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Chapter 6 Proteins in Action
1. Hemoglobin is a multisubunit allosteric protein
that carries O2 in erythrocyte.
1.1 Hemoglobin is a well-studied and well-understood
protein.
1.1.1 It was one of the first proteins to have its
molecular mass accurately determined.
1.1.2 The first protein to be characterized by
ultracentrifuge.
1.1.3 The first protein to be associated with a
specific physiological function.
1.1.4 The first protein with a single amino
acid substitution being related to a genetic
disease (the beginning of molecular medicine).
1.1.5 The first multisubunit protein with
its detailed atomic structure determined by Xray crystallography.
1.1.6 The best understood allosteric
protein.
1.2 Determination of the atomic structure of
hemoglobin A (from normal adult) is very revealing.
1.2.1 The protein molecule exists as a a2b2
tetramer.
1.2.2 Each subunit has a structure strikingly
and unexpectedly similar to each other and to that
of myoglobin, indicating quite different amino acid
sequences can specify very similar 3-D structures.
1.2.3 Extensive interactions exist between the
unlike subunits through noncovalent interactions.
1.2.4 Quaternary structure changes markedly
when O2 binds. Crystals of deoxyhemoglobin shatter
(break) when exposed to O2.
1.2.5 O2 binds to the sixth coordination
position of the ferrous iron (as in myoglobin).
1.3 Hemoglobin is a much more intricate and sentient
(sensitive) molecule than is myoglobin.
1.3.1 The oxygen-binding (dissociation) curve of
hemoglobin is sigmoidal and that of myoglobin is
hyperbolic.
1.3.2 Myoglobin has a higher affinity for O2,
evolved for O2 storage.
1.3.3 Hemoglobin releases O2 efficiently at low
oxygen level tissues (evolved for O2 delivery), myoglobin
does not.
1.3.4 Oxygen binding of hemoglobin shows positive
cooperativity. The binding of O2 (the ligand) at one heme
facilitates the binding of O2 at the other hemes on the
same tetramer (vice versa, unloading of oxygen at one
heme facilitates the unloading of oxygen at the others).
(Negative cooperativity refers to a decrease of activity.)
1.3.5 Increasing concentrations of H+ (with a
decrease of pH) or CO2 lowers the O2 affinity of
hemoglobin (H+ and CO2 has no effect on O2 affinity of
myoglobin). This is called Bohr effect, which helps the
release of O2 in the capillaries of actively metabolizing
tissues. (melecular mechanism?)
1.3.6 One molecule of 2,3-diphosphoglycerate
(BPG) binds to the central cavity of one tetramer of
hemoglobin, which lowers its O2 affinity.
1.3.7 Fetal hemoglobin (HbF) binds BPG less
strongly than does hemoglobin A (adult) and
consequently has a higher oxygen affinity. (physiological
function? Extraction of O2 from the mother)
1.4 Cooperativity is a particular case of an
allosteric effect
1.4.1 Allosteric effect refers to the
phenomenon in which a molecule (allosteric
effector) bound to one site on a protein causes a
conformational change in the protein such that
the activity of another site on the protein is
altered (increased or decreased).
1.4.2 H+, CO2, and BPG all show an
allosteric effect (heterotrophic?) for the O2
binding process of hemoglobin.
1.5 Two models have been proposed to explain the
allosteric regulation phenomena
1.5.1 The sequential model (proposed by
Daniel Koshland, Jr.) hypothesizes that the binding
of one ligand to one subunit changes the
conformation of that particular subunit from the T
state (with a low activity) to the R state (with a high
acitvity), a transition that increases the activity of
the other subunits for the ligand.
1.5.2 The sequential model can be analogized
to the tearing process of postage stamps. (see fig.)
1.5.3 The concerted model (proposed by Monod,
Wyman, and Changeux) hypothesizes that symmetry
is conserved in allosteric transitions (all subunits are
in the same conformation) and the binding of each
ligand increases the probability that all subunits in
that molecule are converted to the R-state (with a high
activity). All-or-none model.
1.5.4 The interplay between these different
ligand-binding sites is mediated primarily by changes
in quaternary structure. The contact region between
two subunits can serve as a switch that transmits
conformational changes from one subunit to another.
T: tense state, circle, less active;
R: relaxed state, square, more active
Concerted, all-or-none
Sequential
1.5.5 The functional characteristics of an
allosteric protein are regulated by specific
molecules in its environment. In another words,
in the evolutionary transition from myoglobin to
hemglobin, a macromolecule capable of
perceiving information from its environment has
emerged.
1.6 Sickle-cell anemia was found to be caused by a single
amino acid change in the b chain of hemoglobin
molecules.
1.6.1 The hemoglobin molecule from sickle-cell
anemia patients (HbS) was found to have a higher pI
value (having more net positive charges).
1.6.2 Peptide fingerprinting (protease digestion +
electrophoresis + chromatography) of HbS and HbA (wt)
revealed that all but one of the peptide spots matched.
1.6.3 Amino acid sequencing revealed that HbS
contains Val instead of Glu is at position 6 of the b chain!
1.6.4 The oxygen binding affinity and allosteric
properties of hemoglobin are virtually unaffected by this
change (the b6 is located at the surface of the protein).
1.6.5 High concentration of deoxygenated HbS
forms fiber participatates, which sickles the red blood
cells, because the fiber formation is a highly concerted
reaction.
1.6.6 Presence of Val6 on the b subunits generates a
hydrophobic patch on the surface which complements
with another hydrophobic patch formed only in
deoxygenated HbS, thus generating the fiber precipitates
(a polymer of HbS).
1.6.7 Sickle cell trait (heterozygote) confers a small
but highly significant degree of protection against the
most lethal form of malaria (probably by accelerating the
destruction of infected erythrocytes, in Africa).
1.6.8 Fetal DNA can be analyzed for the presence of
the HbS gene (prenatal DNA diagnosis).
1.7 Thalassemias are genetic disorders
characterized by defective synthesis of one or
more hemoglobin chains.
1.7.1 This can be caused by a missing gene,
impaired RNA synthesis or processing,
generation of grossly abnormal proteins.
binding sites occupied
[L]
 = ------------------------------- = ------------total binding sites
[L] + Kd
 is a measurable quantity in experiment.
[L] is the free ligand concentration.
The ordinate = n log[L] - log Kd
Bohr’s effect and its molecular mechanism
Release of O2 in
peripheral tissues
Binding of O2 in lungs with
release of H+.
Stabilization of T state??
legend???
Cavity in the tetramer hole
Mutation and molecular interaction’s changes
2. Immunoglobulin superfamily members,
found on cell surfaces or secreted, are widely
used for specific molecular recognition.
2.1 An immunoglobulin G (IgG) molecule (of ~150 kD)
was found to contain two light and two heavy chains,
connecting to each other through disulfide bonds.
2.1.1 Papain digestion convert the IgG molecule
into two Fab and one Fc fragments.
2.1.2 Each Fab fragment (containing one
complete light chain and half of a single heavy chain)
binds one molecule of antigen in a similar manner to
the original immunoglobulin molecule.
2.1.3 Each IgG molecule binds to two molecules of
antigens (thus called bivalent).
2.2 Complete amino acid sequence analysis of purified
myeloma patient’s immunoglobulins revealed strikingly
that the L and H chains consist of variable and constant
regions.
2.2.1 Residues 1 to 108 in the L chains are
relatively variable, and 109 to 204 relatively constant.
2.2.2 Residues 1 to 108 in the H chains are
variable, and 109 to 446 relatively constant.
2.2.3 Three segments in the L chain and three in
the H chain display far more variability than do others,
which are thus named as hypervariable segments (also
called complementary-determining regions, or CDRs,
because they determine antibody specificity).
2.2.4 Amino acid sequence analysis revealed that
the variable region of the light chain (VL) is similar in
sequence to that of the heavy chain (VH).
2.2.5 The constant region of each heavy chain
can divided into three parts (CH1, CH2, CH3) of
similar sequences.
2.2.6 The amino acid sequence of the constant
region of each light chain (CL) is similar to that of the
three parts in the constant region of the heavy chains.
Rodney Porter and Gerry Edelman were
awarded the Nobel Prize in 1972 in Medicine or
Physiology for their structure-function studies on the
antibody molecules.
2.3 X-ray crystallography studies revealed, strikingly,
that the immunoglobulin molecules are made of 12
structurally similar domains.
2.3.1 Each domain has a recurring structural
motif, called the immunoglobulin fold consisting of two
broad sheets of antiparallel b-strands joined by a
disulfide bond(?).
2.3.2 The CDRs of both VL and VH are located
in loops at one end of the sandwich made of the two bsheets that come together to form one antigen binding
site.
2.3.3 The immunoglobulin core serves as a
framework that allows almost indefinite variation of
the CDR loops, corresponding to various antigen
specificity of various antibodies.
2.4 T cell receptors, Class I and Class II Major
histocompatibility complex (MHC) proteins, and
intracellular adhesion molecules (ICAMs) all contain
domains similar to the immunoglobulin domains, thus
belonging to the immunoglobulin superfamily.
2.4.1 The immunoglobulin domains (folds) are
widely observed in these protein molecules, revealed by
sequence homology (~20% identity) and similar foldings
in some are confirmed by X-ray structure determination.
2.4.2 All these proteins are involved in molecular
recognition (antigen recognition by antibodies, TCR, and
MHC proteins; cell-cell interactions by ICAMs).
2.4.3 Most of the immunoglobulin superfamily
members are found on cell surfaces (e.g., IgM, TCR,
MHC) or secreted (IgG).
CDR1, CDR2, CDR3, CDR=complementarity determining region
3. ATP-driven conformational cycles lead to muscle
contraction.
3.1 Striated muscles contain overlapping arrays
of thick and thin filaments.
3.1.1 The thick filaments are primarily
made of myosin protein.
3.1.2 The thin filaments are primarily made
of actin, tropomyosin, and the troponin complex.
3.1.3 Thick and thin filaments slide past
each other in muscle contraction (a model
proposed based on X-ray, light microscope, and
EM studies, fig.)
3.2 The force of muscle contraction arises from
the interplay of myosin, actin, and ATP.
3.2.1 Myosin consists of two globular
heads joined to a long a-helical coiled coil tail.
3.2.2 The myosin molecule can be cleaved
by trypsin into two partially functional
fragments, with one being able to form
filaments, and the other being ATPase and able
to bind actin.
3.2.3 Myosin molecules spontaneously
assemble into filaments in solutions of
physiological ionic strength and pH.
3.2.4 Actin molecule exist in monomer form (Gactin) at low ionic strength and polymerizes into a
fibrous form (F-actin, very similar to the thin filaments)
at physiological (higher) ionic strength.
3.2.5 Threads of actomyosin complex are formed
when mixing actin and myosin in solution.
3.2.6 Addition of ATP dissociates actomyosin into
actin and myosin.
3.2.7 The actomyosin threads contracts when
immersed in a solution containing ATP, K+, and Mg2+,
whereas threads formed from myosin alone does not.
3.2.8 Myosin, being an ATPase, can be
regarded as an mechanoenzyme catalyzing the
conversion of chemical bond energy into
mechanical energy.
3.2.9 The ATPase activity of myosin is
markedly enhanced when it binds to the
polymerized form of actin (F-actin).
3.2.10 The hydrolysis of ATP drives the
cyclic association and dissociation of actin and
myosin. (model, fig.)
3.3 The power stroke in contraction is driven by
conformational changes in the myosin head.
3.3.1 In resting muscle, the myosin heads, with
bound ADP and Pi, are unable to interact with the actin
units in thin filaments because of steric interference by
tropomyosin, a regulatory protein.
3.3.2 When muscle is stimulated, tropomyosin
shifts position, and the myosin head (with bound ADP
and Pi) reaches out from the thick filament and interact
with the actin units on thin filaments.
3.3.3 The binding of myosin-ADP-Pi to actin leads
to the release of ADP and Pi, which induces a major
conformational change in the myosin head pulling the
actin filament forward for about 100 Angstroms.
3.3.4 Subsequently, ATP binds to the
myosin head (myosin prefers binding to ATP than
to actin) and thus detaches it from actin.
3.3.5 Finally, the bound ATP is hydrolyzed
by the free myosin head, resetting it for the next
interaction with the thin filament.
3.3.6 The essence of the process is a cyclic
change both in the conformation of the myosin
head and its affinity for actin.
3.3.7 The control of protein-protein
interactions by bound nucleotides (ATP, GTP, etc)
is a recurring theme in biochemistry.
4. Troponin and tropomyosin mediate the
regulation of muscle contraction by Ca2+.
4.1 Ca2+ is released from sarcoplasmic
reticulum (fig.) in muscle cells at the
stimulation of a nerve impulse.
4.2 Ca2+ controls muscle contraction by an
allosteric mechanism (through conformational
changes) in which the flow of information is in
the following order: Ca2+, the troponin complex,
tropomyosin, actin, myosin. (are all their
structures determined?)
5. Other accessory proteins maintain the
architecture on the myofibril and provide it with
elasticity.
5.1 Springlike titin molecules, the largest protein
so far found in nature (~3000 kD?), extend from
the thick filaments to the Z disc.
5.2 Nebulin, another large protein molecule, is
closely associated with the actin thin filaments,
and consists of almost entirely of a repeating 35amino-acid acting-binding motif.