Transcript Protein Function and Evolution

Protein Function and Evolution
Role of Globins in O2 Transport and Storage
Red Blood Cells (Erythrocytes)
Myoglobin (Mb) and Hemoglobin (Hb)
Structures of Porphyrins
Ferrous iron, Fe2+,
in heme binds O2.
Heme also has high
affinity for other
molecules, such as
carbon monoxide.
This is why CO is
toxic.
The Heme Prosthetic Group
Geometry of Iron in Oxyhemoglobin
Iron in Oxyhemoglobin
Heme bound to protein
as prosthetic group
(tightly bound co-factor)
protects heme iron from
oxidation (from Fe2+ to
Fe3+ oxidation state),
lowers heme’s
extremely high affinity
for CO, and allows for
regulation of O2 binding
affinity in hemoglobin.
His93 (F8) = proximal His
His64 (E7) = distal His
Binding of Oxygen and Carbon Monoxide
to Heme Iron
O2 Binding Curve for Myoglobin
Equations for Myoglobin Binding O2
Mb + O2 <-> MbO2
K (association constant) = [MbO2]/([Mb][O2])
 (theta; fractional occupancy) = sites occupied/total available sites
 = [MbO2]/([Mb]+[MbO2]) = K[Mb][O2]/([Mb]+ K[Mb][O2])
 = K[O2]/(1+ K[O2]) = [O2]/(1/K + [O2])
1/K = Kd (dissociation constant) = P50 (O2 partial pressure for halfmaximal saturation), so
 = PO2/(P50 + PO2)
Dynamics of 02 Release by Myoglobin
Kd = 1/K = ([Mb][O2])/[MbO2] =
koff/kon
von (on rate) = kon[O2][Mb]
voff (off rate) = koff[MbO2]
Binding Curve Required for a Transport
Protein
Cooperative Binding and Allostery
allostery = “other site”
• T (“tense”) conformational state: low-affinity ligand-binding state
of protein.
• R (“relaxed”) conformational state: high-affinity binding binding
state of protein.
• Homotropic allosteric interaction: effector and ligand regulated
by the effector are the same molecule (e.g., O2 binding affects
subsequent O2 binding).
• Heterotropic allosteric interaction: effector and ligand are
different molecules (e.g., H+ or BPG binding affects O2 binding).
• Positive allosteric interaction: effector binding increases affinity
for ligand.
• Negative allosteric interaction: effector binding decreases
affinity for ligand.
Evaluating Cooperativity
Fractional O2 occupancy:
 = PO2n/(P50n + PO2n)
Rearrange and take logarithms for Hill plot:
log(/(1-)) = nlogP02 - nlogP50
(assumes n = # of O2 binding sites & all O2 bind simultaneously)
In fact, experimentally determined “n” value is Hill coefficient (nH).
nH  n except for hypothetical, wholly cooperative process where all ligand
molecules would bind simultaneously.
nH < n for all real systems.
Hill coefficient very useful in describing cooperativity, since:
nH = 1: non-cooperative process
nH > 1: positive cooperativity
nH < 1: negative cooperativity
Archibald Hill (1910)
Hill Plots for O2 Binding for Mb and Hb
For Mb:
nH = 1
This indicates non-cooperative
process.
For Hb:
nH (max slope) = 3.0-3.5
This indicates positive cooperativity
(since nH > 1) with at least 4 binding
sites for O2 (since n always > nH).
•Intercepts with broken black line at the 0
value for log(/(1-)) indicate P50 and so O2
binding affinity (lower P50 = higher affinity)
•Hb high-affinity 02-binding logP50 = upper
asymptote intercept
•Hb low-affinity 02-binding logP50 = lower
asymptote intercept
Scatchard Plots
Bound ligand/free ligand vs. bound ligand
X-axis intercept indicates maximum
amount of ligand bound (Bmax) or total
number of ligand binding sites (n),
e.g., 1 for Mb, 4 for Hb.
Slope = -K = -1/Kd (or -1/P50)
Shape of curve gives indication
of whether there is
cooperativity (positive or
negative) or not.
Note: Scatchard plots and other data plotting methods
in biochemistry are used a great deal for
visual/graphical representation even today.
Biochemical parameters used to be determined by
manual plotting but now computers are used, since
regression analysis on a computer is much more
accurate for determining n, Kd, etc.
Subunit Interactions in deoxyHb
(T State)
Some Major Interactions in deoxyHb that Are
Disrupted in T -> R Transition to oxyHb
Some Major Interactions in deoxyHb that Are
Disrupted in T -> R Transition to oxyHb
Changes at a1-b2 and a2-b1 Interface in T > R Transition in Hb
Change in Hb 4o Structure with O2 Binding
Some major changes:
•Rotation of a1b1 relative
to a2b2.
•Change in size of
central cavity.
•Shift of C-termini and
FG corners of b chains
relative to C helices of a
chains
•C-termini of b chains
interact with C helices of
a chains in T (deoxy)
state. These
interactions are
broken in transition to
R (oxy) state.
Mechanism of T -> R Transition: Iron
Pulled into Heme Plane when O2 Binds
His F8 (proximal His) also dragged along in T -> R transition, pulling F
helix and shifting subunits relative to one another, increasing O2 affinity of
binding sites on other subunits.
Perutz Model (1970)
Movement of Heme and F Helix in T -> R
Transition in Hb
Effect of Replacing Proximal His in Hb with Gly
and Adding Imidazole
Normal Hb: Cooperativity
Replacement:
No cooperativity
Negative Allosteric Effectors of O2 Binding in
Hb: Stabilizers of T State of Hb
• H+ (“The Bohr effect,” Christian Bohr, 1904)
• 2,3-bisphosphoglycerate (BPG)
• Carbon dioxide (transported in blood as bicarbonate and
carbamates):
– Bicarbonate formation: CO2 + H20 <-> HCO3- + H+
– Carbamate formation:
Hb-NH3+ + HCO3- <=> Hb-NH-COO- + H+ + H2O
– CO2 lowers O2 binding affinity through H+ released (contributing to
Bohr effect) and formation of carbamate at N-termini of Hb b
subunits, stabilizing T state interactions between a and b chains.
Bohr Effect on Hb: Protonation of Certain
Groups on Hb Decreases Affinity for O2
Protonation of a number of
groups favors T state. For
instance:
Protonation of His146 (HC3)
on b chain allows for formation
of T (deoxy) state salt bridge
with Asp94.
Networks of Ion Pairs and Hydrogen
Bonds in DeoxyHb
All of these interactions are broken in T -> R transition.
(White + signs: groups protonated in Bohr effect, stabilizing deoxyHb
T state.)
2,3-Bisphosphoglycerate (BPG)
In mammals
In birds
Binding of BPG to DeoxyHb: Stabilization of T
State of Hb
Combined Effects of CO2 and BPG on O2
Binding by Hb
Role of Globins in O2 Transport and Storage
Release of CO2 in lungs
(or gills in fish).
CO2 carried in veins as
bicarbonate. Also,
deoxyHb carries CO2 as
carbamates.
CO2, H+ and BPG
decrease Hb’s affinity
for O2 and so favor
release of O2 in tissues.
Oxygenation of Hb in lungs.
OxyHb carries O2 in
arteries.
[CO2] (and [H+]) high in tissues
as a result of respiration.
Two Models of Allostery
Koshland, Nemethy, Filmer (KNF) Model (1966): Sequential
or Induced Fit Model
•Ligand binding at one site causes protein conformational
change (induced fit), shifting binding affinity in adjacent
subunits only, so complete T -> R transition is a sequential
process.
•Can account for both positive and negative cooperativity.
Monod, Wyman, Changeux (MWC) Model (1965):
Concerted or Symmetry Model
•Equilibrium between T and R states.
•Transition is a concerted process, affecting all
subunits simultaneously in the same way.
•In absence of ligand, equilibrium favors T state.
•Ligand binding shifts equilibrium toward R state.
•Only models positive cooperativity.
Two Models of Allostery
Two Models of Allostery
Koshland, Nemethy,
Filmer (KNF) Model
(1966): Sequential
or Induced Fit
Model
Monod, Wyman,
Changeux (MWC)
Model (1965):
Concerted or
Symmetry Model
Recent Model for Cooperative Transition of
Hb
If both a1b1 and a2b2 each contain at least one O2 bound, T -> R transition occurs.
Protein Evolution and Diversity
Coding and Noncoding Regions of bHemoglobin Gene
Some Mutagenic Agents
Types of Mutations
Comparison of Sequences of Mb and the
a and b Chains of Hb
Evolutionary Conservation of the Globin
Folding Pattern
Evolution of the Globin Genes
Expression of Human Globin Genes at
Different Stages of Development
Fetal Hb (a2g2) has
low affinity for BPG,
which facilitates
transfer of O2 to fetus,
since in the presence
of BPG, fetal a2g2 Hb
has higher affinity for
O2 than does adult
a2b2 Hb.
Some Missense Mutations in Human
Hemoglobins
In addition to missense mutations in human hemoglobins, there are other
hemoglobin diseases called thalassemias in which a or b chains are not
produced at all or produced in insufficient quantities.
Distribution of Mutations in Human
Hemoglobins
Inheritance of Normal and Variant
Proteins in Heterozygous Cross
Sickle-Cell Anemia
Red blood cells become abnormally elongated and sickle-shaped. Sickled
cells block capillaries and die prematurely.
Sickle-Cell Hemoglobin (HbS)
Sickle-cell anemia:
First disease for which a
plausible molecular explanation
was put forward (Pauling and
coworkers, 1949 - "Sickle cell
anemia: a molecular disease").
b6 (A3) Glu -> Val substitution
(Ingram and Hunt, 1956):
DeoxyHbS forms abnormal
polymer, causing red blood
cells to sickle.
Homozygosity for sickle-cell
hemoglobin (HbS/HbS) is
lethal in childhood.
Heterozygosity (HbA/HbS)
increases resistence to
malaria, which explains its
prevalence in tropical areas of
the world.
Structure of Sickle-Cell Hemoglobin (HbS)
FIbers
Immunoglobulins (Antibodies):
Diversity in Structure and Binding
Antigenic Determinants
Most antigens are foreign proteins or polysaccharides.
Interactions of Antigen with Antibody
Schematic Model of Antibody Molecule
Model of X-Ray Structure of IgG
The Immunoglobulin Fold
Generation of Antibody Diversity
VDJ Recombination
Generation of Antibody Diversity
Somatic hypermutation (point mutations)
Clonal Selection Theory of the Immune Response
>107 distinct antibodies generated in humans through variable recombination of exons and
somatic hypermutation in antibody genes in B cells. A single B cell makes a single type of
antibody. Those B cells producing antibodies that bind to a foreign antigen that is present (e.g.,
following infection) are selectively amplified to form large numbers of clones through cell division,
so then more antibodies are produced against that antigen.
Two Developmental Pathways for
Stimulated B Lymphocytes
Antibody attached to B cell
membrane = B-cell receptor
Soluble antibodies released
from effector B cells
(plasma cells).
Most abundant circulating
antibodies: immunglobulin
G (IgG).
Humoral and Cellular Immune Responses
Humoral: secreted
antibodies (mainly IgG)
Cellular: B-cell
receptor on B cells
and T-cell receptor
on killer T cells.
Human Immunodeficiency Virus
HIV binds to a specific cellsurface protein (CD4) on
helper T cells, enters these
cells and kills them, leading
to immunodeficiency.
Technical Applications of Antibodies
Preparation of Polyclonal Antibodies
Production of Monoclonal Antibodies
Enzyme-Linked Immunosorbent Assay
(ELISA)
Western Blot Analysis
Conceptually related
techniques:
•Immunoprecipitation (IP
useful for pull-down or co- IP)
•Immunofluorescence
microscopy