Ch 35 Lecture 1
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Most molecular motor protiens are members of the __.
What are blast cells?
How do you induce differentiation?
P loop NTPase family
still capable of dividing and proliferating
Adding a differentiaiton signal, such as MyoD, which signals production of transcription factors, which deactivate genes for proliferation and activate muscle specific protiens. They fuse together cell contents within cells.
Explain a sarcomere.
nuclei migrate to the periphery and control a function of the cell
several in a single muscle fiber
it is the functional unit; gets smaller upon contraction
What are the two proteins in the sarcomere?
- actin: filamentous structural protein; cells form long thin fibers
- myosin: mechanoenzyme; member of P-loop ATPase family; uses enzymatic activity to do mechanical work; thicker than actin
actin and myosin overlap in the A band
What are the theories of contraction?
thy can ball up
or they can slide relative to each other
What experiment did they do to test the relationship with actin and myosin?
took a beaker with actin and a beaker with myosin and poured them together
the solution became viscous because they were bonding.
Took the viscous solution and added ATP--> viscosity went down because ATP allowed a reaction between actin and myosin to reverse itself
What is the cycle between actin, myosin, and ATP?
ATP displaces actin--> ATP-myosin
Hydrolysis of ATP: ADP-P-Myosin
Actin attaches: Actin-ADP-P-Myosin
ADP-P leaves: Actin-Myosin
myosin in buffer; add ATP; observe conformational changes
Third experiment performed
And result of this
cleaved using papain and trypsin:
got four segments
S1 and S2, the globlar heads
- HMM: contains the S1 and S2 domains
- LMM: forms the alpha helical coiled coil. This coil has no kinks (no proline); there's a repeat of seven amino acids; less mass
What do trypsin and papain do myosin? What is the original structure of myosin?
what do the globular heads do?
What does the Ploop do?
Trypsin separates the LLMM from the HMM
myosin: 520 kD molecule made of 6 polypeptide chains
papain separates the active site from the other chain
ATPase activity limited to globular domain; doesn't form filaments, but hydrolyzes ATP
Ploop has sequence that allows ATP to move out
Explain in detail the seven repeating amino acids.
- A and D: hydrophobic
- BCF: charged
A and D always end up on the same side, allowing them to hide in the helix; hyrophobic interactions allow tighter binding
The charged ones lie outside.
What does the neck region of myosn consist of?
the essential and regulatory lght chains, whcih reinforce and give strength during conformational changes
Myosin is a __ moleule with ___ and __.
- + end being the heads
- - ends being the tails
How does actin exist?
as individual subunits called g actin (globular actin)
g actin polyerizes into a filament--> F actin, which is an ATPase, but doesn't use it to do work. It uses it to help polymerize
It is also unidirectional and polar
What is the optical trap experiment?
- there are beads that a laser beam can monitor the movement/ distance of and a thin filament stretched over the beads (ceramic)
- there is a middle bead that has the HMM
ATP is added and actin begins to hydrolyze it; the beads move in stepwise fashion due to myosin pushing along actin in stepwise fashion
What effect does ATP binding have on myosin?
What effect does ATP hydrolysis have?
it decreases it's affinity for actin, causing it to let go and undergo a conformational change
affinity for actin increases; binds in a new place and release of inorganic phosphate
What are the two different conformations?
Myosin's lever arm has two conformations depending on whether ATP is bound or absent.
When ATP is bound, the lever arm reorients itself, performing a power stroke.
Explain what a small movement in the P loop region can do.
P loop attaches to switches, which attach to relay helix, which attach to lever arm
A little movement in the P loop translates into slightly larger movement in the swtich 1 adn slightly larger change in switch II, relayed to relay helix, movement of lever arm.
How is the myosin and actin association controlled?
tropomyosin and the troponin complex;
tropomyosin wraps around actin, covering the binding site and preventing myosin from bindning.
Explain in detail the troponin complex.
It has the TnC, TnI, and TnT regions
TnT is a tropomyosin-binding subunit which regulates the interaction of troponin complex with thin filaments;
- TnI inhibits ATP-ase activity of acto-myosin;
- TnC is a Ca2+-binding subunit, playing the main role in Ca2+ dependent regulation of muscle contraction
Explain muscle contraction in terms of action potential.
- in cells, the SR is where calcium is sequestered.
- Action potential depolarizes cells, causing a release of Ca, which binds to TnC subunit of the troponin complex
TnC changes conf
TnI changes conf, moving tropomyosin away and revealing a binding site that myosin can now access
Why is the cytoskeleton complex?
- gives structure to cell with no cell wall
- railway or communication system/ cell unit
Since everything is connected, we can do what?
send info and elements and move things in the cell
What are the components of the cytoskeleton?
microtubules: made of alpha and beta tubulin, whcih polymerize to form a hollow tube with a hollow core
microfilaments: made of actin
intermediate filaments: made of keratin; comes together in multisubunit helices like collagen
Why are microtubules directional?
they have a plus and minus end; organized at the microtubule organizing centers; binding of the first subunit allows polymerization outward
What is the motivation to assemble and disassemble?
- GTP in active site--> polymerization
- GDP: depolymerization
Eukaryotic flagella are anchred by __. Explain it.
axoneme; bundle of microtubule fibers adn other associated fibers that is continuous with the membrane; there is a nine plus two aray, and subfiber A is continuous; subfiber B is not
__ is the __, located all the way up and down the flagella.
__ connects pairs of microtubules together. Everything in an axoneme must __.
- stay connected
- multiple binding sites for ATP
- gets ATP in the active site; reaches out to next microtubule to grab on; hydrolyzes ATP--> conformational change that allows the power stroke to occur
What are three facts about dynein?
it is an ATPase
uses energy associated with hydrolysis to do work
has an affinity for MT; and, that affinity changes whether ATP or ADP is bound
As dynein walks along the microtubule, what do the microtubules do? How about when dynein is released?
- they bend relative to each other
- they spring back
Explian the structure of kinesin.
fairly large protein with four subunits; globular motor domain; large helical coiled coil domain
different from myosin in that the binding site for vesicles is the tail
Explain the optical trap experiment using kinesin?
- single kinesin bound to a ceramin bead
- substrate kinesin walks on is a MT from teh minus end to the plus end
Over time, bead was displaced in stepwise fashion.
What does kinesin have?
it has a ploop, switch I and II, relay helix attached to neck linker
small changes in the middle lead to a big change outside
similarities between myosin and kinesin
use atp to hydrolyze bond--> eergy converted into conformational change--> work
proteins bound to MT not actin; subtle difference in attaching
- in kinesin: ATP bound leads to greater affinity for actin
- in myosin: ATP bound leads to release
Bacterial flagellar motion
counterclockwise rotation allows swimming
Explain bacterial flagella
made of protein called flagellin; comes together to form hollow tube; the flagellum rotates--> different motor and energy source: proton gradient
Flagella is anchored by __; come throguh __ and anchors self in _, which is made of __.
Which has lower pH, MotA or MotB?
Mot B thanks to pumps that pump protons out of cell
Explain MOtA and MOtB
one is a half channel taht connects to outside of membrane (MotA) and one connects inside (MotB); they get halfway; and FLiG allows acces to other half channel
Explain the rotation
proton hops to ms ring, spins around in counterclockwise direction; goes to other channel
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