12 miller chap.3 lecture

Chap.3 Protein Structure & Function
Topics
• Hierarchical Structure of
Proteins
• Protein Folding
• Examples of Protein Function-
Ligand-binding Proteins &
Enzymes
• Regulating Protein Function by
Protein Degradation
• Regulating Protein Function by
Noncovalent and Covalent
Modifications
Goals
Learn the basic structure and
properties of proteins and
enzymes, which carry out most
of the work in cells (Fig. 3.1).

Overview of Protein Structure Hierarchy
The four levels of
protein structure are
illustrated in Fig. 3.2.
A detailed discussion
of each of these
levels is presented in
the next few slides.
Experiments have
shown that the final
3D tertiary structure
of a protein ultimately
is determined by the
primary structure
(amino acid sequence).
The 3D fold (shape) of
the protein determines
its function.

Primary Structure
The primary structure of a
protein refers to its amino acid
sequence. Amino acids in peptides
(<30 aas) and proteins (typically
200 to 1,000 aas) are joined
together by peptide bonds (amide
bonds) between the carboxyl and
amino groups of adjacent amino
acids (Fig. 3.3). The backbone of
all proteins consists of a [-N-
Cα(R)-C(O)-] repetitive unit.
Only the R-group side-chains
vary. By convention, protein
sequences are written from left-
to-right, from the protein’s N-
to C-terminus. The average yeast
protein contains 466 amino acids.
Because the average molecular
weight of an amino acid is 113
daltons (Da), the average N
molecular weight of a yeast Cα(R)
protein is 52,728 Da. Note that
1 Da = 1 a.m.u. (1 proton mass).

Secondary Structure: α Helix
Secondary structure refers to
short-range, periodic folding
elements that are common in
proteins. These include the α helix,
the β sheet, and turns. In the α
helix (Fig. 3.4), the backbone
adopts a cylindrical spiral structure
in which there are 3.6 aas per
turn. The R-groups point out from
the helix, and mediate contacts to
other structure elements in the
folded protein. The α helix is
stabilized by H-bonds between
backbone carbonyl oxygen and amide
nitrogen atoms that are oriented
parallel to the helix axis. H-bonds
occur between residues located in
the n and n + 4 positions relative to
one another.

Secondary Structure: β Sheets & Turns
In β sheets (a.k.a. “pleated
sheets”), each β strand adopts
an extended conformation (Fig.
3.5). ß strands tend to occur in
pairs or multiple copies in β
sheets that interact with one
another via H-bonds directed
perpendicular to the axis of each
strand. Carbonyl oxygens and
amide nitrogens in the strands
form the H-bonds. Strands can
orient antiparallel (Fig. 3.5a) or
parallel (not shown) to one
another in β sheets. R-groups of
every other amino acid point up
or down relative to the sheet
(Fig. 3.5b). Most ß strands in
proteins are 5 to 8 aas long. ß
Turns consist of 3-4 amino acids
that form tight bends (Fig. 3.6).
Glycine and proline are common in
turns. Longer connecting ß turn
segments between ß strands are
called loops.

Tertiary Structure
Tertiary structure refers to the
folded 3D structure of a protein.
It is also known as the native
structure or active conformation.
Tertiary structure mostly is
stabilized by noncovalent
interactions between secondary
structure elements and other
internal sequence regions that
cannot be classified as a particular
type of secondary structure. The
folding of proteins is thought to
be driven by the need to place the
most hydrophobic regions in the
interior out of contact with water
(Fig. 3.7). The structures of
hundreds of proteins have been
determined by techniques such as
x-ray crystallography and NMR.
Different methods of representing
structures are shown in Fig. 3.8.
Keep in mind that most proteins are somewhat flexible and
undergo subtle conformational changes while carrying out their
functions.

Secondary Structure Motifs
Secondary structure motifs are evolutionarily conserved
collections of secondary structure elements which have a defined
conformation. They also have a consensus sequence because the
aa sequence ultimately determines structure. A given motif can
occur in a number of proteins where it carries out the same or
similar functions. Some well known examples such as the coiled-
coil, EF hand/helix-loop-helix, and zinc-finger motifs are
illustrated in Fig. 3.9. These motifs typically mediate protein-
protein association, calcium/DNA binding, and DNA or RNA
binding, respectively.

Quaternary Structure
Multisubunit (multimeric)
proteins have another level
of structural organization
known as quaternary
structure. Quaternary
structure refers to the
number of subunits, their
relative positions, and
contacts between the
individual monomers in a
multimeric protein. The
quaternary structure of
the trimeric hemagglutinin
surface protein of
influenza virus is shown in
Fig. 3.10b. The tertiary
structure of a
hemagglutinin monomer is
shown in Fig. 3.10a.

Modular Domain Structure of Proteins
Domains are independently folding and functionally specialized
tertiary structure units within a protein. The respective
globular and fibrous structural domains of the hemagglutinin
monomer (which happen to be individual polypeptide chains) are
illustrated above in Fig. 3.10a. Domains (such as the EGF
domain) also may be encoded within a single polypeptide chain,
as illustrated in Fig. 3.11. Domains still perform their
standard functions although fused together in a longer
polypeptide (e.g., DNA binding and ATPase domains of a
transcription factor). The modular domain structure of many
proteins has resulted from the shuffling and splicing together
of their coding sequences within longer genes.

Epidermal growth
factor (EGF) domain

Supramolecular Structure
In many cases, multimeric proteins
achieve extremely large sizes,
e.g., 10s-100s of subunits. Such
complexes exhibit the highest level
of structural organization known as
supramolecular structure. Examples
include mRNA transcription
preinitiation complexes (Fig. 3.12),
ribosomes, proteasomes, and
spliceosomes. Typically,
supramolecular complexes function
as ”macromolecular machines" in
reference to the fact that the
activities of individual subunits are
coordinated in the performance of
some overall task (e.g., protein
synthesis by the ribosome).

Evolution of Protein Families
Through genome sequencing
and classical gene cloning
approaches, the sequences
of an enormous number of
proteins have been compiled.
Comparison of sequences
shows that most proteins
belong to larger families
that have evolved over time
from a common ancestor
protein, as illustrated for
the globin family of O2
binding proteins (Fig. 3.13).
Proteins that have a common
ancestor are called
homologs. The members of a
protein family often show
>30% sequence ID, have a
common 3D fold, and usually
perform closely related
functions.

Structure of the Globin Proteins
These globular proteins are composed of mostly α helical
secondary structure. The similar folds of the globins can be
readily seen by comparing the structures of the β chain of
hemoglobin, myoglobin, and leghemoglobin (Fig. 3.13). The closely
similar structures of mammalian myoglobin and the hemoglobin β
subunit might be expected, but the resemblance of the distantly
related plant leghemoglobin is
striking. Comparison of the
sequences of the members of
protein families has brought
to light the fact that amino
acids within a given class
exhibit a large degree of
functional redundancy. In
this regard, the 3 proteins
discussed here exhibit less
than 20% identity in their
sequences, yet have the
same structure. Lastly, in
hemoglobin 2 different globin
chains have combined to form
a multisubunit protein.

Overview of Protein Folding
Many experiments have shown that
proteins can spontaneously fold
from an unfolded state to their
folded native state. This proves
that the amino acid sequence
contains enough information to
specify tertiary structure. Bonds
within the peptide backbone seek
out different possible
conformations as the final tertiary
structure is achieved (Fig. 3.14).
Folding tends to occur via
successive conformational changes
leading to secondary and then
tertiary structure elements (Fig.
3.15). The native conformation of
a protein typically is its lowest
free energy, and therefore, most
stable structure. The unfolded
(denatured) conformation of a
protein can be generated by
heating or treatment with certain
organic solvents.

Chaperone-assisted Protein Folding
The folding of many proteins, particularly large ones, is
kinetically slow and is assisted in vivo by folding agents known as
chaperones. These proteins are found in all organisms and even in
different organelles of eukaryotic cells. Chaperones assist in 1)
folding of nascent polypeptides made by translation, and 2) re-
folding of proteins denatured by environmental damage, such as
heat shock. Molecular chaperones bind to unfolded nascent
polypeptide chains as they
emerge from the ribosome,
and prevent aggregation,
misfolding, and degradation
(Fig. 3.16). The hydrolysis
of ATP by the chaperone
drives conformational
changes that prevent
aggregation and help drive
protein folding. Accessory
proteins participate in the
process. Eukaryotic
molecular chaperones such
as Hsp 70 (cytosol & mito
matrix) and BiP (ER) are
related to the bacterial
protein DnaK.

Chaperonins
Eukaryotic chaperonins such as the TriC complex are large
multimeric complexes related to the bacterial GroEL and GroES
proteins. These complexes take up unfolded proteins into an
internal chamber for folding (Fig. 3.17). ATP hydrolysis drives
folding.

Neurodegenerative Diseases
In neurodegenerative diseases
such as Alzheimer's disease and
transmissible spongiform
encephalopathy (mad cow),
insoluble misfolded proteins
accumulate in the brain in
pathological lesions known as
plaques, resulting in
neurodegeneration (Fig. 3.18).
In Alzheimer's disease, the
protein known as amyloid
precursor protein is cleaved into
a peptide product (β-amyloid)
that aggregates and precipitates
in amyloid filaments. The
misfolding of β-amyloid, which
involves a transition from α
helical to β sheet conformation
leads to filament formation. In
mad cow disease, prion proteins
precipitate causing lesions.

Ligand-binding Proteins
The term ligand refers to any molecule that can be bound by a
protein. Ligands may be hormones, metabolites, or even other
proteins. Ligand binding requires molecular complementarity. The
greater the degree of complementarity, the higher the specificity
and affinity of the interaction. Affinity is reflected in the Kd for
binding. Protein-ligand binding is illustrated here for antibodies
(Fig. 3.19a). The complementarity-determining regions (CDRs) of
the antibody make highly specific contacts with epitopes in the
antigen (Fig. 3.19b).
CDR Epitope
(a)

Overview of Enzyme Catalysis I
Enzymes are proteins (a few are RNAs called ribozymes) that
catalyze chemical reactions within living organisms. Enzyme-
catalyzed reactions typically are highly specific, and rate
enhancements of 106-1012 are common. In an enzyme-catalyzed
reaction, the reactant (the substrate) is converted into the
product. Like all catalysts, enzymes are not consumed in a
reaction. Further, they do not change the ∆G0' or Keq for the
reaction, only its rate.
Rate enhancement is
achieved due to the
fact that enzymes are
most complementary to
the transition state
structure formed in
the reaction. This
results in stabilization
of the transition state
and lowering of the
activation energy
barrier (∆G‡) for the
reaction (Fig. 3.20).

Overview of Enzyme Catalysis II
The transformation of a substrate to the
product occurs in the active site of an
enzyme. The active site can be subdivided
into a catalytic site wherein amino acids
that catalyze the reaction reside, and a
binding pocket that recognizes a specific
feature of the substrate, conferring
specificity to the enzyme-substrate
interaction. A schematic model for an
enzyme catalyzed reaction is shown in Fig.
3.23. The kinetic equation describing the
reaction E + S ⇔ ES → E + P. A reaction
coordinate diagram showing the binding and
catalytic steps of an enzyme catalyzed
reaction is shown in Fig. 3.24.

Enzyme Kinetics: Enzyme Concentration
The velocity of an enzyme-catalyzed reaction reaches a maximal
rate (Vmax) at high concentrations of substrate (Fig. 3.22a). V max
is achieved when all enzyme molecules have bound the substrate
and are engaged in catalysis (saturation). The French
mathematicians Michaelis and Menten developed a kinetic
equation to explain the behavior of most enzymes. They showed
that the maximal rate of an enzyme-catalyzed reaction (V max)
depends on the concentration of enzyme (Fig. 3.22a) and the
rate constant for the rate-limiting step of the reaction.

MM equation:
x 1.0 Vmax [S]
V0 =
x [S] + KM

x 0.5

x

Enzyme Kinetics: Substrate Affinity
Michaelis and Menten also derived a kinetic constant, the
Michaelis constant (KM), that is indicative of the affinity of most
enzymes for their substrates. The lower the KM the higher the
affinity of the enzyme for the substrate (Fig. 3.22b). The K M
happens to be the concentration of substrate at which the
reaction rate is half-maximal. The concentrations of cellular
metabolites usually are set near the KMs of the enzymes that
carry out their metabolism. This allows cells to respond to
changes in substrate concentration.

1/2 Vmax

Mechanism of Serine Proteases I
Proteases are enzymes that cleave peptide bonds in other
proteins. The serine proteases, which are important for
digestion and blood coagulation, contain reactive serine residues
in their catalytic sites. Also present are aspartate and
histidine residues that together with serine make up what is
called the catalytic triad. The active sites of serine proteases
also contain binding pockets that confer specificity by
positioning the peptide bond that is to be cleaved next to the
reactive serine (Fig. 3.25a, trypsin). The digestive proteases
trypsin, chymotrypsin, and elastase select cleavage sites based
on the features of their binding pockets (Fig. 3.25b).

Gly
X

Specificity
Trypsin-basic aas
Chymotrypsin-aromatic aas
Elastase-small side-chain aas

Mechanism of Serine Proteases II
In the serine protease reaction mechanism, an acyl enzyme
intermediate is formed transiently after peptide bond cleavage
by serine (Fig. 3.26). Subsequently, the acyl group is hydrolyzed
off the serine later in the reaction. Both acid-base catalysis
(Steps a,c,d,& f) and transition state stabilization (Steps b & e)
occur during the reaction. The reaction mechanism is inhibited at
low pH due to protonation of His-57 (inset). The pH optimum of
serine protease reactions therefore occurs at or slightly above
neutrality.

Multifunctional Enzymes
Most metabolic pathways
occur via multiple enzyme-
catalyzed steps. As illustrated
in Fig. 3.28, the rates of
pathway reactions can be
increased if the substrates
and products of each step are
channeled to the next enzyme
in the pathway. Channeling is
enhanced in multisubunit
enzyme complexes and by
attachment of enzymes to
scaffolds (Fig. 3.28b), or
even by fusion of encoded
enzymes into a single
polypeptide chain (Fig. 3.28c).

Regulating Protein Function by Degradation
The proteolytic degradation (turnover) of proteins is important for
regulatory processes, cell renewal, and disposal of denatured and
damaged proteins. Lysosomes carry out degradation of endocytosed
proteins and retired organelles.
Cytoplasmic protein degradation
is performed largely by the
molecular machine called the
proteasome. Proteasomes
recognize and degrade
ubiquinated proteins (Fig.
3.29). Ubiquitin is a 76-amino-
acid protein that after
conjugation to the protein,
targets it to the proteasome.
In ATP-dependent steps, the
C-terminus of ubiquitin is
covalently attached to a lysine
residue in the protein.
Polyubiquitination then takes
place. The proteasome
degrades the protein to
peptides, and released ubiquitin
molecules are recycling.

Regulating Function by Ligand Binding
The binding of a ligand to a
protein typically triggers an
allosteric ("other shape")
conformational change resulting
in the modification of its
activity. An overview of
regulation via allosteric
transitions is presented here in
the context of the tetrameric
O2 binding protein, hemoglobin
(Hb). As shown in Fig. 3.30,
the O2 binding curve for Hb
does not show the simple
hyperbolic shape exhibited by
proteins that bind a ligand with
the same affinity regardless of ligand concentration. Instead,
the Hb O2-binding curve is sigmoidal which indicates that the
affinity for O2 molecules increases after the first 1 or 2 have
bound. In this case, binding displays positive cooperativity.
Negative cooperativity is observed with other protein-ligand
systems. The reduced O2 binding affinity of Hb at low O2
tensions favors release of O2 to peripheral tissues.

Calmodulin-mediated Switching
Many proteins play switching
functions in cell signaling. Calcium
ion (Ca2+) is a very important
messenger in cell signaling. Cells
maintain cytoplasmic calcium
concentration at about 10-7 M.
When calcium concentration rises
above this level due to hormone-
receptor signaling processes, etc., Ca2+
it binds to a protein known as
calmodulin (Kd = 10-6 M) triggering
conformational changes that result
in its activation. Calmodulin
contains 4 helix-loop-helix motifs
(EF hands) each of which can bind
calcium (Fig. 3.31). Calcium
binding causes a major allosteric
transition in calmodulin. In its
alternate conformation, calmodulin
binds to target proteins, changing
their activity.

GTPase-mediated Switching
Proteins belonging to the GTPase superfamily, such as Ras and G
proteins, serve as guanine nucleotide-dependent regulatory
switches that control of the activity of specific target proteins
(Fig. 3.32). When bound to GTP, these proteins adopt an active
conformation that modulates target protein function. When bound
to GDP, their activity is turned off. The time-frame of activation
depends on the intrinsic GTPase activity (the timer function) of
these proteins. In addition, GTP and GDP binding (and thus
activity) may be regulated by other factors. Examples of such
regulation will be covered later.
Target protein
function

Regulation by Kinase/Phosphatase Switching
Protein function also can be regulated by allosteric transitions
caused by covalent modification via phosphorylation (Fig. 3.33).
Phosphorylation typically occurs on serine, threonine, and tyrosine
residues. Enzymes known as kinases carry out phosphorylation.
Their activity is opposed by phosphatases, which hydrolyze
phosphates off of the modified amino acid. Some proteins are
turned on by phosphorylation; others are turned off.

12 miller chap.3 lecture

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12 miller chap.3 lecture