Myosin

Michael N. Metzmaker 2-27-07

Molecular Machines Myosin and Actin

One of the best ways to improve today’s machines, and to come up with completely new ones, is not necessarily by thinking big. As a matter of fact, in recent years, thinking small has become much more important.

Nanotechnology, even though it is a new field, has been researched with great fervor and it shows almost limitless potential. Its scope extends most notably through various fields of medicine, where bionanotechnology is experiencing many breakthroughs. However, it can also be used in other fashions, most notably in ways to aid in the dilemma of energy scarcity in the modern world. Eco-friendly molecular machines could be the key to many of the problems with energy usage across the world. They could create new sources of energy, and new machines to use them. They could generate more efficient forms of already existing processes, such as photo- and chemosynthesis. Others could be modified to work as engines.

Many existing biological molecular machines already work as motors. For example, the molecular walkers, such as dyneins and kinesins pull organelles and vesicles along microtubules inside cells. There are outboard motors with propellers called flagella, and tiny stroking “arms” called cilia, that propel bacteria through solution. All of these have significant applications for nanotechnology. However, one molecular motor has potential to be used at a variety of scales: myosin.

Myosin is a protein involved in one of the most large scale movement mechanisms of the animal kingdom: muscle movement. Every muscle contraction, from the skeletal muscle contraction, which extends the knee while running, to the cardiac muscle contraction of the heart that pumps blood, requires myosin. It is one of the predominant biological molecular machines whose effects can be seen at the macroscopic scale.

Everyone can see how muscles work as a tissue, pulling on joints and causing motion. The way a muscle contraction works at the molecular level, however, is much more complicated. While different types of muscles work in slightly different ways, the molecular processes behind skeletal muscle contraction form the basis of the other types of muscle contraction. The other forms (cardiac and smooth muscle contraction) also use the myosin machine in conjunction with an actin filament, but skeletal muscle’s obvious organization of muscle fibers into sarcomeres is the easiest to see with a microscope. Skeletal muscle is easier to access and stimulate to contract in a lab environment. It has therefore been studied more extensively than the other types of muscle.

Muscles cells are organized into hundreds of thin, cordlike myofibrils, arranged longitudinally, which are sectioned into many cylindrical sarcomeres. Each sarcomere contains the working molecular portions of the muscle, and is the basic contractile unit of the muscle (Figure 1.0). In each sarcomere, under a light microscope, one can see thin filaments and thick filaments, which overlap. This overlap is what allows the sliding filament theory of muscle contraction to work.

The sliding filament theory was proposed by Andrew Huxley in 1969. It is based on the idea that in a sarcomere, there are two types of filaments visible through a microscope. The thick filaments are made up of the bundles of myosin, while the thin filaments are made up of actin. The H-Zone is an area in which there are only myosin filaments, with no overlap of actin. The I-Band is an area containing only actin. The A-Band is the area containing all of the myosin, all the way out to where there is actin overlap. The M-Line is the middle line toward which the myosin heads pull.

At rest, the thick filaments overlap the thin filaments slightly. After partial contraction, they overlap more, and after full contraction the two filaments overlap completely, until they are lined up in a very neat column. The opposite occurs with during relaxation (Figure 1.1).

It seems difficult to believe, but during muscle contraction, no filaments change in length. Instead, molecular interactions within the sarcomere cause the filaments to slide past each other longitudinally, producing an overall shortening and thickening of the tissue.

The interaction of myosin and actin in the presence of ATP (adenosine triphosphate) allows muscles to contract at the molecular level. Each myosin molecule consists of a long tail and a globular head region. All the tails of the separate myosin proteins bundle together in the middle of a sarcomere, and the heads remain at intervals closer to the outside to form the thick filaments. The actin proteins are formed into a twisted double chain of the many globular actin subunits. Each actin has a small space that easily connects to the myosin head called the “myosin-binding site”.

The myosin head is the driving force behind the bioenergetic reaction of the actin-myosin complex. It can bind ATP and hydrolyze it into ADP (adenosine diphosphate) and a Pi (inorganic phosphate). The hydrolysis of ATP raises myosin into a high-energy configuration, so that it can more easily bind to the actin, forming what is called a cross-bridge. Then, as myosin releases ADP and Pi – moving toward its low energy configuration – it slides the actin filament toward the center of the sarcomere. Once this release is complete, the myosin is ready to attach a new ATP and go through the process again (Figure 1.2).

Other structures involved in the process of muscle contraction are tropomyosin and troponin. However, these most likely would not be present in commercial uses of the molecular machine. Tropomyosin is a long, cordlike protein that blocks the myosin binding sites on the actin proteins. It is attached to many troponin molecules that, when bound to calcium ions, pull the tropomyosin off the binding sites, and allow contraction to begin. Therefore, in biological contractions, calcium is crucial for troponin and tropomyosin. Its presence allows the process of muscle contraction to occur (Figure 1.3).

Fortunately, if the molecular machines were engineered correctly, they could be created without the presence of troponin and tropomyosin. Because myosin and actin can be attached to surfaces inexpensively using histidines, it would be very easy to simply omit putting troponin and tropomyosin in the solution prior to attachment. The proteins would be cloned from an organism into bacteria such as E. coli which could then manufacture only the specific protein desired.

It is assumed that a large obstacle in commercializing the use of biological molecular machines is implementing them outside of a cellular or biological environment. Fortunately, this is not necessarily the case. Kinesins have been implemented in vitro since as early as 1985. Several other molecular motors have been implemented outside of biological systems more recently as well.

The actin-myosin complex has great potential for commercial use. It has already been developed into a motor of sorts, and is patented and up for sale to the highest bidder. It involves myosin, attached to a substrate, and actin, attached to another substrate. The motor would require a fuel, but it would be the low cost, low pollution, energy efficient fuel. Simply enough, ATP runs the myosin motor, just like in muscle cells. And, without the added interference of tropomyosin, calcium is not required. The proteins can be attached to a variety of surfaces. Actin, for example, seems to attach nicely to nickel-coated surfaces. Precise lithographic positioning might allow attachment of the proteins in very organized arrangements.

The structure of the motor can vary slightly. The main idea is that the two surfaces – one with actin and one with myosin – would be surfaces of a cylinder. For example, the myosin would probably coat the inner surface of a slightly larger cylinder, and the actin would coat the outer surface of a slightly smaller cylinder. The two surfaces would have to be very close together to allow interaction between the proteins, but far enough apart to allow rotation. The proteins would have to be arranged along the curved surface in such a way to produce a pull all in a predetermined direction. The inner cylinder can then have a gear protruding from one end, which can drive another gear and be involved in powering a very small machine (Figure 1.4). It also has the potential to be scaled up to macroscopic proportions. Also, the inner cylinder could be attached to a variety of other simple machines, such as a pulley system, a propeller, a wheel, or a lever-arm.

The rotational formation provides a huge advantage over the biological sliding filament formation in that relaxation would not have to take place; the gear could be in almost perpetual motion, provided a constant energy source was available. The energy that runs the device would be ATP, which is, of course, an extremely clean and renewable resource. The eco-friendly advantages of ATP as a resource are obvious, including abundance, no pollution, and simple manufacture. The advantage to this particular system is just as important. ATP would have a regulatory effect on the motor in that the concentration of ATP in the system would determine the rate at which it would run. The ATP could simply be flooded into the space between the two cylinders to facilitate rotation.

Potential uses for the motor are wide ranging. They could be used in animals that have had nerve damage and had lost motor function in a limb or digit. It could be used in a robot, to facilitate movement of limbs or any other parts of the machine. The potential of having multiple cylinders built inside one another to make a very large motor gives the machine astronomical potential. It could be used in a car to replace the internal combustion engine. Multiple cylinders being stacked inside of one another could increase the speed of the motor by a huge margin. The imagination is the only limit to the application of this machine.

The molecular machine of the actin-myosin complex has a very useful aspect: cheap motion. Whether discussing its biological properties in muscle contraction or its potential commercial use in driving gears, the machine produces quick motion that is reproducible, inexpensive, efficient, sustainable, and widely applicable. The machine can solve the problems involved with burning fossil fuels in transportation and may protect the environment in years to come.

ATP Synthase paper

Trey Dyer

FYS- Molecular Machines

Prof. Jed Macosko

2/12/07

The Body’s Rotary Based Generator- ATP Synthase

Mankind has been studying the human body since, well, the beginning of mankind. Over time, man has gradually come to grasp what makes our bodies move or our blood pump. In the last century or so, scientists have been making landmark discoveries such as cells and DNA. Theses discoveries have created sub-branches of biology including bionanotechnology. Bionanotechnology is the study of molecular machines inside the cell and how they work. Every cell in our body is made up of molecular machines. The potential of machines in bionanotechnology can shape and change our world. Eco-friendly machines such as the energy producing ATP synthase can - and hopefully - will be harnessed for the improvement of our environment. In order to fully harness machines such as ATP synthase humanity must first understand its structure and function. Understanding these concepts is crucial to the development of bionanotechnology.

In order to understand ATP synthase’s function and how to harness its power, we must first understand and grasp its structure. ATP synthase is found embedded in the membranes of micro-organisms such as bacteria, chloroplast (thylakoid membrane), and in the inner mitochondrial membrane of eukaryotic cells. It is a large “mushroom-shaped” enzyme. This portion of ATP synthase is water soluble. Its crystal structure has been deciphered where as the F₀ portion has not. It exists as a complex of at least 24 proteins, depending upon the organism.

ATP synthase is divided into two motor-like sections along an axel- the F₀ and F₁ motors. The F₁ motor is the part of the enzyme that is located on the outside of the membrane. It is sometimes called the “cap” of the mushroom shape. It is made up of 5 different types of subunits: 3a (alpha), 3b (beta), 1g (gamma), 1d (sigma), and 1e (epsilon). The three alpha and the three beta subunits form a ring of alternating subunits. This is what is known as the cap of the mushroom. This is the catalytic portion of ATP synthase. These six alternating subunits form a ring around the gamma subunit. The gamma subunit is the axel of the motor of ATP synthase extending down into the F₀ motor. The epsilon is connected to the gamma subunit towards the bottom of the axel where it disappears inside of the cell membrane. The sigma particle is connected to the top of the hexamer of alpha and beta particles. The sigma particle is connected to the stator which is used down in the F₀ portion of ATP synthase. The three beta subunits in the hexamer or “cap” of the mushroom of ATP are the binding sites where ATP is catalyzed. The F₁ motor is turned due in large part to the F₀ motor.

The F₀ motor is embedded in the membrane of the cell. It is made up of 3 subunits: a, b, and c. Six c subunits form yet another hexamer located in the cell membrane. This hexamer is formed around the very same gamma subunit or axel as the F₁ motor. These c subunits are strongly hydrophobic. The rotation of these six proteins rotates the gamma subunit, which in turn rotates the F₁ motor. Loosely connected on the outside of these three proteins is the a subunit. The translocation of protons takes places between the a subunit and the rotating c subunits. Connected to the a subunit is the b subunit. The b subunit is what’s known as the stator. It guides the protons in order for the electrochemical gradient to cause the F₀ motor to rotate or run. The b subunit connects to the sigma subunit of the F₁ motor which connects to the hexamer as well. The structure of ATP synthase is a complex structure of proteins essentially forming two motors connected through one axel. This structure enables ATP synthase to accomplish its true function- generating energy for the cell.

As stated earlier, ATP synthase is composed of two rotary type motors sharing the same axel. The question now is how do these two motors turn into one super-efficient generator. The first thing to understand with any motor is what its fuel is, what does it run on? The F₁ motor runs on ATP or the catalyzation of ATP. However, this motor is usually turned by the F₀ motor which causes the catalyzation of ATP. Producing ATP is this machine’s main purpose since ATP is the core source and form of energy used by nearly every organism in the body. The fuel for the F₀ motor is an electrochemical gradient created with the flow of protons across the cell membrane.

These motors are bidirectional. Cleavage of ATP can cause the F₁ motor to rotate and in turn creating an electrochemical gradient. Also, the movement of protons through the cell membrane can cause the F₀ motor to spin which in turn causes rotation in the F₁ motor and thus catalyzes the reaction that synthesizes ATP. It is essential to see how these two motors work together to make ATP synthase an effective generator.

The F₀ motor is the starting point of ATP synthesis. First the hexamer of c subunits must rotate to turn the gamma subunit, which turns the F₁ motor. The F₀ motor is driven by an electrochemical gradient of protons within the cell membrane. The stator of the F₀ motor, the b subunit, guides protons in the membrane in between the a and c subunits. This creates an electrochemical gradient, which in turn causes the hexamer of c subunits to rotate. This rotates the gamma subunit or axel, which in turn rotates the F₁ motor. The movement of protons across the membrane is due to a simple electrochemical gradient. The stator then guides some of these proton in between a and c, which leads to rotation.

The rotation of the F₀ motor also causes the rotation of the F₁ motor, which causes the catalyzation of ATP. When the gamma subunit is rotated, the active sites or beta subunits undergo a change in binding affinity for the reactants of the ATP catalyzation. These reactants are inorganic phosphate and ADP. The three sections of the hexamer (one alpha and one beta subunit) have different affinities to the nucleotides. One section, the loose section, binds the ADP and phosphate together. The second section, the tight section, then binds these reactants so tightly that ATP is formed. The third section, the open section, then releases the newly formed ATP. As the F₁ motor rotates, these sections of three change into these different affinity levels: loose changes to tight, tight to open, and open to loose again. ATP is then transported throughout the cell to where energy is needed. ATP synthase is now an effective generator that turns an electrochemical gradient into a viable source of energy in ATP. ATP works as an enzyme really. It catalyzes the following reaction: ATP^4- + H₂O <=> ADP^3- + P_i^2- + H⁺.

The central axel, the gamma subunit, of ATP Synthase rotates 50-100 times a second. Daily, the human body generates over 100 kg of ATP a day. The actual precise name for ATP synthase is ATP phosphohydrolase (H⁺-transporting). There are four main types of ATP synthase; all of which are very similar. A big difference, though, is the V-ATPase which can use Na⁺ or sodium to create its electrochemical gradient to run the F₀ motor. Overall, ATP synthase is an incredible machine with incredible powers.

ATP synthase is just one of many molecular machines that have the potential to change and shape the world. ATP synthase is a very complex and efficient motor and energy generator for the body. As technology advances and mankind continues to make breakthroughs in the field of bionanotechnology, ATP synthase will be an essential machine to harness because its endless possibilities. Mankind is in constant need of energy. Humanity continues to use up our resources and our environment in search of energy. If harnessed, a molecular machine such as ATP synthase could do wonders for mankind and the world around us.

Aptamers

Ashley Edwards

Molecular Machines FYS

Aptamers

Aptamers show important promise in the medical field; eventually they might be able to treat a myriad of diseases from macular degeneration to HIV to cancer. Already there are drugs on the market that utilize aptamers, and there are many companies that are starting to take interest in these new potential treatment options because they are highly specialized and can be custom-made in a mere matter of months to target almost any molecule in the body. Aptamers are similar to antibodies in that they target protein-protein interactions, but differ in how they are made, the functions they perform, and the way in which they are used in medicines today.

Aptamers are micromolecules made up of nucleic acid that bind to a specific target molecule. They work by disrupting a specific protein interaction by direct competition with what usually binds to the target protein. They use the full range of bonding methods that proteins use, including hydrophobic interactions, charge-charge interactions, and hydrogen bonding. Aptamers are chemically synthesized and combine the best properties of antibodies and small molecules such as chemical stability, high affinity, and low immunogenicity.

In order to make an aptamer, a pool of individual nucleotides is placed in a test tube with the appropriate RNA or DNA polymerase. Since the aptamers are made up of nucleic acid, they can be written out symbolically on paper in a series of letters representing the nucleic acid (adenosine, thymine, guanine, cytosine, and uracil), just like DNA and RNA. Aptamers have individual three dimensional shapes so that they can fit tightly to a receptor protein like a lock and key. Once you have an incredibly large group of aptamers (in the millions), they must be selected for the specific target protein. They can show high specificity for their target protein, and can therefore discriminate between similar proteins.

The Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is the method used to select aptamers for the target proteins. First, a starting library of nucleic acids is incubated with the protein target of interest. Next, the aptamers that bind to the target, which is usually a protein, but can be almost anything (even ATP), are separated from those which did not bond. The aptamers that bind are then amplified to create a library with those specific aptamers. The process is then repeated, since originally multiple aptamers might bind to the target protein, but after a couple of trials, there will usually be very few that consistently bind. Antibodies, on the other hand, are biologically expressed and not chemically synthesized like aptamers are which makes them more expensive and does not provide them with high stability in harsh environments. Antibodies are also not usually tested intracellularly, which makes them more likely to change once they are inserted into the body. Aptamers work on the outside of the cells, which makes it easier for their environment to be controlled and makes them easier to make, whereas antibodies enter the cell and therefore create many more obstacles to consider.

Once an aptamer that will bind to the target molecule is found, it can be used in a practical manner. Aptamers are usually used for one of two things; either function-blocking or escorting. Function-blocking aptamers disrupt the protein interaction by direct competition and can therefore interrupt the process of disease. HIV, for example, uses TAR RNA to help replicate itself and recruit other cells. An aptamer that mimics the TAR RNA, can compete with it for binding positions on the tat protein, which is what the TAR RNA of HIV binds to. By sequestering the tat protein from the HIV RNA, the aptamer can inhibit the replication of the virus and slow down or even stop the progression.

Escort aptamers are designed to deliver radionuclides, toxins, or cytotoxin agents to diseased tissues. In order to make an escort aptamer, an existing aptamer that has been shown, through the SELEX process to bind to the target protein, is used. The aptamer is truncated in a way that it will still bind to the target protein but will not have any effect. On the truncated parts, it is possible to attach almost anything organic that will react with the cell. In this way, aptamers can deliver many different things to the outside of the target molecules. They also show great promise in this area because of their rapid blood clearance and their high level of adaptability. The rapid blood clearance helps aptamers quickly leave the body. In tissues, aptamers can accumulate into high concentrations because they are taken in rapidly and the tissues retain them very well. The aptamers have a high level of specificity; they can differentiate normal from abnormal tissues, which shows that they could be capable of targeting cancerous cells.

Already, Eyetech Pharmaceuticals has come up with macugen, which is FDA approved and takes advantage of aptamers in treating macular degeneration (the disease that destroys the vision in the elderly). The drug uses an anti-VEGF aptamer that binds to the VEGF 165 protein that signals the growth of abnormal blood cells in the eye. Macugen blocks the binding of the VEGF to its receptor, and therefore prevents the growth of abnormal blood cells that cloud vision.

Many other companies are working on using aptamers in their drugs as well. The leading company in aptamer research is Archemix, which holds a number of patents on aptamer technique, and therefore has made quite a few handsome deals with various drug companies such as Pfizer, Nuvelo, Elan, and Merck KGaA. Basically, Archemix uses SELEX to select aptamers that bind to the proteins that are involved in the diseases that the drug companies wish to treat. Archemix then gives the drug company the right to mass-produce and market the drug in exchange for research funding upfront and milestone recognition, along with more compensation if the drug does well on the market.

Archemix recently announced the initiation of a clinical trial for a new anti-platelet aptamer that stops platelet-dependent clotting in only the targeted sites of the body, while keeping the rest of the platelet clotting normal. This could potentially help patients with life-threatening thrombosis, which causes the formations of clots in the bloodstream, inhibiting the flow of blood in the circulatory system. The formation of clots in the bloodstream can cause strokes and if it goes untreated, can cause death. This breakthrough drug could have enormous potential if the clinical trials go well, and would help promote the use of aptamers in many more medications that treat many different diseases. Archemix’s cooperation with Merck KGaA is all about finding treatments and/or identification of cancerous cells. If aptamers could be used to target every type of cancer cell, then the terminal cases could decrease dramatically, if not disappear completely.

Obviously, aptamers are on the up-and-coming medicinal wave, and in another decade, it is quite possible that almost every new drug will be made from aptamers. They show great promise in binding to almost any target molecule, and once targets are identified for diseases, the selection process for aptamers can begin. Unfortunately, some diseases seem to be more complex, and therefore simple competition with the target site might not be enough to stop the progression. Hopefully though, aptamers can make a huge difference in the diseases that do have target cells, and also be a key component in identifying diseased tissue through its escort capabilities. Archemix has already made huge leaps in the field of aptamer drugs, and the contracts with various drug companies will provide them with enough research funding to continue finding aptamers that will work to combat some serious and common diseases.

Literature Cited

"Aptamer Therapeutics." Archemix. Jan. 2007. Archemix Aptamer Therapeutics Company. 9 Feb. 2007 .

"Aptamer." Wikipedia. 7 Feb. 2007. 9 Feb. 2007 .

P. Schultze, R.F. Macaya, and J. Feigon: "Three-dimensional solution structure of the

thrombin-binding DNA aptamer d(GGTTGGTGTGGTTGG)", J. Mol. Biol. 235, 1532-1547 (1994).

White, Rebekah R., Bruce A. Sullenger, and Christopher P. Rusconi. "Developing Aptamers Into Therapeutics." J Clin Invest. 106 (2000): 929-934. PubMed. 9 Feb. 2007.

review of textbook

The Bionanotechnology textbook is a good book for beginners who are looking to learn more about the field. A basic background of biology or chemistry would be very beneficial to the reader but it is not required to understand the concepts. Bionanotechnology could be one of the leading fields of research and discovery in the coming years and this book provides a solid basis of understanding about the field.

The Alternative Pathway

Michael Farrell

Prof. Macosko

27 February 2007

The Alternative Pathway and the Molecular Machines Used in It

The human body uses a very complex system called the Immune System in order to heal and protect itself from injuries and infections. One of the components of this intricate system is called the Complement System. The Complement System is a “biochemical cascade… that helps clear pathogens from an organism, and promote healing” (“The Complement System”). The Complement System is composed of three different pathways, each of which has its own function. The pathway which this paper focuses on is named the Alternative Pathway. The Alternative Pathway is also a biochemical cascade, which is “a series of chemical reactions in which the products of one reaction are consumed in the next reaction” (“Biochemical Cascade”), whose purpose is to find pathogens in the body and protect the body from possible harm using molecular machines.

The Alternative Pathway can be broken down into four distinct steps, each one as important as the previous step. The first step “begins with the activation of C3 and requires Factors B and D and Mg++ cation, all present in normal serum” (“Immunology- Chapter 2: Complement”). C3 is a protein produced in the liver, and Factors B and D are proteins which are also found in the body and are essential to the Alternative Pathway. Serum is just another name for blood and it where all these molecules are found. In this serum, hydrolysis of C3 is occurring and the C3i complex is formed. C3i is simply the name for the hydrolyzed form of C3. After C3i is formed, Factor B then binds to it, which makes the substance susceptible to cleavage by Factor D. Not surprisingly, Factor D then cleaves Factor B into Bb. This cleavage forms a new complex named C3iBb. C3iBb acts then acts as a convertase for C3. A convertase is “an enzyme that catalyzes the conversion of a substance to its active state” (“Convertase”). This C3iBb complex will then cleave C3 from the surrounding serum into C3a and C3b. This newly formed C3b will also bind the Factor B protein and become susceptible to cleavage by Factor D, similar to the process C3iB went through. Now, C3bB will be cleaved by Factor D resulting in the C3bBb complex. C3bBb is also a C3 convertase, like C3iBb, and cleave the surrounding C3 into even more C3a and C3b. It is important to note that in this first step, unlike the other two pathways of the Complement System, the Alternative Pathway is “triggered by C3 hydrolysis directly on the surface of the pathogen, it does not rely on pathogen-binding proteins like the other pathways” (“The Complement System”).

During the first step of the Alternative Pathway, the C3 that is being produced binds to the surface of a pathogen in the blood. If there is no pathogen present, the C3b will be deactivated and will end the biochemical cascade. As the C3 binds to the pathogen it interacts with the Decay Accelerating Factor (DAF). The DAF can perform a number of tasks but they all result in the same outcome: stopping the production and formation of additional C3b in the blood. One way it could do this is by “blocking the association of Factor B with C3b thereby preventing the formation of additional C3 convertase” (“Immunology- Chapter 2: Complement”). Another possible function of the DAF is to “accelerate the dissociation of Bb from C3b in C3 convertase that has already formed, thereby stopping the production of additional C3b” (“Immunology- Chapter 2: Complement”). An additional way the amount of C3b is controlled is by the “enzymatic degradation of C3b by Factor I” (“Immunology- Chapter 2: Complement”), which is facilitated by complement receptor 1 (CR1). Not every cell has CR1, but it also helps in controlling the levels of C3b inside a cell. It may not seem important to control the levels of C3b, since it provides a good service to the immune system, but studies have shown that people with surpluses of C3b are actually more prone to contract certain infections (“Immunology- Chapter 2: Complement”). This process of controlling the amount of C3b comprises the second step of the Alternative Pathway.

The third step continues the biochemical cascade with more reactions. Now that the C3b has stopped being produced, the previously mentioned C3bBb comes to the forefront of the cascade. It has been “‘hooked’ onto the surface of the pathogen, (and) will then act like a ‘chainsaw’, catalyzing the hydrolysis of C3… which positively effects the number of C3bBb hooked onto a pathogen” (“The Complement System”). The C3bBb complex is a very unstable substance, but when it binds to the pathogen, or “activator surface” (“Immunology- Chapter 2: Complement”), it stabilizes. C3bBb is also stabilized by the protein Factor P, or properdin, binding to it. Some of the pathogens that it can bind to are bacterial, yeast or plant polysaccharides, fungi, bacteria and viruses. Once it stabilizes, it is able to create more C3b by cleaving C3. This stabilization of the C3bBb complex is the third step of the Alternative Pathway.

At this point in the process, there is an abundance of C3b and C3bBb just outside of the pathogen. These two combine to form the C3bBbC3b complex, also known as C5. This generation of C5 is where the Alternative Pathway ends. From here, C5 will cleave into C5a and C5b and C5b will help form the Membrane Attack Complex (MAC). The MAC punches a hole into the membrane of the pathogen and initiates cell lysis. In addition the MAC, the Alternative Pathway can also lead to mast cell degranulation, another form of protecting the human body from disease and infection.

The Alternative Pathway is the most basic of the three pathways of the Complement System and scientists believe that the other pathways may have derived from it (“Immunology- Chapter 2: Complement”). The Alternative Pathway is more of a general defense mechanism against disease and infections, as compared to the other two pathways which target specific pathogens. Although it is primitive, many molecular machines are still involved. All of the enzymes that cleave proteins in the pathway are considered molecular machines. This list includes Factor D, C3iBb and C3bBb. These machines could theoretically be used to cleave other substances in practical life and be useful to humans in other fields besides immunology.

Other than the protein C3, the structures of the complexes named above are unknown and a distant goal of scientists currently. This is a main reason why not much is known about the possibilities of these molecular machines and how they could help humans in the future.

When looking at the Alternative Pathway, it is important to understand the dynamic of the biochemical cascade. Without a previous reaction, the next cannot be completed. So even though it may seem like C3 doesn’t need to be cleaved twice during the pathway, it needs to because of the order of the cascade. It is important to keep this in mind when trying to understand the process of the Alternative Pathway.

The Alternative Pathway is a main aspect of the body’s defense against pathogens and molecular machines play a vital role in its application. These microscopic wonders protect people from disease by triggering certain chemical responses, without which diseases and infections would run rampant in our bodies.

Works Cited

“The Alternative Complement Pathway.” Microbiology @ Leicester: Infection and

Immunity. 2004. 26 February 2007.

http://www-micro.msb.le.ac.uk/MBChB/Merralls/Alternative.html

“Biochemical Cascade.” Wikipedia. 29 January 2006. 26 February 2007.

http://en.wikipedia.org/wiki/Biochemical_cascade

“Blood Plasma.” Wikipedia. 24 February 2007. 26 February 2007.

http://en.wikipedia.org/wiki/Blood_plasma

“The Complement System.” Wikipedia. 25 February 2007. 26 February 2007.

http://en.wikipedia.org/wiki/Complement_system

“Convertase.” The American Heritage Stedman's Medical Dictionary. 2nd ed. 2004.

“Immunology- Chapter 2: Complement.” Microbiology and Immunology On-Line.

11 September 2006. University of South Carolina. 26 February 2007.

http://pathmicro.med.sc.edu/ghaffar/complement.htm

Outline

1) Introduction
2) Complement System
a) Classical Pathway
b) Alternative Pathway
c) Lectin Pathway
3) MAC Components
a) C5
b) C6
c) C7
d) C8
e) C9
4) Formation of the MAC
a) Steps Involved
b) Interaction between components
5) MAC Regulation
a) S-protein
b) CD59
6) Conclusion

Paper

Kyle Cubin
2-12-06
Paper #1
Membrane Attack Complex


Membrane Attack Complex: The Oil Drill Molecular Machine


A human’s immune system has the ability to protect the body from nearly all dangers detrimental to its health. The complement system is one of the most crucial mechanisms of the immune system because of its ability to drill through the membrane of an antigen, or infected cell, and effectively destroy it. The system has various pathways and processes, but in the end it all leads to one destructive machine, the membrane attack complex which effectively drills through the cell’s membrane and induces cell lysis. The membrane attack complex is a combination of five different proteins that come together to form the final mechanism for killing the antigen, each protein serving a different purpose in creating a new molecule that has a new independent role. The membrane attack complex is a natural molecular machine that is a great example of the potential of bionanotechnology.
The complement system has three different pathways that all lead to the membrane attack complex. The first pathway is the Classical Pathway, which is part of the specific immune response. This means is that this pathway is very specific to what it attacks because it is looking for a unique antigen that triggers the response. The initial step for this pathway is for antibodies to recognize the foreign cell and attaching to the cells surface, allowing the C1-complex to bind to the antigens. From this point, the new C1-complex begins to cleave subsequent proteins in a cascade of events that leads to the formation of C3-convertase. Now the Alternative Pathway starts, which is also capable of beginning in the innate immune response without the proceeding Classical Pathway. So in this pathway the initial protein does not need the antigen to be recognized by antibodies to bind to the surface. The starting point of this process is C3 hydrolosis, which breaks the C3 protein into C3a and C3b. C3b then cleaves C5 into C5a and C5b, and C5b then becomes the starting point of the membrane attack complex (MAC). The third pathway, or the Lectin Pathway, is essentially the classical pathway, but instead of utilizing the C1-complex it uses opsonin, mannan-binding lectin (MBL) and ficolins. The pathway also utilizes different serine proteases than the other pathways do.
The pathways involved in the complement system serve two main purposes. The first is to put protein segments into the bloodstream to draw more lymphocytes, or white blood cells, to the site of infection. The second purpose is to form the membrane attack complex. The membrane attack complex will basically drill a hole in the antigen cell, which allows free diffusion of molecules in and out of the cell, leading to the death of the cell. The membrane attack complex is formed by five different kinds of proteins; C5b, C6, C7, C8, and C9. The structure of these proteins is crucial to the formation of the MAC. C5b possesses a metastable binding site, most likely caused by the expression of a hydrophobic site that is specific to C6. C6 and C7 have very similar molecular structures; they are both single-chained glycoproteins and considered to be serine proteases. C8 is made up of three non-identical chains; the alpha and gamma chains are disulfide linked and the beta chain is covalently associated with the alpha-gamma complex. C9 has a structure such that if cleaved a hydrophilic segment, C9a, and a hydrophobic segment, C9b, is produced. C9a represents the NH2-terminal end of the molecule and the C9b is the NOOH-terminal segment. It is thought that the hydrophilic nature of C9a is important for binding to the C5b-8 complex, while the hydrophobic C9b site is important for anchoring the MAC into the cell membrane.
The actual process of the formation of the MAC is a cascade of steps each having to occur in a given order. The process begins with the C5 protein being cleaved into C5a and C5b, C5a is released into the blood as a chemokine to attract neutrophils, a type of innate white blood cells, to the site of infection. Alternatively, the C5b segment attaches to the cell surface which exposes the metastable binding site for the C6 protein. After the C6 protein has bound to the C5b protein, the C7 protein can come and attach to the C5b-6 complex forming the C5b-7 complex. This complex now expresses hydrophobic regions that are capable of inserting into the membrane, thus forming the anchoring device of the MAC. The C7 also forms a site with specificity for the C8 protein that binds to the entire complex. The new C5b-8 complex has a receptor for numerous C9 proteins and once the initial C9 protein binds the process of C9 oligomerisation starts. Essentially, C9 oligomerisation is the binding of at least twelve C9 proteins in a circular shape as each protein punches into the cell membrane. It is at this point that the MAC is finished and the cell lysis begins, leading to the ultimate death of the cell.
It is apparent that the membrane attack complex is an effective cell killer, but it has potential to damage healthy cells if left unregulated. The complement system has been proven to have direct relationships with autoimmune syndromes, including issues as severe as the destruction of vascular integrity in a lung allograft and causing secondary injuries following spinal cord trauma. There are certain proteins involved in the regulation of the MAC to prevent from autoimmune attacks; the first is the S-protein. This is the primary inhibitor of the attachment of the MAC to the cell membrane. The S-protein will actually bind to the metastable site on the C5b-7 complex that would normally bind to the lipid membrane. This prevents the complex from ever getting anchored into the cell and beginning to destroy it. The S-protein allows the rest of the formation of the MAC to be completed off the cell surface and stops short of C9 polymerization. C9 polymerization stops short because of the S-protein, which also regulates the formation of the ring of C9 proteins that will puncture the hole in the cell membrane. The next regulatory protein is on the cell surface and is called CD59. CD59 is considered to be the most effective inhibitor of the complement process. CD59 exists on human cells and will bind to the C5b-8 or C5b-9 complexes and not allow the complexes to secure themselves to the cell surface directly; therefore the hole is never punctured into the cell.
Without the membrane attack complex, the human immune system would not be nearly as effective. The complement cascade is involved in both the innate and adaptive immune response and is the ultimate killer of infected cells. The structure of the MAC is very particular and results completely from the configuration of the proteins that it is comprised of. Each protein has a specific site to bind to the next protein in the cascade and that site is only exposed once the previous event in the cascade occurs. The conformational changes of the proteins turn seemingly useless proteins, if left unaltered and alone, into one of the most effective molecular machines in the human body. The MAC is like an oil drill that drills through the infected cells membrane and allows the free flow of molecules. However, the power of the machine is also seen in autoimmune attacks on the body, and because of the power of the MAC it is very effective at killing healthy cells. This is why it is very important to have regulatory proteins that prevent the formation of the MAC in certain situations.

Works Cited
“Complement Membrane Attack Complex”. Wikipedia. http://en.wikipedia.org/wiki/Membrane_attack_complex. 4 December 2006.

“The Complement System”. http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/Complement.html#The_Classical_Pathway. 20 September 2006.

“The Membrane Attack Complex of Complement”. H J Muller-Eberhard. Annual Review of Immunology. Vol. 4. 503-528. April 1986.

“The Role of Complement in the Elimination of Microorganisms”. http://www-micro.msb.le.ac.uk/MBChB/Merralls/Merralls.html. Microbiology @ Leicester. 2004.

Textbook Review

Overall, I think the textbook was an excellent introduction into the world of bionanotechnology. Although at times concepts were a little difficult for me to grasp, the use of multiple figures really added another dimension to the book. I would recommend the textbook to anyone wishing to explore the miniature world of molecular machines and the wonders of bionanotechnology.

Review Bionanotechnology

Goodsell does an excellent job describing the uses of biological machines in nanotechnology. He talks about an already existing assembler can be used to build molecular machines. He also describes the mechanisms by which these machines are structurally assembled and the different molecules that are used to form the machines. He describes the vast benefits of these machines in today's world and also the limitations of bionanotechnology. The illustrations in this book show the structures of naturally occuring molecular machines which help the reader better understand the complicated material. Overall this book is difficult but interesting read that can provide an insight into the world of bionanotechnology which continues to be expanded upon today.

Text Review

Overall, the textbook did a good job explaining its various concepts. It made use of a variety of diagrams and explained each one well.

Bionanotech showed me that molecular machines play a vital role in everyday life. They also have great potential in benefiting humanity if they are harnessed properly.

Textbook Review

The textbook was conceptually very sound. I thought it did a good job of explaining all aspects of the technology it described. I would not recommend this book for use in a freshman seminar class because it expounds on concepts relative to upper level biology and chemistry that are extremely frustrating even to those who are science majors. It is just hard to see a big picture when significant chunks are missing. I also do not like the diagrams in this book. The pictures are of course all the same colors and the detail is so vague that they do not do justice to the amazing bionanomachines they depict. Overall, Dr. Goodsell's book was very informative and opened my eyes toward the field of bionanotechnology.

Review

The text book does an excellent job of thoroughly explaining the topics. The diagrams in the text are very helpful in understanding the text.

Also, the text book would be difficult to understand without a strong background in biology. Some topics discussed in the text are very complicated and the author assumes that the reader understands the many of the scientific terms in the text.

Review of textbook

I thought Bionanotechnology was a very informative book. David Goodsell does an excellent job of portraying the subject of bionanotechnology. I found his descriptions of molecular machines quite intriguing and I felt that they were very well written. I recommend reading Goodsell's novel to learn about the possibilities of bionanotechnology. After reading this book, Goodsell has me believing that these molecular machines are the future of technology and its influence on our way of life.

Molecular Machines

I recently came arcoss a pretty cool, real life applicatin of Biophotolysis, which is the topic that I wrote my paper on. Hummer is currently developing a car called the "Hummer O2" that has body pannels that contain water and algae and use the photosynthetic process to clean the air and convert CO2 to hydrogen and oxygen. The hydrogen can then be used to power the car. Pretty neat. The article is in the March 2007 Popular Science on page 26.

The molecular machine that I researched was the C1 protein complex in the complement system. This protein may end up being very important in the cure of Alzheimer's disease. Alzheimer's patients have increased levels of C1 protein. The protein excess causes the neurons in the patient's brain to take up more oxygen. The increased intake of oxygen causes neural injuries that increase the symptoms of Alzheimer's disease.

Molecular machine

The molecular machine that I researched was extremely interesting in many medically related aspects. The molecule is rotaxane. It consists of a dumbbell shaped conglomeration of molecules with a ring called a macrocycle that fits like a ring on a pencil in between the two "dumbbell" ends. Electron transfer stimulated by light input allows the ring (called a macrocycle) to shuttle up and down the axle between two electron accepting stations. Drug companies are hoping to synthesize a rotaxane with an axle that can be inserted into the membrane of a cell so that the ring may be used as a delivery system across the cell membrane. Need I explain how useful that would be if drugs were efficiently attached to the ring and could be transported directly inside the cell. I think this idea will eventually be helpful with research in several drugs/diseases. Rotaxanes are already efficiently synthesized according to templates in laboratories., and the project has been underway since the 1990's. This is fairly rapid progress.

Computer Tech

R. Stanley Williams, a researcher at Hewlett-Packard is heading a team in an effort to develop computer memory that is just a couple hundred atoms wide. He and his team of researchers are trying to perfect a molecular memory capable of holding just 16 bits of data--about 16 letters' worth. The memory will consist of nano-scale wires laid out in a grid, with molecular "switches" at the points where the wires cross. The switches will determine how information is stored and routed on the grid. The best part, says Williams, is that the memory could be created by throwing the tiny wire grids and the switches together in a chemical soup. The switches would attach themselves to the grid, eliminating what would otherwise be a costly manufacturing process. Pretty crazy idea and could lead to computers that are way smaller and more powerful than anything around today.

Prof. Macosko's work

I found Prof. Macosko's work very interesting. I think that the way the drugs bind to the beads will be very helpful on finding a drug to treat Breast cancer especially the growth factor receptor. I think the possibilities with these beads and PNA are endless. As we find more specific causes of diseases the more uses and treatments we will find with these beads. I found these ideas very intriguing.

Breast cancer cures and cloning

I thought Professor Macosko's approach to research on the breast cancer growth factor receptors was very interesting. I had no idea that it was possible to place DNA primers on small beads that would allow a scientist to essentially "grow" a DNA strand from a small starter DNA piece. Moreover, the beads carrying different DNA can be separated and stored like a collection of books in the library. The PNA can then attach to the DNA while simultaneously attaching a drug to that DNA strand. When the bead bounces off of the protein target, which simulates the cancer growth factor receptors, and the drug that works may be detected by the fluorescence of the bead. By facilitating more efficient drug testing, this method increases the chance that the "wonder drug" will be discovered. I believe this brings a very positive perspective to the battle against breast cancer as well as other threatening diseases on the forefront of research. On the other hand, i think that the science of cloning is far from the point where I could legitimately take a stance in favor or against. Especially in relation to scientific discovery, I like to see the finished product and understand all details before I speculate as to how that would affect me and the world. Out of curiosity I would like to see somebody create a fully functional human clone and present it to the world. I like drama.

prof m's lab and cloning

The research that Professor Macosko is doing seems cutting edge and has potential to help cancer research, and research for many diseases that are currently only moderately effectively treated using receptor blockers, by finding the exact drug necessary to treat the disease and block the receptor. It has the potential to solve the problem we have with having a drug library of millions of drugs, but no diseases with which to treat them. Cloning seems like a very problematic area of science. On the one hand, who has the right to tell mourning parents that they cannot have a child that has the potential to be an exact younger copy of their own, yet how can anyone allow such a violation of the laws of nature? It seems ambiguous at this point, and I think it is good that it is not so effective or developed that it is inexpensive and becomes a problem.

myosin

I am using the machine based on the interaction between actin and myosin in my report. It seems like a really useful machine, and has great potential for further use in motors. Apparently, it is highly effective biologically, as it is the basis for our and all animal muscle contraction, and I expect that it will be very useful in making motors powered purely by ATP and controlled very precisely purely by concentrations of ATP and calcium ions (Ca2+). This machine has a huge amount of potential for low energy cost motors, and can even use the ATP made my my partner's machines.

C3bBb Machine

The molecular machine that I am doing my report on is called the C3bBb complex. It is involved in the immunization process that Kyle talked about last period and is known as the "chainsaw" in the process. It catalyzes the hydrolysis of C3 in the blood into C3a and C3b. This action triggers a series of events until mast cell degranulation occurs, which is part of the immunization process.

Molecular Machines in Developing Countries

I think that it is very important for the United States to use its research on molecular machines to help developing countries. For example, many third world countries are sticken with the HIV virus and many other deadly diseases. With the new research conducted about molecular machines, it may be possible to treat or even cure diseases such as AIDS. Thus, I think that it is very important for the wealthier and more advanced countries to help third world countries.

mc machine

I am not quite sure if this is what I am supposed to be blogging about, but I thought this was a cool topic. The medically related groups have way better stuff to research haha. I read about this molecular machine called the medusa sequencer. It is composed of a DNA or RNA polymerase attached to 4 flexible arms. It is cheap and very accurate at reading DNA and RNA strands. Essentially, its function is to count individual DNA's and RNA's. It may be used in the future to analyze genetic disorders and perhaps create treatments.

Molecular Machines. for Developing Countries

I feel that the responsibility to provide health care in the form of molecular machines is on the United States as long as the machines are being produced in a cost effective manner. It also depends on if our own country is having a similar health problem as the developing country because we are responsible for taking care of ourselves. If we can't afford the resources to do good abroad without hurting ourselves at home the responsibility is at home first.

Molecular Machines in Developing Countries

I think we definitely have a responsibility to help provide cheap, effective medical care to developing countries in the form of molecular machines because the ability to treat diseases would allow the country to focus its attention on ways to develop the country's exports, economy, etc... As a well-developed country, the United States has the resources to help other countries in need, and when we develop molecular machines that would be incredibly beneficial to the developing countries, we should help them out by allowing them to use our technology to further their development.

Restriction Enzymes

Restriction enzymes can be used to cut strands of DNA at specific sequences. If there is a known mutation in the DNA, these restriction enzymes can cut it out. Then using the sequences of plasmids to clone DNA, we can insert specific sequences. Other enzymes, ligases, can glue at the recognition sequences where the restriction enzyme cut. By harnessing this molecular machine, genetic engineers can modify DNA, possibly eliminating mutations. Especially now since the human genome is known, this technology can really do a lot for harmful mutations.

developing countries

Molecular Machines could be very beneficial to developing countries because they face many problems that people in developed countries do not have to deal with. Many people in these countries face diseases that we can cure using molecular machines. It's our responsibility to use our knowledge and resources to help them when we can. Improving their health and living conditions would greatly improve their life and country as a whole. Molecular machines should be used to help these countries since it would be so beneficial to their people.

developing countries

It is essential that nanotechnology continue to be developed and made available to developing countries. Nanotechnology has the potential to bring extremely inexpensive medical care and solve many other problems in developing countries as well, such as water purification, as someone already mentioned. A large value of nanotechnology to developing countries is its potential for effectiveness, relative inexpense, and potential ease of use. Suppose, in s few years, that there is a nano machine that can be injected into the bloodstream and released the medicine that the patient needs, without a diagnosis from an experience doctor. There are many more potential ways that nanotechnology could help, and it seems as if the possibilities are endless.

molecular machine

A molecular machine we have not yet discussed in class is AGG01 which is actually a tentative name of a new antibiotic from peptides. It sets up new trans-membrane protein channels which induce ion transportation out of the cell. Many essential molecules are lost in this transportation, and apparently the excess of the molecules outside of the cell kills bacteria, but can only kill pathogenic cells. It has enormous medical potential and is known in some circles as the new Penicillin. AGG01 comes from the mother's milk of kangaroos and has shown promise in treating e. coli and many other diseases; in fact, in many cases has shown to be more effective than Penicillin.