Questions

Why does myosin bind actin and kinesin/dyenin bind microtubules?

Differences in structure reveal how myosin binds actin and kinesin/dyenin bind microtubules.

If all motor proteins use ATP hydrolysis for movement, how is diversity generated?

Differences in rate constants for the reaction steps and different morphologies lead to diversity of speeds and step sizes in motor proteins.

How do motor proteins  interact with cargo?  

Through specific bonds or through other proteins.

How is transport regulated?

How do they transport such a wide array of cargo? (such as protein complexes, protein-nucleic acid complexes, organelles?

Differences in their tail domains  allow them to  evolve into adaptors, linking them to cargo through interaction with receptor proteins on the cargo surface.

What type of cargo binds the three classes of motor proteins?

How do you measure the forces exerted by single motors?

How do you determine step size and ATP consumption per step?

What regulates the activity of motors and how they bind cargo?

Why does movement take place through diffusion or passive transport? Why does movement mediated  by motor proteins take place via- active transport?

Cargo often consists of large organelles such as synaptic vesicles or melanosomes and not single molecules. For small distances and small particles like nucleotides passive transport due to diffusion is faster and less complicated than active transport by motor proteins. For large particles, the cell is gel- like rather than liquid like due to extended cytoskeleton and high protein content. Therefore, diffusion constants are smaller than in liquids and decrease sharply as particle size increases. Motor proteins have thus evolved to carry cargo through this gel- like cytoplasm.

How  do neurons regulate which materials get transported, and in which direction, or how materials  are routed to proper destinations?

• one possibility is that all motor proteins bind to every vesicle or organelle, but that the neuron can activate or inactivate motor molecules individually
• vesicles in which only kinesin activated would move anterograde, vesicles in which only dynein activated would move retrograde
• kinesin may be regulated by phosphorylation, when kinesin phosphorylated, transport is inhibited
• ensuring vesicles reach proper place may involve specialized proteins that identify and catch a particular organelle as it moves by a specific site in neuron

How is motor activity regulated in vivo?

How is a specific cargo loaded?

What is the nature of coupling between the chemical step(ATP binding or hydrolysis ) and subsequent conformational change?

What are the critical residues that regulate such coupling and how  do they achieve that?

What is meant by processivity of motor proteins?

Processive means a single motor protein can take several steps along its track (microtubule/ microfilament)   without dissociating.

What triggers the forward step?  

ADP release

Why is it difficult to decipher the mechanism of motor proteins?

Molecular motors are extremely small and transitions in their ATPase cycle occur in milliseconds. Thus methods for measuring motor movements and conformational changes must be compatible with these spatial and temporal parameters. The problem with measuring large number of molecules is that the discrete actions executed by individual molecules become blurred. Single molecule analyses along with EM studies and high resolution structural studies can provide information about the working of motor proteins.

How is futile cycling/ATP hydrolysis without a step forward prevented?

Studies in myosin, have showed that tension mediated regulation of the leading head slows down ADP dissociation or decreases the rate of ATP binding at the empty site. This prevents the leading head from dissociating prematurely from actin and thus curtails futile cycling.  

Motor Proteins in vitro

What are the limitations associated with the application of motor proteins in vitro?

• These proteins have to be kept in an environment similar to the cytosol. This is much more complicated than an aqueous environment, since pH, salt content and temperature have to stay within a certain range and denaturing substances have to be avoided.
• Concentration of fuel ATP and exhaust ADP and Pi  have an influence on enzyme activity.
• Contact between proteins and a bare synthetic surface often leads to denaturation of the protein so surface properties have to be controlled.
• Proteins if not kept frozen/lyophilized degrade due to oxidation and action of proteases present in trace amounts.

What are the problems associated with the construction of molecular shuttles?

Guiding the direction of  movement, controlling the speed( ATP hydrolysis), loading and unloading of cargo , coupling of cargo to the shuttle – by a strong and specific link are some of the problems that need to be addressed while constructing molecular shuttles.

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