Fundamentals: 10:00-12:00 Scribe: Audrey Thompson

Fundamentals: 10:00-12:00 Scribe: Audrey Thompson Thursday, August 13, 2009 Proof: Susanna Pischek Dr. Miller Biochemistry Page 1 of 8 I. Amino Acids [S1]: (answers question from previous lecture) II. [S37 from previous lecture] a. QUESTION: Why does ion exchange chromatography allow you to separate glycine from alanine in an amino acid analysis when they have identical charges? b. ANSWER: i. There is no pure type of chromatography; ion exchange chromatography is predominate. There is also a binding phenomenon problem. ii. The stationary phase has hydrophobic character because the sulfonic groups are placed on a polystyrene matrix. Polystyrene is a hydrophobic substance. iii. Going down the column, the amino acids that have almost identical charges will have different side chains. iv. Alanine has a more hydrophobic side chain than glycine. Therefore, alanine is going to be retarded since it binds to polystyrene. v. From amino acid analysis, those with big hydrophobic side chains (i.e. valine, leucine, isoleucine, phenylalanine) elute later down the column. vi. Basically a predominate chromatography exists, but there are other types going on as well. vii. Ion exchange and absorption chromatography are both occurring.

I. Proteins [S1]: (gives review of Protein Intro lecture because we did not have the PowerPoint during the lecture) II. General Considerations [S3] a. Protein synthesis is expensive. b. Proteins are very fragile (be “nice” to them), quite unstable, and do not last forever with the exception of some collagen and structural proteins. But once you feel the pains of arthritis or osteoporosis, you will see that even these proteins are not forever either. c. Proteins are entrusted with crucial roles of almost all physiological functions. d. We have a way of talking about proteins. The molecular weight is MR with no units. i. Molecular mass is given in kDa. Da is the mass of one hydrogen atom. 220,000 Da means the protein is as heavy as 220,000 hydrogen atoms. III. Protein Diversity [S4] a. There is a great diversity among proteins. b. Potential diversity: the objective is to make one example of all possible proteins which have 65 amino acids in the primary structure. i. That is you can make the number of possible molecules to be (1x10^20)^65. You have 20 amino acids that you could put in at any given time and 65 possible places to put them along the polypeptide. ii. If you did this the mass of molecules that you would make would be along the lines of 4.4x10^64 grams which is 7x the weight of the earth. iii. Large diversity of proteins can be made. iv. The molecular weight of a 65 amino acid chain is 7150. This is because 110 is the actual average molecular weight of an amino acid. Instead of going though the chain and finding the weight of each amino acid, you can assume the molecular weight is 110 and then multiply that by the number of amino acids in the chain (65). This will give you the molecular weight of the polypeptide chain. IV. Protein Function [S7] a. The red markings are the proteins that will be discussed in greater length in the next few lectures. ***DID NOT FINISH PREVIOUS DAY’S LECTURE, SO HE PICKED UP AT SLIDE 19 OF WEDNESDAY’S LECTURE.

I. Primary Structure of Proteins [S1]: II. Separation of Fragments [S19] a. We isolated a protein and fragmented it using chemical agents. b. Trypsin is used in enzymatic fragmentation, and cyanogen bromide is used in chemical fragmentation. c. Start with an intact polypeptide chain; after fragmentation you have a mixture of smaller peptides. The difference between peptide of this size and protein is not ridged. In some fragmentations you will have a fragment which is maybe 70000 molecular weight because there wasn’t anything in the mixture to cleave it. Do not get hung up on what a peptide is and what a protein is. In this case all of these substances are peptides that came from the parent protein. d. With this mixture you must separate each peptide. This is done by ion exchange chromatography which is analogous to what we saw with the amino acid analysis. You can do this because every peptide will have distinct features. Fundamentals: 10:00-12:00 Scribe: Audrey Thompson Thursday, August 13, 2009 Proof: Susanna Pischek Dr. Miller Biochemistry Page 2 of 8 e. EXAMPLE: At pH 5 we have one peptide that was formed by cleaving with Cyanogen bromide. Go through peptide. By looking at the peptide and knowing the amino acid, you can determine the charge. i. Glycine will have a positive charge. Every peptide will start with an amino acid that has a free amino group. Every peptide/protein will begin with an amino group because it will be attached by its carboxyl group. The amino group will be there on every polypeptide. This will be a positive charge at pH5. Aspartic acid has a side chain with a carboxyl that when ionized has a negative charge. Valine has no charge. Asparagine has no charge. Lysine has a positive charge, methionine has no charge, isoleucine and serine no charge, arginine the last amino acid in polypeptide will have a positive charge on the side chain and a negative charge on its carboxyl group. ii. Notice there are 3 positive and 2 negative charges with an overall charge of +1. Go through the other in the same way; it has an overall charge of +3. iii. The stationary phase in this instance is different because we are dealing with large molecule rather than individual amino acids. It is a polycellulose with minus grouping. Same principle with the sulfonic group in cation exchange chromatography in amino acids. We use a matrix with more openness because we will push large substances through it. This way you can separate the individual peptides and purify them. iv. There are other ways to separate the large molecules/peptides/fragments. III. Affinity Chromatography [S20] a. If you have fragments with special affinity/binding affinity for a given substance then you can isolate one or two peptides by affinity chromatography which allows you to specifically bind to the column one or more fragments and then selectively isolate them that way. IV. Ion-Exchange and Affinity Chromatography [S21] a. Positive charge proteins bind to negative bead and the negative charged beads come out quickly. Those that are highly positive will be retained longer. Lightly positive or negative will be pushed through the column more quickly. b. With affinity chromatography, substances that have a special affinity to glucose are separated. In this case you could use bead covered in glucose molecules. The peptide would bind to that strongly. You would then insert large amounts of the binding material like glucose that would knock the protein off the beads by competition. The protein would be released in this manner. i. A neat way to work in this fashion is to have an antibody against the protein or the peptide. The peptide will bind to the bead because it recognizes the antigen that has been made against it. c. There are several ways in which you can isolate proteins. V. Size Exclusion Chromatography [22] a. Does not depend on binding but on size. Sometimes you will have two proteins that will have identical charges. On can be very large and have a positive charge of 5; another can be very small and also have an overall positive charge of 5. Ion exchange would hardly differentiate between these two. You can take advantage of their differences in size. This is done by size. b. Whereby the stationary phase is made of particles of a gel which have varying opening and as molecules come down thorough there. c. Large molecules will travel quickly because they cannot enter the pores of the column; therefore, they are prohibited from entering into all of the volume of the column and therefore come out very quickly; these are the large yellow molecules in the picture. d. The small green molecules have some availability within the gel particles, and therefore must pass though a large volume of the column than the yellow molecules. And they will go though much more slowly e. The smaller red molecules can enter freely into all the gel particles and will travel through a large volume of the column and come out much later. f. This is another method in chromatography that can be done to eventually get purified fragments. g. This can be a very difficult task. Fragments can be in high volume. CNBr cleavage is very helpful because there are less methinine residues and you will get smaller amounts of larger sequences. These are more difficult to sequence. VI. Electrophoresis: SDS-PAGE [S23] a. Sodium Dodecyl Sulfate (SDS) Polyacrylamide gel electrophoresis. b. SDS is a large hydrocarbon molecule with a sulfate group &12 carbons in a row. The final molecules are charged by sulfate and have some sodium molecules attached to it. i. Binds to proteins because of hydrophobic bonding of this large hydrocarbon tail of 12 carbons. It is similar to the Fmoc in that it changes the structure by changing all the amino acids to be negatively charged. Because it has a strong negative charge it binds to a large number of proteins. All are changed to a negative charge. Can be placed in an electrophoresis apparatus usually a slab gel. The gel is made of micelles and acts like a sieving arrangement whereby the small molecules pass though quickly and the large molecules are held up. It is the opposite of the size exclusion method. And all of the proteins will Fundamentals: 10:00-12:00 Scribe: Audrey Thompson Thursday, August 13, 2009 Proof: Susanna Pischek Dr. Miller Biochemistry Page 3 of 8 migrate to the positive cathode because they are negatively charged. They will be separated according to their molecular weights. The problem is that it cannot be quantified in this manner. If you need to isolate certain amounts of a protein you won’t get it this way because it is done on very small quantities. Valuable in determining purity. You can take any fragment and go though several isolation procedures but you never know your success in isolating it in the pure form. But if you use SDS-PAGE and see a nice clean band as in the photos it will tell you that you have isolated it in a pure form. c. Student Question: Can you explain again how the SDS causes the amino acid to become negative? i. Answer: You have a polypeptide chain that has several hydrophobic domains, and the SDS binds all along the polypeptide chain. One protein may take up 50-60 SDS molecules and each SDS has two negative charges so it overwhelms the charges of the native protein. d. Student Question: Will the size of the SDS molecules affect the overall mass of the complex? i. Answer: the SDS molecule is small compared to the protein so the SDS adds very little to the length of the molecule. The molecules are denatured and form spears. The SDS does not add significant molecular weight. VII. [S24] a. Next you chemically sequence the peptide. This is done by Edman degradation; named for the person who first described it. b. React the polypeptide with a reagent called phenylisothiocyanate. This looks to the polypeptide chain, binds and makes a reaction with amino group of the chain. i. It essentially makes a thiopeptide bond with the amino group on the polypeptide chain. It is covalently bound by the splitting off of water. It looks just like the peptide bond but the oxygen is replaced by a sulfur atom. c. We now have the peptide chain derived by phenylisothiocyanate.. d. If you then approach the system and add TFA. It acts as a catalytic reagent and causes the Sulfur atom of the thiocyanate to make a nucleophilic attack on the carboxyl of the polypeptide chain. When that occurs the Sulfur becomes the reagent and is bound to the C atom. The leaving atom then becomes the nitrogen atom. The first amino acid in the polypeptide has been plucked away from the rest of the chain in the form of a thizaolinone as depicted here. You treat it with a weak aqueous acid and get the PTH (phenylthiohydantoin) derivative of the amino acid. e. When you take away the amino acid you convert to the PTH derivative will have the original amino acid (including the amino group, carbon, alpha carbon, R-group and carboxyl group). Every time you do this you can then repeat the complexing and take off the second acid. Every time you do this you have the PTH derivative. You use the PTH derivative to determine the amino acid. This tells you the primary structure of the protein. f. Very straight forward process that is automated today. You can set it up go on vacation for a week, come back, and pretty much read the amino acid sequence. g. Look in book and notice a chromatography where amino acids are separated in reverse phase chromatography as with the Fmoc derivative. You can also do this with the PTH derivative of amino acid. Same principle of adding a very hydrophobic group to the amino acid and then chromatograph them in reverse phase fashion. VIII. Reconstructing the Sequence [S25] a. Once you have done this you can put the chain together. This is a very simple example where we have cleaved the chain with Trypsin and staphylococcal protease. i. Trypsin cleaved in such a way that is has left a lysine at the C-terminus of both fragments. This tells you that Lysine is the last amino acid/C-terminal amino acid of the peptide. If not then you would have cleaved there and had another polypeptide. You had two lysines in the peptide that gave rise to two peptides. If lysine had been in the middle you would have had three. ii. Staphylococcal protease has been used as a cleavage agent and it cleaves after a glutamic acid residue. In this case between glutamic acid and phenylalanine. b. You can tell now by looking at these that the primary structure (audio cut out) c. So that the real primary structure of the molecule is essentially this then combined up here and the primary structure is L-V-G-K-A-E-F-S-G-I-T-P-K. (explaining the fragments given on slide) d. This is how you put polypeptide together and decide the order of the polypeptide fragments before the cleavage. e. Told story about him and his colleagues doing a similar process as described above with a collagen molecule. IX. Laboratory Synthesis of Peptides [S26] a. The next thing you may want to do is make a peptide. b. The best and most used way to do this is outlined on the slide. This particular procedure involves derivitizing amino acids and protecting a variety of functional groups.

X. [S27] a. The first thing you do is attach the first amino acid. Fundamentals: 10:00-12:00 Scribe: Audrey Thompson Thursday, August 13, 2009 Proof: Susanna Pischek Dr. Miller Biochemistry Page 4 of 8 i. In this case the amino acid will be the C-terminal amino acid. You attach that to a stationary phase; an insoluble styrene bead. It is attached to the styrene bead because we want to do these reactions and get rid of the reagents but don’t won’t to handle the product. The product will stay attached to the insoluble beads. b. In order to make this type of addition we now need to put the second amino acid on, the penultimate amino acid in the polypeptide chain. i. To do that we need to protect it with the Fmoc group (identified Fmoc group in diagram on slide). It has a carboxyl group that conveniently makes a peptide bond with the amino group on the second amino acid. This is amino acid number 2 that we are going to add to the chain. (identifies the amino acids that will be joined) c. We want to protect the N-terminus because we don’t want to make a polymer of that second amino acid. We want the second amino acid to be placed on the chain and that’s it. So we have to then protect the N-terminus of that amino acid so that is doesn’t constantly polymerize and make a long chain of residue number 2. d. In addition to that protection we want to enhance the availability of carbon atom in the carboxyl group of the second amino acid. i. We do that by reacting it with a carbodiimide type reagent. It has a lot of electronegative groups in it and gives rise to this type of configuration (identifies figure on slide). It has somewhat of an ester bond arrangement. The ester bond simply denies electrons to the carboxyl carbon so that (audio cut out) e. …peptide linkage with residue number one f. So now by that procedure we have been able to attach a known amino acid to the first amino acid. So we have now made a dipeptide of which we know the primary structure g. Then you repeat this process. i. Take the derivatized dipeptide, remove the Fmoc to get the free dipeptide, and then bring in a new amino acid that is protected with Fmoc and react is with carbodiimide. ii. This will react with the dipeptide to give you a derivatized tripeptide. iii. It is possible to do this very precisely about 50 times. You can’t make a 2,000 residue polypeptide chain this way because not every reaction is complete. So if you have one tenth of one percent deactivation or removal of the protecting group properly arranged. You can add a polymer in the middle of your polypeptide chain. So there are problems with that and can make very big difficulties if you are intending to go out many, many amino acid. h. It’s valuable to make the peptide that you want and in this way you can also add amino acid that normally won’t be in proteins. i. Example: You could make a polypeptide with valeric acid, lactic acid, or some other kind of reagent which would be very valuable for certain studies. ii. Also it you can add D amino acid and a variety of other things to the polypeptide. Therefore this method is valuable tool in that way. XI. Uses of Amino Acid Sequences [S28] a. We can use amino acid sequences in many ways. b. They give rise to some ideas about the function of the protein and they reveal a domain structure. c. In the polypeptide chain there will be a sequence theme and then it will change to another theme. i. You will have triplets in collagen molecules: Gly-X-Y, Gly-X-Y, and then all the sudden it will change to be a different type of sequence. ii. That is a very informative situation because it’s allowed the prediction that all proteins have been created by mixing around 5,000 or 6,000 basic sequence themes. There is a great deal of evidence for that. Some of the very complex proteins that we know are really made up of a mosaic of various themes. iii. The function depends on what kind of theme is in the protein. You can also see the mosaicism of the primary structure. d. You can facilitate predictions on higher orders of structure. You can prediction secondary structure but tertiary and quaternary structure is still a guessing game as to what it is. e. You can facilitate construction of probes for genes. Once you have the primary amino acid sequence you can discern what the appropriate gene might be for that. f. Student Question: What does mosaicism mean? i. Answer: have you ever gone to a museum and seen a mosaic? A mosaic is a painting or construction made from tiny pieces of something like stone. A mosaicism can be a variety of (audio cut out)…the next one looks like a collagen sequence, the next one looks like thyroxin, the next one looks like calmodulin, that would be a polypeptide that would be a mosaic in terms of its themes or sequence. g. You can make probes for genes you can look at evaluation between proteins. Fundamentals: 10:00-12:00 Scribe: Audrey Thompson Thursday, August 13, 2009 Proof: Susanna Pischek Dr. Miller Biochemistry Page 5 of 8 i. If you have homologous genes within a species they are called paralogs. For instance you have Type II collagen in your cartilage, you have Type I collagen in your skeletal system. Type I and Type II collagen in each individual would be paralogs. ii. The same protein in different species is called orthologs. So you have Type II collagen in you cartilage; the Type II collagen in a baboon would be an otholog of your collagen. XII. Cytochrome C [S29] a. Cytochrome C is a protein sequence which has been known for many years and actually it turns out to be a well preserved protein throughout the animal kingdom and plant kingdom. i. There are very few changes from species to species with respect to cytochrome C. If you go through and compare the human cytochrome C and with a chimpanzee cytochrome C you will see no differences in the 104 protein residues. XIII. Cytochrome C [S30] a. Reads through the chart on the slide. XIV. Globin [S31] a. You can also look at the relationships between globins. b. They are the myoglobin, alpha-globin, and beta globin; they are quite highly related. If you look at the relationships between them you can see the commonalities that are outlined in blue. XV. Evolutionary Tree of Globins [S32] a. Described information on the slide XVI. The Unit Evolutionary Period [S33] a. The unit evolutionary period is the time required for an amino acid sequence to change by 1% after divergence of species. i. It takes cytochrome C 21 million years to have a 1% change in its sequence. ii. Globins evolve more quickly and take 6.1 million years, iii. Fibrinopeptides are peptides that are involved in clotting, making fibrin, and preservation of the circulatory system. They evolve much more quickly in 1.2 million years. XVII. Apparently Different Proteins May Share a Common Ancestry [S34] and [S35] a. We can see various relationships between proteins. i. For instance lysozyme is found in tears, saliva, and various tissues that would be exposed to a lot of bacteria. ii. Lysozyme is able to hydrolyze bacterial proteoglycans and bacterial polyglycolytic material. b. Human milk α-lactalbumin is a protein in milk along with albumin and other proteins. c. Lysozyme and α -lactalbumin have very similar amino acid compositions, structures, primary sequences but have very different functions. d. You can see these relations between structures, especially if you know the primary structure.

I. Protein Secondary Structure [S1] a. Relatively easy because there are very few secondary structures. b. They are short-range in globular proteins, meaning you will have small amounts of secondary structures in globular proteins. c. In fibrous proteins, secondary structure reigns supreme. They are long-range and in many cases the entire proteins is fibrous/secondary structure. d. All Φ bond angles are equal and all Ψ bond angles are equal providing a repetitive structure. e. Stability attained through H-bonds. II. Title [S2] a. This is a simple polypeptide bond. Identifies carboxyl carbon with N atom attached and the peptide bonds in the figure. b. If you look at these planes, they can rotate relative to each other and there are bond angles for instance the Φ angles essentially represents the spacing between the carboxyl…(audio cut out)…not going to go into great deal about this now. i. But I think you can imagine if you knew all the angles of the Φ and Ψ bonds along a polypeptide chain you would know what that polypeptide chain looks like in its native structure. III. Title [S3] a. If you bend one of the plates you will note that you will have problems with continuing the bending because the spheres of oxygens will hit each other and not allow the bending. b. This is basically the reason why I told you the other day that when you make a peptide bond you actually impose a great deal of structure on this random coiled material. It is really not a freely rotating material. c. You can see all the different arrangements you can bend the bond into. Fundamentals: 10:00-12:00 Scribe: Audrey Thompson Thursday, August 13, 2009 Proof: Susanna Pischek Dr. Miller Biochemistry Page 6 of 8 d. In the second figure the interference is between the hydrogens. e. These examples show what effects the folding of a polypeptide chain. IV. Classes of Secondary Structure [S4] a. You can have loops which don’t have a repetitive structure but are important structurally. V. Title [S5] a. Alpha helices are the most famous of all protein structures. b. The second figure shows how the planes will curve around in making this particular helix. It’s easier to see what an alpha helix looks like by looking at the last figure which only shows the backbone. i. This is a right handed helix. Our L amino acids are all capable of forming a right handed alpha helix except Proline. Proline is a breaker of alpha helices. ii. If you put your thumb along the axis of this helix you will notice that the fingers of your right hand will show the direction of the turn of the helix. iii. All proteins composed of D amino acid will form a left handed helix. You can only form a right handed helix with L amino acid. c. (audio cut out)….where all of the Φ bonds are -60°and all the Ψ bonds are -45° , The pitch of the helix is 5.4Å. This means that along the axis of the helix that the pitch for one complete turn is 5.4Å. The distance of 1.5Å is the distance occupied by one amino acid. There are 3.6 amino acid in one complete turn of the helix. d. A variety of things are possible with an alpha helix. When you have an alpha helix you have a cylinder. i. The amino acids form the backbone and the R groups protrude from the sides of the cylinder. ii. This R groups give it the character. By judicial placement of R groups in the primary structure you can have one side that is very hydrophobic and the other side that is very hydrophilic. iii. This can create a great deal a difference in chemistry depending on the side you are looking at. VI. Title [S6] a. Another advantage is it has a dipole moment and is such that the c-terminus is negative and N-terminus is positive. b. Prominent in globular proteins namely β- hemoglobin subunit i. It is essentially all alpha helices and a few loops in a non-helical confirmation. c. Myoherythrin is found in lower organisms such as invertebrates. i. Carries oxygen similar to hemoglobin. VII. Title [S7] a. Hydrogen bonding is the agent that keeps protein stable b. Every carboxyl group is Hydrogen bonding with an amino group above it; every carboxyl group shares a hydrogen with the N above it. Arrangement of the molecule has stability built into it. i. Even though hydrogen bonds are weak there are many of them that constitute a very stabilizing structure for a protein especially secondary structure. c. (audio cut out)…Amino acids such as Glutamine and Asparagine have the ability to H bond with these agents with Hydrogen atoms. You have no clear carboxyl group below to Hydrogen bond with. At the top the carboxyl group it will generally have nothing to H bond with and this is where you will find Histidine that will give Hydrogen bond capabilities to this part of the helix. They are caps; Asparagine serves as N-cap and Histidine serves as the C-cap. (I tried to typed what he said but it didn’t make much sense. See the slide for more clear explanation.) d. Without good hydrogen bonding you will no longer have the helix. e. Hydrogen bond capabilities determine how long the helix will be. VIII. The Polyproline Helix [S8] a. More open helix and more difficult to see. Points out features of the helix as see in the figure on the slide. b. This is a left handed helix. It is a 3:10 helix because there are three amino acids and ten atoms per turn of the helix. The distance is 3.3 and therefore the pitch is 10Å. In contract an alpha helix is about 5.5Å to make a turn. c. Student Question: Since this is a left handed helix does it require D amino acid? i. Answer: No. This require Proline. It is made of L-Proline. It forms this way because Proline is a unique amino acid. d. Imagine the helix is like the spiral in a notebook. If you were to take the alpha helix and stretch it out it would be like the polyproline helix. IX. The Beta-Pleated Sheet [S9] a. Formed by the side to side alignment of polypeptide chains. i. Chains can be anti-parallel or parallel ii. stability comes from hydrogen bonds. iii. Distance of the anti-parallel strands is 3.5Å and for the parallel strands is it 3.3Å. X. Title [S10] a. Formed as anti-parallel because adjacent chains are running in different directions. Fundamentals: 10:00-12:00 Scribe: Audrey Thompson Thursday, August 13, 2009 Proof: Susanna Pischek Dr. Miller Biochemistry Page 7 of 8 i. One chain is running N-terminus to C-terminus while the other is C-terminus to N-terminus. ii. Comes about easily because all you have to do is to have a polypeptide chain turns around on itself. b. R groups are in orange. Notice that every other row of R groups is below the plane of the beta sheet and others are above the plane. That gives rise to ability to have the lower part of the plane have one configuration/chemistry and the ones above have another configuration/chemistry. This is a happy arrangement. XI. Title [S11] a. Parallel run in same direction. i. N-terminus to C-terminus, N-terminus to C-terminus, etc. ii. In this situation hydrogen bonding is not so direct. There can only be H bonding at an oblique angle. iii. These sheets are typically large and full of hydrophobic amino acid and represent parts of a globular protein that will come down to be the skeleton or central domain of the globular protein once it’s been folded. b. Anti-parallel sheet i. Typically fewer of them are involved in a given complex. Usually only 2-3 strands wide. ii. Usually amphipathic in that the R groups below the plane can be hydrophobic and the R groups above the plane can be hydrophilic. iii. These will be found around the surface of the protein where the hydrophilic side can be on the outside and the hydrophobic side can be on the inside. c. Both types are very stable because the strands are connected very readily by hydrogen bonds. XII. Title [S12] a. Comparing alpha helices with beta strands you will notice that the alpha helix is a tight arrangement (3.6 amino acids in 5.5Å) while in the beta sheet you only have two amino acids in one complete turn of the polypeptide chain. In order to count the number of amino acids in a turn you count from one N then go to N of 2nd amino acid. If takes two amino acids to go from one N to another in a beta strand. b. Beta strands are typically represented as ribbons. c. Helices will be represented by either cylinders or helices. XIII. The Beta Turn [S13] a. This allows the polypeptide chain to reverse direction; this is the kind of turn you need when you are creating a beta pleated sheet in anti-parallel because you can just actually turn the polypeptide chain on itself and come right back along side of the other. b. Carbonyl C of one residue is H-bonded to the amide proton of a residue three residues away c. Proline and glycine are prevalent in beta turns. XIV. Title [S14] a. It takes four amino acid to make a beta turn. You can have beta turns in the two configurations seen on the slide. b. You can generally expected amino acid number two in this turn to be Proline. This is because the unusually side chain configuration has the carboxyl group pointing towards its side. Proline always causes a change in direction . XV. Fibrous Proteins [S15] a. Much or most of the polypeptide chain is organized approximately parallel to a single axis b. Fibrous proteins are often mechanically strong, usually insoluble, and play a structural role in nature. c. Making a fibrous protein can me thought of similar to making a helix. i. Describing Figure a: If you take a general sequence note that you start with amino acid number 1, 2, 3, etc. to form the helix. Each of these will have a side chain along the helix surface. This structure is an amphiphilic helix. Note that one side of the helix is mostly hydrophilic amino acids (blue). The other side of the helix, because of the way the amino acids are sequenced is rather hydrophobic (yellow). ii. You can arrange the amino acids in such a way that the helix will have different chemistry on its different sides. d. Figure b: citrate cynthase is a completely non polar helix. Ever amino acid along helix is hydrophobic. e. Figure c: calmodulin is a polar helix and every amino acid has a polar side chain. XVI. Title [S16] a. You can have a helix that is going to become a coiled coil or essentially what you have with fibrous proteins. b. Drawing on board. i. Remember that an alpha helix has 3.6 amino acid in every turn, there are 7.2 in every two turns. Suppose we wanted to make a sequence: the 1st, 8th, 15th, and every other 7th amino acid is leucine. However it takes 7.2 to make a complete turn. You will get a staggered arrangement of hydrophobic groups around a single cylinder. When they are made this way you can combine them as illustrated in diagram. It is suitable for making a coiled coil. Fundamentals: 10:00-12:00 Scribe: Audrey Thompson Thursday, August 13, 2009 Proof: Susanna Pischek Dr. Miller Biochemistry Page 8 of 8 1. This is the same configuration taken by myosin chains in muscle. 2. Myosin has some heads and an extremely long tail of coiled coils; called supramolecular helices. ii. When a hydrophobic interaction takes place between two molecules like this they want to bind around or hug each other. They hydrophobic interactions will demand this type of reaction. XVII. Title [S17] a. Lots of different configurations can be made with an alpha helix b. Some large elements of alpha helix will be found in things other than structural proteins such as the influenza hemagglutinin. i. Causes lots of infections. ii. H1N1 is one of the hemagglutinin that the virus uses to attach to cells. c. DNA polymerase, tRNA synthetase, and catabolite activator protein have lengthy helices. XVIII. Alpha Keratin [S18] a. Found in hair and wool. b. Has a large domain c. XIX. Title [S19] a. Central part is an alpha helix that forms coiled coils that can coil around other coiled coils. We don’t have much of this. XX. Beta Keratin [S20] a. All beta sheets. b. We don’t have much. XXI. [S21] a. Silk is made of beta keratin. b. Anti-parallel polypeptide chains. i. That can be stacked because the pleat will accept side chains of beta sheet above/below. ii. All amino acids can be small and hydrophobic. iii. The only amino acids in pure silk are glycine and alanine. iv. Water just beads on the surface of pure silk because of the strong hydrophobic nature. XXII. [S22] a. Spider web is made of alpha helices surrounded by beta pleated sheets.

I. Tertiary Structure of Proteins [S1] a. Long range, overcomes secondary structure, incorporates secondary structure and is the overall leader of the proteins. b. The overall spatial relationships between segments of primary and secondary structure c. Structure determined by x-ray diffraction and NMR d. II. Tertiary Structure [S2] a. Globular proteins create maximum internal bonds and minimize solvent contact. i. Goes from random coiled molecules to a globular molecule. Lengthy to tight. b. Fibrous Proteins create maximum intermolecular bonds and maximize molecular to molecular contact. i. Start as random coil that will usually pair up with another molecule to give you a dihelix or triple helix ii. There is no collapse or folding back on the molecule iii. Extensive contact with a solvent mitigated only a little bit with a companion peptide. This tells you that these things occur because of the hydrophobic/hydrophilic nature of the molecule. III. Globular Proteins a. Read slide b. Globular proteins have about the same density as water. i. The empty spaces are often filled with water.

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