein Folding
Molecular chaperones such as Hsp70, bind to regions of unfolded
polypeptides that are rich in hydrophobic residues. They protect
both proteins denatured by heat and newly synthesized and not
yet folded proteins from aggregation. They also block the folding
of certain proteins that must remain unfolded until they have
been translocated across a membrane. Some chaperones also
facilitate the quaternary assembly of oligomeric proteins.
The Hsp70 proteins bind to and
release polypeptides in a cycle
that uses energy from ATP
hydrolysis and involves several
other proteins, such as Hsp40 and
NEF (nucleotide-exchange factor)
(Fig. 4-30). When the bound
polypeptide is released after a
cycle of ATP hydrolysis, it has a
chance of folding properly. If not,
the protein may be bound again,
and the process repeated. Hsp70
can also deliver unfolded
polypeptides to chaperonins.
Chaperonin-assisted Protein Folding (I)
Chaperonins are elaborate protein complexes required for the
folding of many cellular proteins that do not fold spontaneously. In
E. coli, 10-to-15% of newly translated proteins require the
GroEL/GroES chaperonin system for folding under normal
conditions. This increases to 30% when cells are subjected to heat
stress. The mechanism of protein folding by the GroEL/GroES
chaperonin is shown in the next slide (Fig. 4-31). Each GroEL
complex consists of two large chambers formed by two heptameric
rings. GroES (also a heptamer) blocks one of the GroEL chambers
after an unfolded protein is bound inside. The chamber with the
unfolded protein is referred to as cis; the opposite one is trans.
Folding occurs within the cis chamber during the time it takes to
hydrolyze the 7 ATPs that are bound to subunits in the heptameric
GroEL ring. The GroES and the ADP molecules then dissociate and
the protein is released. The two chambers of the GroEL system
alternate in the binding and facilitated folding of client proteins.
Inside the chamber, a protein has about 10 seconds to fold.
Constraining the protein within the chamber prevents inappropriate
protein aggregation and also restricts the conformational space
that a polypeptide chain can explore as it folds.
Chaperonin-assisted Protein Folding (II)
Protein Folding Disorders: Amyloidoses
In a number of human disorders
(amyloidoses; including Alzheimer
disease), a soluble protein that is
normally produced by a cell adopts a
misfolded state and converts into an
insoluble extracellular amyloid fiber
(Fig. 4-32a). Commonly, fiber
formation is promoted by the
aggregation of regions of proteins that
have a ß conformation. In Alzheimer
disease, proteolytic cleavage of a
neuronal membrane protein (amyloid-ß
precursor protein, APP) produces an helical membrane-spanning peptide
(amyloid-ß peptide) that converts to
the ß conformation and aggregates into
amyloid fibrils (Fig. 4-32 b & c). The
extracellular deposition of amyloid
fibrils is associated with plaque
formation and ultimately death of the
nearby neurons.
Protein Folding Disorders: Prion Diseases (I)
In the neurodegenerative diseases known as spongiform
encephalopathies, a misfolded form of a normal neuronal protein
PrP is responsible for disease. Spongiform encephalopathies occur
in many species of animals. In humans, the disorders are known as
kuru and Creutzfeld-Jacob disease. In cows, the disorder is
known as mad cow disease. In sheep it is called scrapie, and in
deer and elk, it is called chronic wasting disease. The diseased
brain becomes riddled with holes (Fig. 1, Box 4-6). Progressive
deterioration of the brain leads to a spectrum of neurological
symptoms, and is always fatal.
Protein Folding Disorders: Prion Diseases (II)
Prion protein (PrP) is a normal constituent
of brain tissue in all mammals. The protein
is thought to possibly function in cell
signaling. Illness occurs when the normal
cellular PrP (PrPC) folds into an altered
conformation called PrPSc (Sc denotes
scrapie) (Fig. 2, Box 4-6). In PrPSc, part
of the -helical region of PrPC is
converted to the ß conformation. PrPSc
monomers then associate forming amyloidlike ß sheets. Importantly, the interaction
of PrPSc with PrPC converts the latter to
PrPSc, initiating a domino effect in which
more and more of the brain protein
converts to the disease-causing form. For
this reason PrPSc is infectious. The
mechanism by which PrPSc leads to
spongiform encephalopathy is not
understood. In inherited forms of prion
diseases, a mutation in the gene encoding
PrP produces a change in one amino acid
residue that is believed to make the
conversion of PrPC to PrPSc more likely.