Moir - Molecular Biomechanics Flashcards
give the width of microfilaments and microtubules
mf: 8nm
mt: 25nm
what are microfilaments and microtubules made up of? which structure can be branched?
mf: actin can be branched
mt: tubulin never branched
how many human actin types are there, name the 3 groups?
4 alpha actin isoforms - musccle types
beta and gamma - non muscle types
what is the biologically active form of actin?
f-actin
by how many a/a’s do the different forms of actin vary by?
4 to 5
what is the pitch of f-actin?
14 monomers (7 monomers either side)
describe the shape of f-actin
helical
name the standard conditions at which f-actin spontaneously polymerises
1mM ATP 2mM Mg 50mM K
how can it be proved that f-actin is polar?
myosin binds at same angle of 45°
name and describe the 3 stages in the actin polymerisation graph (g-actin polymerising to f-actin)
lag: energetically unfavourable
extension: trimer production reaches critical level, production of f-actin energetically favourable
equilibrium: rate of the forward and backward reaction are the same
at which end of f-actin is g-actin added?
barbed(+)
which molecules/ions have to be bound to f-actin in order for it be stable?
ATP/Mg
what do capping proteins do and name the 2 used to cap the barbed and the pointed end
stop f-actin unraveling
CAPZ caps barbed
tropomodulin caps pointed end
what do monomer binding proteins do and name and state the function of 2 of them
isolates actin to build filaments without destroying them
thymosin beta 4 - binds ATP-G-actin forming a Tβ4-ATP-G-actin complex. When free ATP-G-actin is added to the F-actin filament the free ATP-G-actin concentration will go down. As this disrupts the free ATP-G-actin/Tβ4-ATP-G-actin equilibrium more Tβ4-ATP-G-actin will dissociate leading to more free ATP-G-actin.
Overall: binds ATP-G-actin to inhibit addition G-actin to F-actin. Important in removing actin clots in blood.
profilin - activated by PIP2. Binds ADP-G-actin which increases the loss of ADP from G-actin. ADP is replaced by ATP. Stops ATP-G-actin binding (-) end so therefore grows from (+) end.
Overall: promotes nucleotide exchange and filament formation.
what does dystrophin do? how much of the genome does it make up?
anchors f-actin to the cell mem via c-terminus
is 0.1% of genome
what is the cause of 90% of human dystrophies?
deletions in the dystrophin gene
describe the severity, molecular basis and impact of Duchenne muscular dystrophy
severe (life expectancy = teens/20s)
result of deletions which causes a frameshift mutation
mutant protein cannot bind actin and cell membrane => muscle weakness
describe the severity, molecular basis and impact of Becker muscular dystrophy
mild severity (life expectancy = 50-60yrs) result of deletion is no frameshift mutation mutant protein is shorter but binds membrane and actin => functional muscle weakness
what kind of protein is fimbrin? describe its effect on actin morphology
actin bundling protein
rod shaped actin filaments
N&C terminal regions both actin binding sites
Allow creation of actin cables and microvilli
what kind of protein is filamin? describe its effect on actin morphology
gelation protein
bent rod shaped actin fibres
n and c terminus both actin binding sites
creates gel: important in cellular movement as it creates networks of f-actin not sheets
describe the function of gelsolin and the impact it has on the structure of actin
binds f actin and cleaves it
binds ‘barbed’ end = dissociation of the filament
important part of serum prevents actin clots in blood
describe the troponin I test for cardiac arrest
cardiac muscle tropinin I increases after heart attack
can’t use actin antibodies to measure amount of cardiac actin as it also recognises smooth muscle actin
TN-I found in striated muscle not smooth therefore its isoform is specific to the heart and can be used to diagnose heart attack
at what angle does actin bind pre-existing actin filaments?
70° from existing filament
how many subclasses of myosin are there? How are these identifiable?
20
Identifiable by ATP/Mg2+ binding site or genome sequence
Is the C or the N terminus the variable region in myosin?
C
Where is myosin II found?
highly ordered sarcomeres
actomyosin contractile bundles in non muscle cells (cell movement, cell division)
what do myosins I and V do?
movement of vesicles
what 2 different types of actin myosin interaction are there?
1) contraction (transient interaction with actin)
2) transport (maintenance of contact with actin)
what unique C terminal feature does myosin II have?
coiled-coil sequence
why does myosin II form a coiled-coil?
myosin II is a dimer made up of 2 alpha-helices.
Residues 1&4 are always located on the same side and are always hydrophobic. this creates a hydrophobic side on both the helices which have to be in contact to minimise disruption from water.
why do some coiled-coil myosins not form a filament?
some myosins (myosin V) have regions of coiled-coil that contain non-helical regions. this is usually because of proline which cannot H bond with its amino group. this results in a dimer and not a coiled coil
which myosin is monomeric?
myosin I
what are nebulin and titin?
giant proteins
regulate length of actin and myosin in skeletal and cardiac muscle
titin is elastic and nebulin determines actin length
why is myosin an ‘incompetent ATPase’
myosin hydrolyses MgATP to MgADP but only in the presence of actin. An incompetent ATPase can hydrolyse ATP to ADP but not remove an actin monomer.
what do tropomyosin and troponin do? how do they do this?
tropomyosin and troponin are regulatory proteins that regulate the contraction of skeletal and cardiac muscle. tropomyosin is wrapped around actin and troponin is bound to tropomyosin. When Ca2+ is released (ie when a muscle contracts) it binds troponin, exposing binding sites on actin. Myosin heads bind these sites and pull actin past it the myosin filament.
describe the changes in Ca2+ concentration in contracted and relaxed muscle and explain why this happens
In contracting muscle Ca2+ is at 10^-5M because the presence of calcium allows myosin heads to bind to actin and hydrolyse MgATP to MgADP. In relaxed muscle the Ca2+ concentration is 10^-8M meaning the actin-activated ATPase properties of myosin are inhibited as there is v little Ca2+.
what is the incidence and effect of myosin cardiac mutations?
1:500
mutations impair cardiac function left ventricle is enlarged and heart failure can occur during stress
what is the cause of myosin related deafness?
stereocilia made up of myosin vibrate and mutations cause impaired function
how are different isoforms of myosin localised in a cell?
myosin antibodies
what is the function of myosin I and II in cell locomotion?
myosin I at leading edge sends out lamelipodia
myosin II at rear of cell pushes the cell
why is it important that actin filaments form branches?
to allow the precise delivery of cargo
whats the difference in stable and transient microtubules?
stable MTs: found in non-differentiating cells
transient MTs: found in dividing cells
give an example of where a stable MT may be found and where a transient MT may be found
stable: neurones, cilia, flagella
transient: dividing cells - essential for organisation of chromosomes in mitosis
what are microtubules composed of?
an alpha beta heterodimer of tubulin
describe the binding of GTP/Mg to alpha and beta tubulin
alpha tubulin: binds GTP/Mg irreversibly
beta tubulin: binds GTP/Mg reversibly and hydrolyses it to GDP/Mg
what is taxol? where does it bind on the tubulin heterodimer?
taxol is an anticancer drug
it binds to beta tubulin
how many protofilaments are there in a microtubule?
13
which end of the microtubule grows?
the beta-tubulin (+) end grows (the heterodimer is added with the alpha monomer on the left and the beta monomer on the right - forms a helix with alpha ring at the bottom and a beta ring at the top)
how long is an alpha-beta heterodimer
8nm
how does an MT grow?
addition of heterodimers to the (+) end, this end grows faster than the (-) end. The dimers added contain GTP and are therefore stable. GTP in beta-tublin hydrolyses to GDP over time and therefore when the GTP cap is lost any GDP bound beta-tubulin will dissociate.
what is the model system for microtubules and why?
axonal transport in neurons is used as a model system. used because they are very stable.
where are proteins synthesised in a neuron? why is this important?
protein synthesis occurs in the cells which allows products to be visualised as they are transported down the axon. this has shown that intact organelles are transported around the neuron and at the end of their life they are taken back to the cell body for re-use.
which two motor proteins travel on microtubules? which direction do they travel?
kinesin (+ directed for delivery - away from the nucleus)
dynein (- directed for return - towards the nucleus)
describe kinesin structure
kinesins contains 2 globular head domains (which are smaller than the myosin globular head domains)
they differ in their tail domain.
composed of a coiled coil
what are the 2 types of kinesin
cytosolic kinesin (carries vesicles and organelles) and mitotic kinesin
what is the function of dyneins
intracellular transport and cell movement (flagella and cilia)
how is cargo delivered precisely?
MTs aren’t branched whereas actin is highly branched. MTs and kinesin and dynein are used for large-scale distribution whereas actin and myosin (V) are used to precisely deliver cargo to locations. Myosin V associates with MT and waits for kinesin carrying cargo. Kinesin picks up myosin and carries it until it reaches actin. It then hands the cargo to myosin which then picks up kinesin and delivers the cargo to the specific place.
what are MT stabilising proteins? describe their structure and name 2
proteins that control the spacing of adjacent microtubules
they have an MT binding domain and a projection arm (which can also bind MTs)
Tau (axons) and MAP2 (dendrons)
which MT stabilising protein is related to Alzheimer’s disease?
Tau protein (abberant polymerisation of Tau is linked to neurodegenerative disorders)
what is the effect and side effects of taxol?
taxol inhibits MT shortening and therefore stops cell division. Taxol works on all cells however and is therefore toxic, it also needs to be administered using organic solvents.
define ‘inchworm’ and ‘hand-over-hand’
inchworm: same globular head of a motor protein always leads and the other drags behind
hand-over-hand: the leading globular head alternates ie walks like a human
how was walking myosin studied? what was the result of the study?
used an optical probe to attach to one myosin head domain. they measured how far the probe traveled (36nm if inchworm and 72nm if hand-over-hand). myosin walks hand-over-hand
is kinesin walking hand-over-hand or inchworm
hand-over-hand
