Lecture 5 Information Flashcards
Why do humans need more hemoglobin than earthworms?
Earthworms do not move as much as humans and therefore have a slower rate of metabolism and do not need as much O2
Is ligand binding always good?
No
if a toxin binds to a protein and is brought into the cell through endocytosis, the toxins can integrate themselves into the cell membrane and can potentially cause paralysis
How does insulin work as a ligand?
insulin binds to protein on the cell’s membrane which triggers the cell to uptake glucose for metabolism
Induced fit
the structure that a protein assumes as it binds its ligand
maxmizes interactions between the ligand and the protein
conformational change
What does induced fit have to be?
reversible
want to be able to reuse the proteins that are binding the ligand
Dissociation constant (Kd)
=reactants / products
*opposite of Keq
What does a lower Kd mean?
a stronger binding affinity
more likely to have products than reactants
Theta
the fraction of binding sites that are occupied
[PL] / [PL] + [L]
how many sites are occupied out of the total number of sites
What happens as ligand concentration increases to theta?
Theta generally increases as more ligand sites are occupied
Eventually theta will reach a max and plateau
How can you graphical find Kd?
plot Theta versus Ligand Concentration
look at where the graph crosses 0.5 theta
Why doesn’t theta ever reach 1 and all binding sites occupied?
this would require so many ligands that you would exceed the solubility of the ligands
the ligands would not be soluble at that high of a concentration and actually start precipitating out
why do we need a delivery system for O2?
O2 is not very soluble in water
will cause harmful bubbles in the blood rather than dissolve
need myoglobin to carry this gas through the blood
Why do we use Fe and Cu to bind O2?
these metals bind O2 reversibly
amino acids do not bind O2 reversibly
How is Fe coordinated in myoglobin?
Fe is attached to histidine residue on one side and directly across the O2 binds
This forms a straight line which allows O2 to bind more tightly
How is O2 allowed into the myoglobin complex?
through H-bonding interactions with histidines
CO does not have this same interaction and cannot get into the complex as easily
What is the shape of the curve of binding of myoglobin to oxygen?
hyperbolic curve
increases, reaches a max, and then flattens out
How are hemoglobin’s 4 subunits held together?
by noncovalent interactions
hydrogen bonds, hydrophobic interactions, ionic bonds, some disulfide, etc.
T state of hemoglobin
low O2 affinity conformation
R state of hemoglobin
high O2 affinity conformation
What does the binding of O2 to hemoglobin do?
changes the conformation of the molecule
Cooperative binding
the binding of one ligand to a subunit affects the structure and the binding affinity of other sites
*same with reverse direction when O2 is lost
What type of curve indicates cooperative binding?
Sigmoid binding curve
Allostery
binding at one site affects the shape of another site
Why do we use hemoglobin and not myoglobin as our primary oxygen carrier?
Myoglobin has a higher affinity for oxygen
Would not release oxygen in deep tissues where it is needed
What 4 things contribute to the binding of hemoglobin to oxygen?
1) pO2
2) pH
3) CO2 concentration in the tissues
4) Binding of BPG
What state does hemoglobin arrive to the lungs in?
arrives in the T-state with BPG attached in central area
What happens when hemoglobin picks up O2 in the lungs?
it switches from T-state to R-state
*BPG is also released before picking up O2
BPG
2,3-bishosphoglycerate
highly negatively charged and binds to positive residues in the central portion of the hemoglobin molecule
allows subunits to be held in the T state
How does pH influence the conformation of hemoglobin?
when CO2 concentration is high (like in the lungs), it becomes HCO3- and H+. These H+ protons lower the pH and can attach to histidine residues on hemoglobin
The histidine residues become positively charged and stabilize a negatively charged aspartate
these interactions help stabilize the T-state
So t-state is favored a lower pHs and O2 will be released
How does CO2 get transported to the lungs?
around 25-30% of CO2 gets transported to the lungs through hemoglobin
CO2 binds to the amino terminus and converts the terminus into a carbonyl group
Why does fetal hemoglobin have a higher affinity for oxygen than maternal hemoglobin?
this facilitates the transfer of oxygen from the mother to the fetus
What is fetal hemoglobin made out of?
gammaglobin, higher O2 affinity
Bohr effect
hemoglobin binds more tightly to oxygen at high pHs
hemoglobin binds less tightly to oxygen at low pHs
pH of the lungs
7.6
pH of the deep tissues
7.2
Altitude and binding affinity
at high altitude, pO2 is much lower and you do not get as much oxygen in the lungs
this means there is less oxygen to drop off in the tissues
need to increase the amount of BPG so the T-state is favored and oxygen will be dropped off
Why do we never give up all the oxygen?
need to keep a reserve for emergency situations
Sickle Cell Anemia
Mutation on the B-subunit of hemoglobin and Glu is converted to Val
Val can generate hydrophobic patches that come together and form a chain of hemoglobin
This distorts the shape of the red blood cell
Are muscle fibers made up of multiple cells?
no
they are actually a single cell with multiple nuclei
Sarcomeres
sections of myofibrils in muscle cells
individual contractile units in muscle fibers
include the tissue between one I-band and another
Thick filament
made of myosin proteins that come together
intertwined dimers make up the thick filament
Thin filament
made up of actin monomers that have polymerized into a chain
ATP is needed to combine actin monomers
I-band
region of thin filament without any thick filament present
contracts in muscle contraction
A-band
center of the thick filament that holds myosins together
Z disks
found on either end of a sacromere
are pulled together as the muscle contracts
What does each actin monomer have on it?
ADP and a region to bind to myosin
Troponin subunits
found on the thin filaments
prevent actin from binding to myosin
Calcium and muscle contraction
Calcium triggers a conformational change in troponin to unbind it and myosin can bind to actin
An electrical signal releases calcium
What state does myosin start in?
begins in a rigor state that has no bound nucleotide and myosin is tightly bound to actin
no movement of muscle
How does myosin work?
ATP binds to myosin and releases it from actin
Myosin then uses ATP to be in an activated state (changes conformation)
Rebinds to another actin filament and releases Pi
Pi triggers a “power stroke” that moves actin one unit forward
Power stroke
conformational change back to the original state that causes ADP to be released
move one actin molecule further along the chain
ADP is released and we return to the rigor state
A prothestic group
is permanently associated with a protein
an example is the heme group
What is the relationship between Ka and Kd?
they are inverses
as one increases, the other decreases
What are the similarities between myoglobin and hemoglobin’s subunits?
myoglobin and hemoglobin’s subunits have a very similar tertiary structure, but different primary structure
What is the defining secondary structure of myosin?
alpha helices
these coil around each other in a left-handed helix
What is the energy released by ATP by actin used for?
actin filament assembly
actin requires energy to polymerize
What binds stronger to the heme group, O2 or CO?
CO
so, have to prevent CO from entering the complex
Which ions/molecules does hemoglobin transport?
CO2, O2, and H+
What converts CO2 to HCO3-?
carbonic anhydrase
What do the nitrogens in the heme group do?
they have electron donating character that prevents Fe2+ from transitioning to Fe3+
Why does the sickle cell anemia trait continue?
the trait prevents people from getting malaria infection
When can you find Kd?
when half the binding sites are occupied
0.5 theta
What would happen if histidine changed into a nonpolar group in heme?
the iron would not be coordinated to histidine
How does CO bind more strongly to the heme group than O2?
CO binds perpendicular to the heme group’s ring
allows maximum overlap between CO and Fe2+
What reduces CO from binding to heme groups?
histidine residues
What triggers a power stroke?
the release of Pi
Titins
the largest single polypeptide chair discovered thus far
link thick filaments to the Z disk
regulates the length of the sacromere itself and prevents overextension
Relationship between Kd and [L]
Kd=[L] when 1/2 of the binding sites are occupied (theta=0.5)
When do you use a Scathard plot?
when you want a more preside measurement of Kd
What does Scathard plot graph?
Bound/Free versus Bound
How to find Kd from Scathard plot?
Slope = -1/Kd
Bmax
all the possible binding sites
[L] + [PL]
How can you express the number of unbound sites?
[P] = Bmax - [PL]
What is the binding pattern of myoglobin to oxygen?
hyperbolic
What is the binding pattern of hemoglobin to oxygen?
sigmoidal
when concentrations of the ligand are low, don’t bind as well as when concentrations of the ligand are high
there is cooperative binding taking place
Why can’t you use Edman degradation in the deep tissues?
CO2 is bound to the N-terminus of hemoglobin
Why do you need capillaries in the muscles?
need them to exchange O2 with CO2
Sarcoplasmic reticulum
stores calcium which can be released to remove troponin
H-zone of the sarcomere
lacks thin filaments when extended
center of the sarcomere
as myosin crawls along the thin filaments, the thin filaments get pulled towards the middle of the H-Zone
