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Fundamentals 1 Dr. Miller Hour 10-11 August 21, 2008 *This is from my personal notes not the audio file because the audio did not record the first part of Miller’s overview -Know about sickle cell -Have superior understanding of myoglobin and hemoglobin (why are they globular) Know their shape and form. Mic comes on….. Overview of what to know: Big distinction between adult and fetal Hb. Know why that is and how that comes about in terms of the chemistry that is necessary to have that phenomena occur. Muscle business – know certain phenomena involved protein chemistry and protein confirmations; take a simple modification of a protein such as phosphrolyating and that is a usual event; involves adding phosphate groups to the side chains of serine and the side chain of serine can be esterified by phosphates groups giving you this figure (draws on board.) This is now the serine side chain with a B carbon, remainder of chain (pointing to picture) and the phosphate group is added to serine or tyrosine or theroinine- all aa side chain that has a hydroxyl group. Phosphorylation of a protein can modify its structure radically o Example: Na/K ATPase system; which is responsible for setting up gradient between the cytosol and the extra cellular membrane with respect to K and Na concentrations. o Na is high extracellularly, K is rather low; reverse is true intracellularly – K is high, Na is low. Those gradients of the ions across a cell membrane require input of energy; that chemical potential is not achieved without having energy put in. Energy comes into that through ATP potential. ATP is hydrolyzing to provide a phosphate group which goes on to the ATPase molecule (protein) which is now in the membrane and modifies the protein so it lets Na out (it lets Na across the membrane) and take K in. Then you desphosphorylate by using a phosphryolase enzyme; take the phosphate away and the enzyme becomes totally reversed; Na goes in the cell, K is pumped to the outside of the cell. The process can then repeat; the enzyme system is dependant on ATP to set up a chemical gradient across the cell membrane. This is important because we are going to see movement across a protein, namely myosin, depending on whether it is phosphorlyated. Slide 1: This is our myosin molecule in the electron microscope; this is a chemical picture (ribbon diagram) of what the head of one these myosin molecules looks like; this is not the tail o Purple and yellow areas are light chains that accompany the myosin head; part of the head that interacts with actin fibers to give muscle contraction. o The arrangement is set up nicely for our system Slide 2: In order to get our coiled-coil arrangement within myosin tails, we have to have the 7 reside heptad repeats and there are more repeats which we have not talked about at great lengths (28 reside repeat which is 4 of these 7 residues and there is a 196 reside repeat which is a multiple of 7 residues.) Within heptads repeats (the 7 aa repeat) is the fundamental repeat. Residues 1 and 4 or A and B will always be hydrophobic and the other residues will be 2, 3 and 6 will be ionic. This gives rise to coiled coil situation (already addressed) Slide 3: On a readily repeatable sequence, the hydrophobic groups will come together (points to picture (this is 1 alpha helix, this is another) and in the myosin molecule, the hydrophobic groups come together and cause the chains to wrap around each other and make the staggered position of the helix. Then the ionic (hydrophilic) regions are on the outside of the coiled-coil. Look at coiled coil again – the hydrophobic domains are buried in the coils and the hydrophilic domains are on the outside to the environment. Those are important for molecule staggering. o They wrap around each other because the hydrophobic regions are covered up and then they interact with each other because of the + and – charges on the outside of the coiled coil. Slide 4: The more repeats that I am talking about are the repeats that you see on the surface of the molecule that cause them to stagger relatively to one another. Slide 5: This is how myosin molecules form a quaternary structure, they come together and aggregate and stagger relative to each other so they have a common origin here where one end of the tail is complexed with the ends of other tails and then the individual myosin molecules (each one of these is a coiled coil) and they are staggered relative to each other to get a tree of myosin heads. o This is trunk of the tree and now the limbs are stretched out. Myosin heads are all bristling on the surface of the quaternary structure. Now the staggering (why the molecules are where they are) relative to each other is dependent on the series of repeats you see outside of each individual myosin molecule. Already introduced to us with collagen; same kind operation; the staggering is based on charge-charge interactions of neighboring molecules with make the large structure. So now what is this big line up of myosin molecules to make a myosin fiber – what are they going to do? They are situated on our sacromere. Slide 6: (gesturing to image) The myosin fibers go from here to here, the M disk is in the center where the myosin molecules were linked together. This makes the M line. Myosin clusters stick out from either side of the M line to (this is where they congregate together and go in different directions) Slide 7: Actin fibers come this way and this way; what is going to happen as muscle contracts: the myosin heads are going to grab onto the actin fibers and grab and pull and pull. The pull on the part of the myosin heads is going to decrease the space where the actin fibers are apart. They decrease the middle part of sacromere and decrease where thin filaments are; size of the thin filaments decrease and the H zone is going to decrease because the myosin heads grab the actin filaments and pull. Slide 8: The sarcomere is going to shrink; where the actin filament is pulled together, the size of the actin filaments is decreased and decrease the H zone. That is why muscles contract – the actin filaments are dragged together. The sacromere decreases in size. Slide 9: This comes about because the myosin head grabs hold of the actin filament and takes a power stroke (a push) and pulls the collection of actin molecules together. The myosin head is phosphorylated and has a resting position at the top and because of the phosphorylation, it will take hold and grab the actin filament and then it will be de phosphorylated. After taking away the phosphate, it goes under radical change back into the power stroke. o In power stroke, the actin filament is pulled here. After the power stroke is done, it will be re- phosphorylated by ATP. ATP binds and the myosin head is re phosphorylated by ATP and then goes back to resting position. This explains in rigor mortis; have very little ATP (no active metabolism). Occurs when myosin head has grabbed a hold of the actin filament and does not let go. Your muscle is tense and can’t relax. o Only when the myosin is phosphorylated again does muscle relaxation occur. Slide 10: Signal to start up muscle operation comes from calcium which is present in the lumen of the sacroplasmic reticulum when the message that the muscle needs to flex comes down the line through the nerve impulse and when the nerve impulse hits the skeletal muscle, calcium is released from the endoplasmic reticulum and comes into the muscle fibers. It is after relaxation when calcium is pumped out back into lumen by a process that requires ATP energy again. Have to have ATP to get muscle to flexing and you have to have ATP to get the promoter of muscle flexing away from the muscle to totally relax. o That is another process. What does calcium have to do? Why is it there? It comes in and destroys the arrangement of the troponin and the tropomyosin around the actin filament. Actin filament is in blue; all the other molecules we talked about our fibers, but you are seeing a cutout of them. They are draped over actin filament to not allow the myosin heads which are in the pink to not react with the myosin molecule. You do not want uncontrolled contraction to take place at random. Introduction of calcium allows those particular molecules to fall away and to stop obstructing the locations when the actin molecules are going to act with the myosin molecules. Once calcium comes in, actin is free to be touched and pulled by myosin head, which is attached there. Once calcium leaves, you go back to protected area and calcium is pumped out. Basic overview of how muscles contract and how we interact with our environment. Slide 11: We can move around using skeletal muscles. Slide 12: Smooth muscle contraction is totally different; contraction mechanism is the same but smooth muscle movements are slow and long term. They are rarely reversed very rapidly. One of the more common forms of smooth muscle contraction include when the smooth muscles of the blood vessels contract and you have to pump a lot of blood through them. o Blood pressures rises because smooth muscle contractions are long term; you don’t have high blood pressure over a couple minutes and then it goes down. o We are constantly pumping blood through ever ever smaller vessels; the same amount of blood going through smaller and smaller vessels causes a rise in blood pressure. That is because smooth muscles have contracted and have remained contracted; there is not a quick method for reversing that. You can take drugs like esonphil (?) and agents that reduce blood pressure by relaxing the vessels. The blood vessels can then expand and then pressure goes down. This is a common thing – blood vessel contract due to hormone called angiotension which is made by kidneys and an enzyme called rennin which modifies a large protein to give rise to angiotensin (is a small peptide which causes blood vessels to contact) and causes smooth muscles cells and vessels to contract; as long as angiotenisin is around, you will have smaller and smaller vessels. Angiotensisin inhibitors or drugs that inhibit the enzyme that makes angiotensin will allow the smooth muscles to expand and reduce blood pressure. Contraction is by same general mechanism by the rapidity of the response and relaxation occurs is generally under hormonally control. Can not control directly like skeletal muscles which are directly controlled; smooth muscles are generally controlled by ANS and hormones. Smooth muscle situation is similar chemically but different in operation. Slide 13 : skipped. THE COLLAGENS Slide 1: Large family of proteins characterized by lengthy segments of triple helix; confirmation made possible my repetitive primary structure, gly-x-y. For the most part the molecules are secreted into extracellular spaces where, in aggregate form, they function as structural elements; not only structural elements, but a variety of other functions; chiefly they are structural elements. The collagens constitute the largest class of proteins in vertebrate organisms. Slide 2: There are a total of 30 types containing as many as 35 genetically distinct chains. The FIBERFORMING collagens are the most quantitatively, most important. They are the predominant collagens composed of 9 unique polypeptide chains; so there are 9 genes involved in the formation of fiber-forming collagens. The chains form five different molecular species. There is a procollagen to collagen conversion, just like a proinsulin to insulin conversion. Fibers are constructed of staggered, side by side parallel associations of molecules (just like in quaternary structures). Slide 3: There are the NON-FIBER FORMING COLLAGENS with interrupted helical domains. There is seldom a procollagen to collagen conversion; instead of being staggered side-by-side in a parallel fashion, these molecules are staggered in an antiparallel fashion for aggregate formation. They DON’T form fibers in the strict sense of the word, but they from aggregates (formulations of collagens outside of the cell). There are fiber associated collagens, network collagens (basement membrane collagens), filament collagens (type 6), anchoring fibril collagens (type 7), and multiplexins, which are membrane bound. The anchoring fibril collagen attached the epidermis to dermis; dependent on collagen; reason you cannot molt your skin like some organisms do! Slide 4: Skin: 90% of derived dermis is collagen molecules Largely type 1 and type 3 collagen in the skin. Slide 5: If you look at the skin more carefully, the epidermis is separated from the dermis by a basement membrane; You also see sebaceous glands (sweat gland); Type 1 and type 3 collagens predominate in the skin. The basement membrane that separates the dermis from the epidermis is composed of type 4 collagen. Slide 6 The entire skeletal system is essentially a collagen matrix which has been calcified. Slide 7 T the blood vessel walls are formed largely of collagen and another protein called elastin for the expandable vessels. Slide 8 The dentition and teeth are essentially formed of a dentin matrix which is calcified; the enamel on the surface of teeth is not really a collagen matrix organ, it has a separate type of protein but the hydroxyapaptite here is the same as in the general bone. The teeth are buried and held in place by bony structure, which is a collagen type tissue, but even more important, the ligaments which hold the teeth into the bone, the periodontal ligaments actually undergo decay and absorption when one has periodontal disease. The ligaments which hold the teeth into the bone are essentially destroyed by periodontal disease and ligaments are collagen fibers. Called ligament because they associate hard tissue with hard tissue. A tendon goes from bone to muscle; a ligament goes from bone to bone. Tendons and ligaments are equivalent chemically, they are both all collagen. If you grandparents are edentulous (lost all their teeth) because periodontal disease has destroyed the connection between the teeth and the bony structures in which the teeth are implanted. Periodontal disease is one of the reasons collagen was first studied at the national institute of health for dental research. Slide 9 Also have collagen in cartilage. Slide 10 Eyeball itself, the white of the eye, is a total collagen material composed of collagen fibers, large & rough collagen fibers so that the eyeball cannot be deformed easily. Can be deformed physiologically after many years but is not easily deformable. The cornea, which represents the beginning of eye is completely transparent normally that lets the light come is a collagen type structure as well; the main pass of the cornea is covered by type 1 and type 5 fibers. The lens is surrounded by a capsule which is a basement membrane which is a type 4 collagen. The vitreous humor is a putty-like or soft-tissue like material composed of type 2 collagen that is really hydrated; the vitreous humor is similar to a cartilage. It is a pseudocartilaginous tissue. The retina has more basement membrane. In the eye, there are more types of collagen than in the dentition. Slide 11 Fiber-forming collagens, the specifics! Type 1 has two alpha 1 chains and one alpha 2 chain; two chains are identical and the third is separate; requires 2 genes to make type 2 collagen; it is a heterotrimer and is the collagen you find in large connective tissues like the skeletal system, tendons, ligaments, the sclera of the eye, most of your large massive connective tissue. There are NO ALPHA HELICES AT ALL, THE NAME OF THE CHAIN IS THE ALPHA CHAIN. Type 2 collagen is a homotrimer; all three chains are identical; they are alpha 1 type 2 chains found in cartilage and related tissues. Another related tissue besides the vitreous humor is a soft, cartilaginous, jelly-like material found between the vertebral bodies in the vertebral column. Type 3 is another homotrimer in soft tissues along with heterotypic fibers of type 1 collagen; the procollagen to collagen conversion is slow and incomplete so that these type three molecules usually contain a non-helical, globular domain at their N-terminal region; so you have a collagen molecule for type 3 with 3 helices and three individual chains that will retain the globular domain which is usually clipped away in the other collagens (type 1 and type 2). Just because the chains are called alphas, does not mean that they are all the same; for one type 1 chain is synthesized by a chromosome #17; alpha 1 type 2 gene is synthesized by chromosome #7, and the type alpha 1 type 3 is synthesized by a gene on chromosome number 4 in humans. They are all genetically distinct chains which have origin in unique genes; Slide 12: Type 5 and 11 Heterotypic fibers (type 1 -5 ) and type 2- 11 Procollagen in collagen conversion yields pN molecules just like type 3 where you have the pro N terminal piece on their and the function here is to monitor fiber size. Collagen fibers in bone and dentin are huge on the order of 800-1000 angstroms in diameter; collagen fibers in the wall of the uterus are tiny and in the vessel walls are tiny because they are tissues that need to expand and contract. Slide 13: Starts referencing diagram below: This chain has 1056 amino acids; a triple helical domain where every third amino acid is glycine; In propeptide domain at the carboxy terminus you have 246 amino acids and at the propeptide amino terminus you have 139. Those domains will be removed. The heavy thick line is the triple helical area and the thin line is thin, non triple helical area. Put three chains together you will have a rigid molecule which has a large globular end at the c-terminus and a smaller globular end at the n-terminus. When you cleave the propeptides away, you are left with a large, asymmetric molecule which has tiny regions of c-terminus and n-terminus left (non-helical regions- the white open box regions). These non helical regions are important in cross linking. Whole molecule is 300 nm in length and 15 angstrom units in diameter; equivalent to having a dowel rod that is one inch in diameter and 17 feet long- very lengthy and rigid, rod-like molecule. Slide 14: How do you get these molecules that bring together three different types of chains and you have them synthesized by ribosomal synthesis within a fibroblast or an osteoblast or a cell making dentin or a cell making the sclera of the eye. These chains are synthesized and while they are being synthesized, the proline is being hydroxylated, and the hydroxylysine is being glycosalated (adding carbohydrate molecules to a polypeptide chain). Notice that we are translating here the polypeptide chains from the ribosomes; the polypeptide chain is being extruded into the endoplasmic reticulum which is a signal that is eventually going to be secreted. Cells that secrete things put them first into the endoplasmic reticulum; while the chain is being synthesized….microphone cuts out and then he dismisses us for our break!