Structural Biochemistry/Protein function/Actin

From Wikibooks, open books for an open world
< Structural Biochemistry
Jump to: navigation, search

Actin is the most abundant protein found in eukaryotic cells. It is a monomeric unit of microfilaments (actin filaments). The globular actin is often called G-actin. It contains a nucleotide-binding site, which can bind to ATP or ADP. The conformation of actin depends on the ATP or ADP in the nucleotide-binding site. Actin filament is often called F-actin. It is twisted helical chains of actins, which the actin monomers orient in the same direction of actin filament. It has polarity that contains different ends in its structure. One end is called barbed (+) while the other end is called pointed (-). They are called by the appearance when myosin S1 fragments are bound to it. An actin has a myosin binding site at every 2.7 nm. Actin filament occur as linear filament and also form structural networks, which plays a major role in muscle contraction, cell movement, cell signaling, cytokinesis, and cell division.

The structure of actin monomer was observed to atomic resolution through x-ray crystallography, which scientists have been determined that the structure of actin as filaments. As actin monomers (G-actin) bind together, they form actin filament (F-actin), which has a helical structure. Each monomer has a distance of 27.5pm with a rotation of 166 degrees around the helical axis. Each actin monomer is orientated in the same direction along the helical structure (F-actin), which makes up a polar character of the structure. One end of the helical structure is called the "barbed" end (+), and the other end is called the "pointed" end (-). Actin filaments are self-assembled, which actin monomers come together as very well-structured, polar helical. Since the aggregation of the first two or three monomers to form actin filament is highly unfavorable, specialized protein complexes, such as Arp2/3, would be required to serve as nuclei for actin assembly in cells. Once the first filament nucleus exists, the addition of subunits is more favorable.

Regulation of Actin Filament[edit]

A skeletal muscle fiber only moves when it is stimulated. Otherwise at rest, the binding sites are blocked. Actin contains two types of regulatory proteins that modulate the binding site. The first type is tropomyosin, a protein chain that lies along actin and covers the binding sites. Troponin C is attached to tropomyosin and directs the position of tropomyosin on actin. Once a Troponin C binds to calcium, it pulls the tropomyosin to unwrap the binding sites. The exposed binding site allow for myosin to bind to actin. Once myosin binds to actin, it forms a "cross bridge" and is called a rigor complex.



Actin-ATP can polymerize to form actin filament. It is more readily to polymerize than actin-ADP, because actin polymerization occurs when bound ATP in actin is hydrolyzed to ADP.

Three Nucleators:


Actin filament is self-assembled spontaneously, but formins can help the formation. Formin binds to the barbed(+) end of actin filament, and actin monomers can be added on it until a plus-end capping protein binds to the barbed end. This mechanism stabilizes the polymerization and consequently produce a linear unbranched fliament due its FH2 domain.

Arp 2/3 Complex[edit]

Branched actin networks can form from polymerization with Arp2/3 complexes. Arp2/3 complex is a seven-subunit protein that includes two actin-related proteins, Arp2 and Arp3, and five other smaller proteins. Nucleation of Arp2/3 complex associated with an activator such as WASp can start the actin polymerization. It binds to the side of an actin filament and nucleates to initiate growing of a new Y-shaped filament branch at a 70° angle. In this formation, the structures of two subunits Arp2 and Arp3 are similar to actin. These two subunits are then triggered by the binding of activator to mimic the barbed end of a filament.


Unbranched actin filaments are produced by spire that has WH2 domains each bind to an actin monomer to complete the nucleation. The mechanism associated with spire is totally different from those with formin and Arp2/3 complex. By organizing actin monomers into a prenucleation complex, spire makes template for filmation formation.

Consider the polymerization reaction in detail. Assume an actin filment with n subunits An. This filment can bind an additional actin monomer, A, to form An+1. The given equation is the following:


Kd is the dissociation constant, which defines the equal concentration of polymers of length n+1 and for the polymers of length n. Therefore, the polymerization reaction will proceed until the monomer concentration is reduced to the value of Kd.


Some major roles of actin include: (1) Being the structural makeup and support of the cytoskeleton.

(2) Dividing and producing cells in order to enable cells to move spontaneously and actively.

(3) Serving as a supportive framework for myosin proteins during muscle contraction.

(4) Acting as a track for the cargo transport myosins in non-muscle cells.

β- and γ- actin Proteins

The mammalian cytoskeleton proteins β- and γ- actin have amino acid sequences that are extremely similar, yet they both have significantly different functions in the cell. β-actin proteins are partly responsible for the cell mobility by pushing the cell forward, whereas γ-actin proteins promote cell adhesion. The β-actin protein structure changes when arginine is added, while γ-actin structure doesn’t change. Scientists have been trying to distinguish between the two proteins from their form and function. They found that slow translation of γ-actin leads to quick degradation by the proteasome in the cell because both arginylation and ubiquitination are allowed. Ubiquitination is a protein post-translation modification (PTM) process which results in labeling of the proteins so that they are sent to the proteasome to be destroyed. On the other hand, the fast translation of β-actin permits only arginylation and stabilizes the protein. γ-actin is less stable than β-actin due to the occurrences of many codons in its gene that slows down the rate of translation. β-actin proteins use different codons that code for the same amino acids. Lysine is an important amino acid found in both β-actin and γ-actin proteins. Researchers found that slowing the rate of translation experimentally allows for the process of ubiquitination by revealing the Lysine to ubiquitin and leads to the quick degradation of the γ-actin proteins.

Actomyosin Powerstroke Pathway[edit]

The powerstroke of actomyosin is linked through three events which lead to the release of products from ATP hydrolysis (inorganic phosphate and ADP): myosin head binding to actin, structural changes in the head causing strong actomyosin interaction, and the swinging of the lever. The study of ATP hydrolysis-linked enzymatic force generation is difficult to perform because efficient force generation requires the powerstroke to occur while myosin is bound to actin. And this process can only begin when myosin is in a low actin-affinity state, so it is quite rare to observe this occurrence.

Myosin has three different parts, a motor domain, the lever and the tail region. The motor domain is what swings the lever during powerstroke of actomyosin, it has three main parts: the nucleotide pocket, the actin-binding region and the relay region. Three loops: P-loop, Switch 1 and Switch 2 are attached to the nucleotide pocket and face the actin-binding and relay region. Weak interactions with actin is begun in the lower part of the actin-binding region, then when the cleft closes, the upper part of the actin-binding region folds over the actin and produces stronger binding interactions. The relay region interprets the conformation of the now folded actin-binding region and swings the lever from the primed “up” position down, the distance traveled by the lever determines the size of the powerstroke.

Kinetics block the ‘futile’ lever swing in an actin-detached state which leads to an ATP-wasting cycle. The ATP binds to myosin rapidly following a quick conformational equilibrium between down-lever and up-lever states (also known as the recovery step); this is followed by the hydrolysis of ATP. ATP can only be hyrdolized by myosin in the up-lever state. When myosin bings to ADP and P, it results in weaker interactions and the release of the P reduces the complexes stability and is rate-limiting in the absence of actin.; this is contradictory to previously thought rate-limiting step: release of inorganic phosphate. Release of inorganic phosphate is only possible during the down-lever state. In the absence of actin, myosin is mostly in the ADP and Pi bound up-state.

Over the past couple decades; many myosin conformations have been identified via crystallization process which teaches us about the allosteric communication pathways between the actin-binding region and the lever region during powerstroke. Experiments have revealed that energy barriers in myosin enzymatic steps, nucleotide binding, ADP release and conformational changes directly depends on the actions of the lever, meaning that the lever controls energy in the myosin complex during powerstroke.

The actin affinity is determined by the nucleotide content of the active site allosterically. Nucleotide-free and ADP-bound forms of myosin have been found to strongly bind actin, but in complexes where the gamma-phosphate sites are occupied with ATP or ADP-Pi, weak actin affinity is found. This is due to the allosteric coupling between the actin-binding region and the nucleotide pocket which is in the more distant regions of the motor domain. The actin affinity is determined by the conformation of the actin-binding region. The affinity depends primarily on the equilibrium of the switch 1 loop of the nucleotide pocket, which can have an open or closed conformation. The actomyosin powerstroke is initiated by myosin at low actin affinity.

An effective powerstroke stems from the pathway of actin-induced acceleration of the lever swing. The lever swing of ADP-Pi-bound myosin is accelerated by actin by over two orders of magnitude. Therefore actin activation is a crucial part in an effective powerstroke, despite the fact that it starts in a weak actin-affinity, or ADP-Pi, state. The reaction flux is brought into the kinetic pathway involving the lever swing cause by the powerstroke. The reaction flux is then brought towards the actin attachment after the futile lever swing is kinetically blocked. This however is not thermodynamically favorable but this non-equilibrium situation is necessary because this pathway has higher free-energy. This is known a kinetic pathway selection and is used to force a reaction through a more efficient pathway rather than a futile one that would be thermodynamically stable.

Another effective powerstroke pathway also begins with a weak actin attachment to a actomyosin complex. But an opening and closing of the actin-binding region, as opposed to just, is what causes the lever swing. In another method, the powerstroke might begin right after the weak binding of the lower actin-region on the myosin. Both of these alternative reaction pathways will result a reaction flux much like the original one described above. This shows that the reaction flux will also undergo kinetic pathway selection, something that scientists have began studying recently in detail to determine how important it is in physiological function.


  1. Berg, Jeremy; "Biochemistry", W.H. Freeman and Company, New York, 2007, Sixth Ed.
  1. Kashina, Anna, Saha, Sougata, Shabalina, Svetlana A., Zhang, Fangliang. "Differential Arginylation of Actin Isoforms Is Regulated by Coding Sequence- Dependent Degradation." Science 17 September 2010: 1534-1537
  1. Málnási-Csizmadia, A. “Emerging complex pathways of the actomyosin powerstroke” Trends in Biochemical Sciences, Volume 35, Issue 12, 684-690, 31 August 2010