Proteins: Myoglobin And Hemoglobin and cytoskeleton 2 Flashcards
1
Q
Globular Proteins
A
- most numerous proteins
- distinguishable from fibrous proteins by the fact that they are soluble in aqueous media.
- core consists of fixed 2’ structures.
- Hydrophobic AA’s are oriented towards interior, Hydrophilic AA’s face the exterior.
- Overall structure is stabilized by H bonds, ionic interactions and less often S-S bonds.
- Because of the high degree of H bonding in the peptide backbone N-H and C=O are neutralized.
2
Q
Myoglobin and Hemoglobin
A
- most studied and best-understood globular proteins
- crucial in conversion of anaerobic life to aerobic life
- aerobic metabolism yields more energy than anaerobic
- due to O limited solubility in water, myoglobin and hemoglobin have evolved to deliver O to tissues in sufficient quantities.
3
Q
Myoglobin
A
- Single polypeptide chain of 153 AA’s
- contains a covalent lay bound heme group
- is the oxygen storage protein of muscle.
4
Q
Hemoglobin
A
- a2b2 tetramer, a subunit is 141 AA’s while b is 146 AA’s
- each polypeptide is structurally similar to myoglobin and each contains a heme
5
Q
Heme prosthetic group
A
O does not bind well to any AA, but it binds to various metals, including Fe and Cu
- Fe is frequently part of a complex with protoporhyrin IX
- porphyrin is composed of 4 pyrrole rings liked by methane bridges
- porphyrin is flat with two open sites for binding to Fe
- porphyrin is a prosthetic group, which helps proteins employ their functions.
- Protoporphyrin IX containing a bound Fe is called a heme
6
Q
Heme group binding
A
- Fe atoms of hemes typically exist as either +2 or +3
- O will only bind to +2 state when O binds the free heme it oxidizes it by one electron resulting in superoxide and ferric heme (+3)
- O bound heme is red, while heme with no O is purple
7
Q
Methemoglobin
A
Hemoglobin that has been oxidized from ferrous to ferric state. Thus unable to bind O.
8
Q
Effect of Protein Structure on O binding
A
- binding is influenced by structure
- cavity where O binds is created by Val (helix E) and Phe (located in the loop between helices C and D)
- There is no clear pathway for O binding to heme. Molecular motions (breathing) of the protein create transient routes to allow O to bind or leave. Rotation of the distal histidine is one important molecular motion
9
Q
Role of the Protein Scaffold
A
- heme cofactors is buried deep in the binding pocket composed primarily of alpha helix 2’ structure.
- the heme is held by numerous binding interactions as well as covalent interaction between the Fe and N atom of the histidine side chain in one of the helices
- coordination of the histidine to the heme completely blocks access of O to this face of the heme, forcing binding to the opposite face. This prevents oxidation of the heme by O and allows for simple reversible binding
10
Q
O binding to Heme
A
- O binding alters myoglobin conformation
- binding of O pulls the heme closer into the porphyrin ring. In turn, the histidine is pulled along, distorting the shape of the alpha helix.
- in hemoglobin O binding has profound effects on the ability of other subunits to bind O.
11
Q
T and R state of Hemoglobin
A
- two major conformations of hemoglobin as predicted by the models for allosteric activation
- O will bind to hemoglobin in either state, has higher affinity for R state
- In the absence of O hemoglobin is more stable in the T state, and is therefore the predominant form of deoxyhemoglobin. R stands for relaxed, T for tense which is stabilized by the greater number of ion pairs.
- transition of T to R causes rotation of alpha2beta2 pairs of subunits 15 degrees relative to the pair of alpha1beta1 subunits.
12
Q
Hill Plots for O2 binding to myoglobin and hemoglobin
A
- if Hill coefficient is 1 there is no cooperativity
- the maximum Hill coefficient is 3 which is less than the number of O binding sites in hemoglobin, which is normal for a protein that exhibits allosteric behavior
13
Q
Allosteric Effects in hemoglobin
A
- structure changes to oxygenation
- Increase in H+ causes Hb to release O2 (Bohr effect)
- 2,3-BPG binds to positively charged groups stabilizing deoxy Hb
14
Q
Carbonic anhydrase
A
- catalyzes rapid interconversion of CO2 and water to bicarbonate and H+ (or vice versa)
- Maintains the acid-base balance in blood and other tissues by transporting CO2 out of the tissues
15
Q
Bohr effect
A
- more O2 is released in tissues with higher absolute and/or relative CO2 values.
16
Q
2,3 - bisphosphoglycerate
A
- dramatically affects Hb’s binding of O2
- stabilizes deoxy Hb
- added during the storage of blood
- increased at high altitudes
17
Q
CO poisoning
A
- CO will bind to heme 20,000 times better than O, 200 times better when heme is bound to myoglobin
- it binds the heme in a bent conformation and a histidine residue makes a favorable H bond with it
- Preferred electronic configuration of CO binding to heme is in a linear conformation. The His residue at E7 steric ally hinders its binding to the heme, reducing its affinity
18
Q
Binding of NO in hemoglobin
A
- NO is a smooth muscle relaxing (hypotensive) agent
- at the tissues, some Hb binds NO instead of one of the 4 O
- NO is then transferred to Cys of beta subunit
- Upon releasing O, Hb passes NO to GSH (as GSNO), stabilizing NO
- NO transferred to receptors in vascular smooth muscle cells, relaxing them, facilitating O passage into tissues
19
Q
Subunit Composition of Hemoglobin Tetramers
A
The P50 values for HbA and HbF are 26 and 20 mm Hg
- in the placenta, this difference enables HbF to extract O from the HbA in the mother’s blood
- HbF is suboptimal postpartum since its high affinity for O2 limit the quantity of O2 delivered to the tissues
20
Q
Sickle cell anemia: HbS
A
- single nucleotide change in beta-glob in gene, Glu—>Val
- only if both genes are affected do you have sickle cell anemia
- normal rbc 120 days; HbS rbc 20 days
- sickling in low blood oxygen, or other conditions favoring deoxy Hb: low pH, high pCO2, high BPG
- fatigue and shortness of breath
- painful crisis
- Possible treatment: Silencing BCL11A normally suppresses the production of fetal hemoglobin
21
Q
Hemoglobin C and SC diseases
A
- Hemoglobin C: Glu6 —-> Lys6 in beta-globin gene
- Homozygous have mild hemolytic anemia; no specific therapy needed
- Hemoglobin SC - One beta-globin gene codes for HbS, and the other for HbC
- Patients often have normal life, until they give birth or have surgery, when they can have infarctive crisis, possibly fatal
22
Q
Aggregation of HbS
A
- DeoxyHbS
- New hydrophobic patch Val6 interacts by Phe85 and Val88 of the beta chain of a neighboring molecule to initiate the aggregation process
23
Q
Methemoglobinemias
A
- Hb with Fe(+3) instead of normal Fe 2+ resulting in a reduced ability to release O to tissues and thereby hypoxia
- caused by benzocaine, dapsone, and nitrates
- caused by reactive O intermediates and some inherited Hb defects
- caused but deficiency in NADH-methemoglobin reductase
- Brownish-blue skin due to tissue hypoxia
24
Q
Thalassemias
A
- deficiency in synthesis of alpha or beta globin
- low levels of alpha2beta2 and abnormal aggregates off alpha or beta globins in rbc
- caused by gene deletion or substitution or deletion of nucleotides in DNA
25
Q
Alpha-thalassemias
A
- normal people have 4 copies of alpha-globin gene
- if 3 genes are defective: HbH, moderately severe hemolytic anemia, gamma Tetramers in newborn or beta tetramers bind oxygen too tightly to deliver to tissue
- if all 4 genes are defective, hydrops fetalis and fetal death since alpha-globin is require for HbF
26
Q
Beta-Thalassemias
A
- alpha-globin synthesis is normal
- alpha-globin aggregates precipitate
- premature death of rbc precursors
- accumulation of HbF and Hb Bart’s
- Causes problems after birth
- minor cases don’t usually require treatment
- major cases - severely anemic in first or second year, required blood transfusions, ion overload, premature death
- marrow replacement helps
27
Q
Intermediate filaments
A
-important for cell structure and localization of cellular processes
-family of proteins
Structure: central rod domain, amino terminal domain and carboxy-terminal domain
Assembly:
1. Forms dimers
2. Two dimers form tetramer (antiparallel, dimers staggered)
3. tetramers form protofilaments
4. 8 protofilaments form a filament
5. Unlike actin, additional tetramers added to both ends of protofilament
28
Q
Intermediate filament organization
A
- Forms network in cell
- Associates with nucleus, plasma membrane, actin, and microtubules
- Anchors cells to each other and to ECM
29
Q
Epidermolysis bullosa simplex
A
- most common IF disease, mildest EB
- defective keratin
- Skin splits in epidermis, causing blisters
30
Q
What type of IF is expressed only in epithelial cells?
A
Keratin
31
Q
Microtubules
A
- largest cytoskeletal protein
- functions in: determining cell shape, cell locomotion, intracellular transport, organelle positioning, separation of chromosomes during mitosis
32
Q
Microtubule Assembly
A
- hollow tubes composed of dimers of alpha and beta tubulin
- Dimers polymerize to form microtubules that have a (+) and (-) end
- Tubulins bind GTP, which regulates polymerization
- GTP-tubulin polymerizes at (+) end
- GTP bound to beta-tubulin cleaved to GDP during or after polymerization
- leads to depolymerization at minus end because GDP-tubulin does not bind to microtubule as well
- (-) is protected to prevent rapid depolymerization
33
Q
Dynamic instability
A
- individual microtubules alternate between growth and shrinkage, determined by rate of tubulin addition relative to GTP hydrolysis
- If GTP-tubulin is added faster than GTP is cleaved, microtubules grow, if the opposite then GDP-tubulin builds at (+) end and microtubules shrink ( if this happens quickly it is a catastrophe)
- growth and shrinkage important during cell division
- drugs that affect microtubule assembly are important to cancer treatment by interfering with cell division
34
Q
Vincristine and vinblastine
A
Bind tubulin and inhibit microtubule polymerization
35
Q
Taxol
A
Stabilizes microtubules and prevents disassembly
36
Q
Centrosome
A
- microtubule organizing center
- initiates microtubule growth
- binds (-) end of microtubule
- microtubules grow toward plasma membrane
- gamma-tubulin key protein for growth of microtubules
- contain two centrioles which are not necessary for microtubule assembly but for formation of cilia and flagella
- cancer cells have multiple cent roscoes, which will cluster
37
Q
Microtubule stability
A
- inherently unstable
- stabilized by: post translational modifications of tubulin so, binding to MAPS
38
Q
MAP’s
A
- cell and tissue specific
- regulate behavior of microtubules
- functions: cap microtubule ends and speed up microtubule assembly by increasing incorporation of GTP-bound tubulin at (+) end
- speed up microtubule disassembly by dissociating GTP-tubulin from (+) end
- Rescue microtubules from catastrophe by stopping disassembly and restarting growth at (+) end by using CLASP
- connect microtubules to IF’s or plasma membrane
- Bind to GTP-tubulin and move growing microtubules to different cellular locations
39
Q
Organization of microtubules in nerve cells
A
-two types: axons (send signals) and dendrites (receive signals)
40
Q
Axons
A
- microtubules have (+) ends away from cell body
- (-) end not anchored in centrosome
- capped at both ends
- contain tau which stabilizes microtubules
41
Q
Dendrites
A
- microtubules oriented in both directions
- contain MAP-2 which crosslinks microtubules to IF’s
42
Q
Microtubule motor proteins
A
- Dynein
- moves along microtubules towards (-) end
- transports macromolecules, membrane vesicles and organelles towards center of cell away from periphery - Kinesin
- moves along microtubules towards (+)
- transports macromolecules, membrane vesicles and organelles away from center of cell towards periphery - ATP hydrolysis required for movement
43
Q
Cilia and Flagella
A
- Microtubule-based projection of plasma membrane
2. Cilia move fluid over cells, and flagella important for sperm movement
44
Q
Cilia and flagella axoneme structure
A
- Microtubules and associated proteins
- Microtubules arranged in 9+2 pattern
- Central pair of microtubules surrounded by 9 our microtubule doublets
- Doublet composed of A tubule (Complete microtubule) and B tubule (incomplete microtubule)
- Microtubule doublets connected to each other by nexin and to central pair of microtubules by radial spokes
- Each A tubule attached to dynein
45
Q
Cilia and flagella basal body structure
A
1) anchors (-) end of microtubules inside cell
2) modified centriole
3) contains 9 triplets of microtubules
4) initiates growth of axonemal microtubules
5) dictates position and orientation of cilia
46
Q
Cilia and Flagella movement
A
- outer microtubule doublets slide relative to each other
- powered by axonemal dyneins
- dynein light chains bind to A tubules
- dynein head groups bind to B tubule
- head groups move towards (-) end which causes A tubule to slide towards the basal end of B tubule
- because the doublets are connected by nexin they bend
47
Q
Microtubule diseases
A
- Microtubule dysfunction may be associated with Alzheimer’s or Parkinson’s
- MAP tau forms aggregates in Alzheimer’s
- Smoking damages cilia in trachea and bronchial tubes
- Primary ciliary dyskinesia - lack or or dysfunctional cilia, manifested as chronic respiratory distress shortly after birth
48
Q
Binding of which of the following to tubulin regulates microtubule assembly?
A
GTP
49
Q
A 47 yo man present with constant coughing. He has been a 3PPD smoker for 30 years. Which of the following cytoskeletal proteins is most likely to be damaged?
A
Microtubules
50
Q
Which of the following is the mechanism of action of the anticancer drug taxol?
A
It stabilizes microtubules and thus inhibits disassembly
