Fundamentals I 10-12Scribe: David Davis

Wednesday, August 12Proof: Name Here

Dr. MillerAmino Acids, Proteins, and Primary StructurePage1 of 5

  1. Introduction [No Slide-On Blackboard]: Summary from Previous Lecture
  2. Two amino acids on board - Aspartic Acid (D) and Lysine (K)
  3. Aspartic Acid - Basic configuration of Amino Acid - Alpha carboxyl group, carboxyl group, an amino group, and hydrogen atom. Makes carbon atom asymmetric.
  4. 3 ionizable groups for D - amino group with pKa of 9, carboxyl group with a pKa of 2, side chain carboxyl group with pKa of 4.
  5. Lower the pKa, the stronger the acid. Notice the alpha carboxyl group has a lower pKa than that of the side chain carboxyl group. Example of when you can never say a group has certain properties, it depends on the environment.
  6. Alpha carboxyl group is a stronger acid because it is adjacent to the positive charge of the amino group. Proton which is leaving has an extra push to leave- positive charges repel each other. Have to know the environment to determine how a substance will act.
  7. Consider K - Amino group on alpha carbon with pKa of 9- strong base but weak acid. Does not give up proton easily. pKa on alpha carbon amino group is SMALLER than the epsilon amino group (side chain amino group-epsilon group because it is on the epsilon carbon-count down alpha, beta gamma, delta, epsilon). Proton on alpha carbon amino group leaves relatively easily compared to side chain amino group due to being next to very electronegative group of atoms (the carboxyl group), the oxygen atoms draw electrons away, makes the bonding between the proton (H) and the nitrogen atom much weaker, so it is a strong acid RELETIVE to the epsilon amino group.
  8. Side chains are important- responsible for carrying out all of the activities of proteins.
  9. Reactions of Amino Acids- Slide #26 of amino acid lecture [S1]
  10. Very Important – amino group reaction- amino group on amino acid reacts with aldehyde which gives rise to a schiff base, a carbon doubled bonded to a nitrogen, nitrogen will still have a couple of extra electrons, nitrogen can be protonated, acid/base situation. In this reaction, water is released. This is the mechanism is how collagen molecules are cross linked to each other and make insoluble fibers.
  11. –of amino acid lectureds- Slide #26 le for carrying outt down alpha,beta gamma, delta, epsilonp due to the 11
  12. Figure 4.11 Reactions of amino acid side-chain functional groups [S2]
  13. Two cysteine molecules combine to make cystine, covalently cross links two polypeptide chains. Two protein chains are involved. Done largely by oxidation. Must remember two electrons are involved in the bond between sulfur and hydrogen. (Oxidation means to remove hydrogen atom not just a proton).
  14. Once both Cysteine molecules have been oxidized, each sulfur is missing an electron from their outer shell, they combine to create a single bond to fill their octet of electrons.
  15. The bond is rather weak. Can be further oxidized to form sulfonic acid groups, or it can be reduced by adding hydrogen back to split the covalent linkage between the sulfur atoms and thus forming two cysteines.
  16. Stereochemistry of Amino Acids [S3]
  17. Every amino acid (except glycine) has a chiral center. Allows molecules to rotate/bend planes of polarized light (Dextro- or Levo- rotatory).
  18. D amino acids are never used by us (eukaryotic organisms). Prokaryotic organisms can use D amino acids.
  19. The Isomeric configuration of amino acids.... [S4]
  20. L- & D- Serine are enantiomers. L-serine is used by humans and all eukaryotic organisms.
  21. The Assignment of (R) and (S) notation for glyceraldehyde and L-alanine [S5]
  22. L and (S) are equivalent, just different naming systems. (R) and D are equivalent.
  23. To determine D (R), or L (S) configuration of amino acids, place chiral carbon of molecule in the center (example in class was to spell C-O-R-N, C-carboxyl group, R group-CH3 for alanine, N for amino group) and then, if it is carboxyl group, R group, then amino group in a COUNTERclockwise manner, that is the (S) configuration, if it is clockwise then you have the (R) configuration.
  24. Figure 4.14 The Stereoisomers of isoleucine and threonine... [S6]
  25. Isoleucine and threonine have two asymmetric centers – the alpha carbon and the beta carbon of the side chain. This means there are 4 isomers for two chiral centers instead of 2 isomers for one chiral center.
  26. Hydroxylysine also has 2 chiral centers.
  27. Spectroscopic Propterties[S7]
  28. Amino acids have certain ultraviolet spectra characteristics. Trp absorbs most readily in the UV range of 270-280. Same for Tyr.
  29. Phe absorbance is around 250-260.
  30. Figure 4.15 The ultraviolet absorption spectra.... [S8]
  31. Proteins lacking Trp, Tyr, and Phe, and ways to detect them is to use a UV range ~190 nm. Looking at absorption characteristics of all peptide bonds at this range.
  32. Amino Acid Analysis [S9]
  33. Done by Chromatographic procedure.
  34. Made possible by fact that if you change the pH environment in which AA’s exist. (Ex. Change pH to 6 for aspartic acid). Carboxyl on alpha carbon loses its proton=COO-
  35. Gamma carbon will also lose its proton=COO-
  36. Alpha amino will retain its protons thus its positive charge and thus, the charge of the molecule will have an overall charge of “-1” on Aspartic Acid at a pH of 6.
  37. Follow the same procedure for Lysine at pH 6 with regard to the pKas of the groups, you would have an overall charge of “+1” at a pH of 6.
  38. This sets up so you can separate AAs based on their charge – done by a process called Cation Exchange Chromatography
  39. Need a column and you put in the column a large number of sulfonic acid groups (the stationary phase) which are negatively charged.
  40. Place mixture of AA’s on a column with the mixture of sulfonic acids ( D and L are used again as an example)
  41. Place in a pH buffer of 6
  42. D has a “-” charge and it will shoot through the column because negative charges repel. Thus, D and E (glutamic acid)-proteins with a “-” charge- will come out first, and on recording chart will see a peak of aspartic acid.
  43. As lysine starts down the column, it will be attached (because of the “+” charge) to the column and be retarded. Won’t come out as quickly.
  44. How to get it out- set up a sodium ion gradient and start to introduce increasing quantities of sodium, and as the sodium ion concentration gets high enough, it will compete with the stationary negative charges and bump off the lysine – the sodium “+” charges will take the place of the lysine.
  45. Called cation exchanged because you are literally exchanging cations. You can also separate proteins that way, because they have the chargeable groups in the proteins.
  46. Student question- What would Histidine look like at a pH of 6?
  47. 50% of histidine side chains would be protonated. It would come out quicker than lysine, as it has a lesser of a negative charge.
  48. Most extreme case is arginine as it has a pKa of 12.5.
  49. All AA’s would fall in between Arginine (most acidic) and Aspartic Acid.
  50. 4.18 Cation.....[S10]
  51. Simply shows types of materials that can be used as cation exchange media..stationary phase molecules.
  52. 4.19 Operation of a.... [S11]
  53. Just a picture of what was just explained in [S9]
  54. Chromatographic fractionation.... [S12]
  55. Final result. Notice aspartic acid is the first one out. Arginine is eluted last.
  56. System is slow, but effective. Can only be done slowly with millimolar and at best micromolar concentrations of the AA's.
  57. Discussion question answered in next lecture. Why does glycine separate from alanine? Why does valine separate from methionine? Why does isoleucine separate from leucine?
  58. Size of peaks correlates to quantity of AA.
  59. Reversed Phase Chromatography of Amino Acids [S13]
  60. Reversed phase-change the stationary phase from a polar stationary phase to a hydrocarbon stationary phase.
  61. Much different from ionic stationary phase. Must change nature of AA because we have a hydrophobic stationary phase. Charge of AA does not matter for this method.
  62. Derivitize ALL of the AA by adding a large hydrophobic molecule called the “F-moc”. Attaches to the alpha amino group of all AA and overrides all of the ionic properties of the AA’s. Now they are hydrophobic. Already hydrophobic molecules are even more hydrophobic (like leucine and isoleucine).
  63. Lysine is extremely hydrophobic as it has 2 amino groups.
  64. More hydrophobic molecules come out first as they are repelled from the static hydrophobic medium.
  65. This is much faster compared to cation exchange. (20-30 min compared to 4-5 hrs).
  66. Very low concentrations can be used (as low as femto- and picomoles.
  67. F-moc is very fluorescent and allows for detection of AAs at a much smaller quantity.
  68. Student question: If you have an initially hydrophobic molecule to begin with, how would those separate out? How would they separate out by weight? Valine used as example. Without derivatizing the molecule, it still has charges because of carboxyl and amino groups so it would shoot right through because of charges. No affinity for hydrophobic medium.
  69. Amino Acid Composition [S14]
  70. Most prevalent AA is leucine (hydrocarbon side chain).
  71. Trp is least used AA.
  72. Amino Acid R Groups [S15]
  73. Globular proteins will curl up and fold because the hydrophobic nonpolar AA are looking to get away from aqueous environment.
  74. Polar and uncharged groups along with hydrophobic groups establish folding patterns for fibrous proteins and promote specific interactions. His- buffering systems, Serine – derivatized by phosphate groups and carbohydrates, Lys-involved in cross linking collagen molecules, Asn involved with carbohydrate groups, Cys involved with oxidative cross linking with cystine.

NEW Powerpoint File - Introduction to Proteins

  1. Proteins [S16]
  2. Proteins essentially fold because they go from a high state of energy to a low state of energy.
  3. Genesis of a Protein [S17]
  4. Realize a protein is translated from mRNA followed by chemical modifications of protein. Then, chaperon binding to help the protein fold. The rest is on the slide.
  5. SKIPPED THIS SLIDE General Considerations [S18]
  6. SKIPPED THIS SLIDE Protein Diversity[S19]
  7. Proteins - Large and Small [S20]
  8. Forget Connectin proteins. They have been renamed as Titins. Proteins found in muscle tissue. Huge proteins.
  9. Examples of the Types of Proteins [S21]
  10. 4 Main types of proteins – Fibrous (Collagen), Myoglobin, Membrane (bacterio rhodopsin-found in the eye), Intrinsically disordered proteins.
  11. Intrinsically disordered proteins- Thought to be impossible. They then become ordered and assume their “business like” structure but only after they are approached by a substrate. Substrate acts as catalyst for the folding of the protein.
  12. Protein Functions [S22]
  13. Proteins do everything
  14. Transducers – receptors (membrane bound proteins that detect high levels of sugar, antibodies, etc and report to the inside of the cell thereby directing cell function.
  15. Protein Functions, Cont. [S23]
  16. Function dependent upon
  17. Specific recognition and binding-a protein may bind to itself and recognize itself and:
  18. Other proteins, substrates, etc
  19. The way a protein works- it must recognize and act upon on a particular substance.
  20. Picture from Book [S24]
  21. Almost half of the proteins we know about, we don’t know the function.
  22. The number of proteins that we have is small in humans
  23. Only about 35,000.
  24. Plants have more at about 150,000.
  25. Interesting Examples [S25]
  26. Albumin-has a large amount of glutamic and aspartic acids.
  27. Plasma protein, about 45 grams/L.
  28. Many hydrophobic sites where drugs travel. Amount of drugs being taken for a day or hour depends on how much is going to be bound up by albumin.
  29. Transports minerals such as Ca.
  30. Alpha 1-antitrypsin-inhibitor of trypsin. If compromised in some way, you have a situation where your lungs are eaten up by endogenous enzymes. They destroy certain proteins. ALPHA 1 controls that activity so that the lung material can remain intact.
  31. Smokers interrupt this activity and the enzyme runs freely and results in emphysema.
  32. P53-major defense against cancer/metastasis. Destroys cells which have lived past their time and stops their normal cell cycle. If this destruction does not occur, metastasis follows.
  33. Alter Egos for Proteins[S26]
  34. Cytochrome c – All oxygen utilizing organisms have and is important in electron transport in mitochondria.
  35. Also responsible for facilitating for apoptosis by activating caspase enzymes which is useful for destroying intracellular particles.
  36. Prolyl hydroxylase – also hydroxylates the side chain of proline.

NEW POWERPOINT FILE - Primary Structure of Proteins

  1. Primary Structure of Proteins[S27]
  2. Polypeptide chain goes from Amino terminus to carboxyl terminus.
  3. Entropy – freedom of movement is lose when amino acids are coupled together.
  4. Energetically expensive to make a protein. Requires ~ 4 ATP per AA 120 sientific calories required to form 1 peptide bond
  5. Establishing a Peptide Bond [S28]
  6. Occurs between two AA’s.
  7. Essentially involves a nucleophilic attack on the part of the nitrogen to a carbon atom- made possible because the carbon resides between two electronegative oxygen atoms, carbon is deprived of electrons, nitrogen has a couple extra electrons, but it isn’t protonated.
  8. Results is a dipeptide, where the original free carboxyl of AA one is attached to the amino group of AA 2.
  9. The peptide bond now has a configuration. Movement is no restricted.
  10. Peptide Bond Example [S29]
  11. Created a product in which is restricted.
  12. Carbon-Nitrogen bond has a double bond aspect. Shorter bond than the normal carbon-nitrogen bond. No rotation around this particulate bond. Never find the Nitrogen spinning relative to the carbon atom.
  13. Since this is a plane, this configuration also cements a total of 6 atoms in space.
  14. Alpha carbon, carboxyl carbon and the oxygen, alpha amino group with its hydrogen of next AA, and the next AA alpha carbon.
  15. The six atoms are held in a particular plane, better viewed on next slide.
  16. The Coplanar Nature of the Peptide Bond [S30]
  17. Polypeptide chain can be viewed as a series of planes which are connected by alpha carbon atoms.
  18. Figure 6.6 Four different....[S31]
  19. Chain of alpha-helix is a series of planes – “bracelet of planes connected by rotating linkage”
  20. By polymerizing AA, introduced intricate structure that travels along the polypeptide chain as long as there exists AA’s.
  21. Controls how protein will fold and what kind of chemistry that can be achieved from different types of folding.
  22. The Sequence of Amino Acids in a Protein [S32]
  23. Sequence of proteins – direct correlation between code and protein sequence.
  24. Figure 5.6 Bovine pancreatic ribonuclease...[S33]
  25. This protein is designed to cleave nucleic acid chains.
  26. Amino terminal (lys) and carboxyl terminal (val)
  27. Certain cysteines come together that are cross-linked in disulfide bonds via oxidative process.
  28. No special treatments are needed for a protein to fold. It does it all on its own. (Some large ones require chaperones.) It does this very quickly.
  29. Sequence determines polar and nonpolar regions which can help in determining the secondary structure.
  30. How are Proteins Isolated and Purified from Cells? [S34]
  31. Talking about cells now.
  32. When talking about proteins, by their nature, are soluble. They are soluble in the cytosol.
  33. Least soluble at their isoelectric point –
  34. At neutral pH, tend to have carboxyl group ionized and amino groups protonated. (Can adjust the pH to get equal amounts of each).
  35. Proteins will be precipitated from solution, especially at the isoelectric point.
  36. Figure 5.11 The Solubility of most Globular....[S35]
  37. Diagram of last slide.
  38. Can increase the solubility of the protein at any pH by adding small concentrations of salt. However at extreme concentrations (4M), the proteins precipitate out. (Just like said in the last slide – all of the water has been taken up)
  39. Solubility of a Fibrous Protein [S36]
  40. Generally “+” charged, more “+”s than “-”s. Add a little bit (0.15-1.0 M) to bring the protein into solution. If you go up to 4.5M, the proteins will precipitate out. Remember, you are starting out with proteins that are insoluble (collagen). Proteins inside the cell are generally soluble, proteins outside the cell are usually insoluble.
  41. This is different because we are cooling the system. Cooling breaks hydrophobic bonds -> collagen goes into solution. Don’t go into solution at or above room temperature. At neutral pH, they follow the general rules.
  42. At pH of 2.5, overall charge is all “+”.
  43. Because all carboxyl groups are all protonated, and all amino groups are all protonated. They go into solution now because they all repel each other.
  44. Will go into solution without the help of salt, precipitated at lower salt concentrations.
  45. How is the Primary Structure of a Protein Determined? [S37]
  46. One easy way – sequence the gene. Need to have a chemical study as well because gene sequence does nto relate the modification of the AA’s in the protein.
  47. First, isolate and denature the polypeptide chain.
  48. Then, use chemical and enzymatic methodology to fragment the chains.
  49. Trypsin is one of the best cleaving agents for protein strands and it leaves at lysine and arginine residues.
  50. CNBr is also helpful – cleaves at Methionine (M)
  51. Once cleaved, fragments are left. If 4 sites for cleavage, 5 segments will be available.
  52. Frederick Sanger was the first [S38]
  53. The hormone insulin consists.... [S39]
  54. Before sequencing of insulin, there was no consensus as to what a protein was.
  55. Showed that every A chain began with lysine and ended with asparagine.
  56. All B chains started with phenylalanine and ended with alanine.
  57. Allowed pharmaceuticals to engineer insulin to circumvent antibody rejection of animal insulin (cows, pigs, etc.)
  58. Insulin is secreted first as proinsulin and not directly as insulin and therefore direct synthesis of the final product is not effective (roughly 1%).
  59. Need to have body cleave proinsulin for proper usage by the body.
  60. Attaining Individual Chains [S40]
  61. Read slide
  62. Fragmentation of the Chains [S41]
  63. Nothing new, read slide
  64. Enzymatic Fragmentation [S42]
  65. Be familiar with all of the enzymes on this slide, but he stressed to know trypsin.
  66. Figure 5.16 Trypsin is a ..... [S43]
  67. Nothing in recording pertaining to this slide
  68. Cyanogen Bromide (CNBr)... [S44]
  69. CNBr-unusual reaction, attaches cyano group to a sulfure. Sulfur along with cyano group is lost and other 2 carbon atoms make bond with a carboxyl group…..recording splits here.
  70. The last fragment will not have any homo-serine and this tells you that it is at the end of the chain, this lets molecular biologists know which one.

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