Proteins: Myoglobin And Hemoglobin and cytoskeleton 2 Flashcards Preview

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Flashcards in Proteins: Myoglobin And Hemoglobin and cytoskeleton 2 Deck (50):

Globular Proteins

-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.


Myoglobin and Hemoglobin

-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.



-Single polypeptide chain of 153 AA's
- contains a covalent lay bound heme group
-is the oxygen storage protein of muscle.



- 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


Heme prosthetic group

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


Heme group binding

- 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



Hemoglobin that has been oxidized from ferrous to ferric state. Thus unable to bind O.


Effect of Protein Structure on O binding

-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


Role of the Protein Scaffold

-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


O binding to Heme

- 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.


T and R state of Hemoglobin

-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.


Hill Plots for O2 binding to myoglobin and hemoglobin

- 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


Allosteric Effects in hemoglobin

-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


Carbonic anhydrase

- 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


Bohr effect

- more O2 is released in tissues with higher absolute and/or relative CO2 values.


2,3 - bisphosphoglycerate

- dramatically affects Hb's binding of O2
- stabilizes deoxy Hb
- added during the storage of blood
- increased at high altitudes


CO poisoning

-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


Binding of NO in hemoglobin

-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


Subunit Composition of Hemoglobin Tetramers

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


Sickle cell anemia: HbS

- 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


Hemoglobin C and SC diseases

- 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


Aggregation of HbS

- New hydrophobic patch Val6 interacts by Phe85 and Val88 of the beta chain of a neighboring molecule to initiate the aggregation process



-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



- 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



-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



-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


Intermediate filaments

-important for cell structure and localization of cellular processes
-family of proteins
Structure: central rod domain, amino terminal domain and carboxy-terminal domain
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


Intermediate filament organization

1. Forms network in cell
2. Associates with nucleus, plasma membrane, actin, and microtubules
3. Anchors cells to each other and to ECM


Epidermolysis bullosa simplex

-most common IF disease, mildest EB
- defective keratin
- Skin splits in epidermis, causing blisters


What type of IF is expressed only in epithelial cells?




-largest cytoskeletal protein
- functions in: determining cell shape, cell locomotion, intracellular transport, organelle positioning, separation of chromosomes during mitosis


Microtubule Assembly

1. hollow tubes composed of dimers of alpha and beta tubulin
2. Dimers polymerize to form microtubules that have a (+) and (-) end
3. 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


Dynamic instability

-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


Vincristine and vinblastine

Bind tubulin and inhibit microtubule polymerization



Stabilizes microtubules and prevents disassembly



-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


Microtubule stability

-inherently unstable
-stabilized by: post translational modifications of tubulin so, binding to MAPS



-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


Organization of microtubules in nerve cells

-two types: axons (send signals) and dendrites (receive signals)



-microtubules have (+) ends away from cell body
- (-) end not anchored in centrosome
- capped at both ends
- contain tau which stabilizes microtubules



-microtubules oriented in both directions
-contain MAP-2 which crosslinks microtubules to IF's


Microtubule motor proteins

1. Dynein
-moves along microtubules towards (-) end
-transports macromolecules, membrane vesicles and organelles towards center of cell away from periphery
2. Kinesin
-moves along microtubules towards (+)
-transports macromolecules, membrane vesicles and organelles away from center of cell towards periphery
3. ATP hydrolysis required for movement


Cilia and Flagella

1. Microtubule-based projection of plasma membrane
2. Cilia move fluid over cells, and flagella important for sperm movement


Cilia and flagella axoneme structure

1. Microtubules and associated proteins
2. Microtubules arranged in 9+2 pattern
3. Central pair of microtubules surrounded by 9 our microtubule doublets
4. Doublet composed of A tubule (Complete microtubule) and B tubule (incomplete microtubule)
5. Microtubule doublets connected to each other by nexin and to central pair of microtubules by radial spokes
6. Each A tubule attached to dynein


Cilia and flagella basal body structure

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


Cilia and Flagella movement

- 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


Microtubule diseases

1. Microtubule dysfunction may be associated with Alzheimer's or Parkinson's
2. MAP tau forms aggregates in Alzheimer's
3. Smoking damages cilia in trachea and bronchial tubes
4. Primary ciliary dyskinesia - lack or or dysfunctional cilia, manifested as chronic respiratory distress shortly after birth


Binding of which of the following to tubulin regulates microtubule assembly?



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?



Which of the following is the mechanism of action of the anticancer drug taxol?

It stabilizes microtubules and thus inhibits disassembly