Ch 35 Lecture 1

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.

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

• 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

thy can ball up 

or they can slide relative to each other

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

Actin-Myosin

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

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

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

ABCDEF

• 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.

the essential and regulatory lght chains, whcih reinforce and give strength during conformational changes

bipolar 

• + end being the heads
• - ends being the tails

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

• 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

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

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.

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.

tropomyosin and the troponin complex; 

tropomyosin wraps around actin, covering the binding site and preventing myosin from bindning.

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

• 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

• gives structure to cell with no cell wall
• railway or communication system/ cell unit

send info and elements and move things in the cell

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

they have a plus and minus end; organized at the microtubule organizing centers; binding of the first subunit allows polymerization outward

GTP

• GTP in active site--> polymerization
• GDP: depolymerization

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

• dynein
• ATPase
• nexin
• 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

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

• they bend relative to each other
• they spring back

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

• 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.

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

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

counterclockwise rotation allows swimming

made of protein called flagellin; comes together to form hollow tube; the flagellum rotates--> different motor and energy source: proton gradient

• rod
• rings
• MS ring
• FliG

Mot B thanks to pumps that pump protons out of cell

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

proton hops to ms ring, spins around in counterclockwise direction; goes to other channel