Amino acids & Protein Structure Chemwiki: Chapter , with most emphasis on 16.3, 16.4 and 16.6

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1 Amino acids & Protein Structure Chemwiki: Chapter , with most emphasis on 16.3, 16.4 and Most jobs (except information storage) in cells are performed by proteins. 2. Proteins usually have only a few possible stable conformations. (Remember staggered and eclipsed ethane from the molecular modeling lab?) 3. Specific protein conformation (structure) is required to maintain function. I. Cellular Functions of Proteins See our previous discussion under Biochemistry II. The α-amino acids (aa) A. Name comes from the structure (amine and carboxylic acid.) The α-c atom is next to the C=O (carbonyl) C. 1) carboxylic acid group 2) α-amino group 3) side chain (a.k.a., R group) 4) Circle the α-carbon! 5) Does this structure contain a chiral (asymmetric) carbon atom? 6) In roughly neutral solutions, amino acids are zwitter ions. B. Except for glycine (where R = H), the 20 common aa all have at least one chiral C atom. Refer to models. 1. L- and D- designations are based on reference to glyceraldehyde. 2. The R- & S- designations used in the modeling lab are geometrically based. Most α-amino acids have a chiral C atom. This means you can have L- and D- isomers. Mirror images L-Alanine D-Alanine C. How do amino acids differ? They differ in the side chains (R group)

2 1. Non-polar 2. Polar but uncharged at normal ph 3. Negatively charged (Remember, we are referring to the side chain.) 4. Positively charged Six of the 20 common amino acids. 2 D. What is important about amino acid side chains? 1. The side chains play a major role in determining protein structure/function. 2. Example: The most common Sickle-cell trait is caused by a valine being substituted for glutamic acid at only one position (out of ~145) in the β-chain of hemoglobin. III. The Peptide Bond (links the monomers to form a polymer) A. Big molecules (a.k.a., macromolecules) 1. Essentially all biological macromolecules are polymers. Polymers are made by linking a large number of monomers together: n A A-A-A-A-A-...-A n-2 -A n-1 -A n O C N H

3 2. In proteins, aa are the monomers. They are linked together by peptide bonds. Note -N-C-C-N-C-C-N-C-C- repeating pattern (Which C is the carbonyl-c, and which is the α-c?) 3 3. Nomenclature a) Two linked aa form a dipeptide (above = glycyl-alanine) b) Three form a tripeptide c) A moderate number (roughly < 30) form an oligopeptide d) A larger number form a polypeptide 4. Peptides can be named by listing their aa sequence, starting from the amino terminal end. What is the amino terminal end?

4 IV. Primary (1 ) Structure of Proteins: the sequence of amino acids A. Specific proteins in your body have specific sequences. That is, every insulin A-chain starts with Gly the amino terminus, then Ile, etc. B. This sequence is called the primary structure of the protein. 4

5 V. Secondary (2 ) Structure of Proteins: repetitive A. Secondary structure is formed from local segments of a protein held in place by intrachain Hydrogen Bonding and comes in two main forms: α-helix and β-sheet α-helix The backbone is twisted in a coil like a spring or slinky. Part of an α-helix from human Hb (beta chain) Can you: 1) Trace the peptide backbone? 2) Find the amino terminal end of the backbone? 3) Locate the Hydrogen Bonds that maintain the α-helical structure? 4) Are these Hydrogen Bonds perfectly aligned? Would the α-helical structure be maintained if the Hydrogen Bonds were disrupted? 2. β-sheet (two forms of this): a) parallel [aligned N C, N C] b) anti-parallel [aligned N C, C N] Can you: 1) Trace the peptide backbones? Drawing of a β-sheet 2) Find the amino terminal ends of the backbones? 3) Is this a parallel or anti-parallel structure? VI. Tertiary (3 o ) Structure of Proteins A. Tertiary (3 ) describes the location of each of the atoms in the protein in 3- dimensional space. This often depends on bends in secondary structure, etc B. Usually, 100% of a given type of protein is in the same 3 o structure; this is a very non-random, highly organized situation (so unfavorable entropy).

6 If something is unfavorable in entropy terms (ΔS), there must be a significant amount of bonding (ΔH) holding it in place. What forces maintain the very non-random structures? Remember: ΔG = ΔH TΔS? 6 1. Peptide bonds (covalent) maintain 1 o structure. 2. Hydrogen bonds maintain 2 o structure. 3. Tertiary structure is maintained by different amounts of a-e different proteins: a) covalent bonds ( S S are a common type) b) hydrogen bonds c) ionic bonds (salt bridges) d) hydrophobic interactions (keep the inside on the inside) (actually, mostly a system ΔS term) Hydrophobic Collapse & protein folding This folding rule, known as nonpolar in, polar out is the most stable arrangement because it allows polar side chains on the surface of the protein to interact with water molecules and allows nonpolar side chains to avoid water. e) London Forces Essentially all proteins do b-e in various amounts. VII. Quaternary (4 o )Structure of Proteins (requires multiple subunits) A. This describes how the subunits of a multi-subunit protein fit together. B. Examples of proteins with multiple subunits: 1. hemoglobin (α2β2) 2. insulin (A chain and B chain) 3. hcg (Why is the β-subunit clinically important? ) hcg structure (pdb: 1hrp) α-subunit: blue; β-subunit: pale green. Red & bright green atoms are carbohydrate (sugar) molecules that are covalently attached to hcg.

7 Space-filling model Ribbon model (backbone) 7 2 o structure is mostly β-sheet with a short α-helix in the alpha subunit. VIII. Overview of Protein Structure & Function A. Protein structure has a massive effect on protein function. Usually alteration in structure radically alters (often destroys) protein function. B. The key to the function of most proteins is the creation of a unique environment (space) where catalysis, transport, or binding can occur. IX. Myoglobin & Hemoglobin A. Myoglobin: single protein chain, 1. Myoglobin meaning: myo globin 2. Found in muscle, oxygen storage 3. Diving mammals and myoglobin. Diving mammals have high concentration of myoglobin in their muscle which allows them to remain under water for longer times. Myoglobin and hemoglobin structures are related. B. Hemoglobin: four subunits, each with a heme. Hemoglobin (Hb) and O2 & CO2 transport. 1. Carries oxygen from lungs (gills) to the rest of the body where it releases the oxygen to allow aerobic respiration, (to get energy from food/fuel for all he body to function.)

8 Consider the job of hemoglobin: it must and yet 8 2. Hb does have more than one stable conformation a) High O2 affinity form is main form present in lungs where [O2] is. b) Low affinity form is present mostly in extremities where [O2] is c) Hb shifts back and forth between these forms as it moves through your circulatory system. 3. Logic: In the lungs: In the presence of high [O2], the first oxygen molecule binds to one subunit. This increases the affinity (attraction) of the other three subunits for oxygen. As each subunit binds oxygen the attraction grows stronger. So in the lungs where oxygen concentration is high, the subunits are all bound to oxygen. In the tissues: The oxygen concentration in the tissues is than the lungs. As the hemoglobin circulates into the tissue, oxygen is released first from one subunit. As the oxygen is released the affinity for oxygen goes down, and more oxygen is released. So in the tissue that is most active and where the oxygen concentration is low, the affinity of Hb for oxygen is at its lowest so a maximum amount of oxygen can be delivered. 4. Other modifiers: a) BPG. Hemoglobin has a binding site for 2,3-bisphophoglycerate (BPG). BPG binding favors formation of low affinity form of hemoglobin. That is, it helps Hb let go of its O2. b) H + and CO2. H + and CO2 also bind to hemoglobin and its affinity for O2. Rapidly metabolizing tissue makes lots of H + and CO2. This lowers the ph of the blood and helps hemoglobin release O2 in the tissues. C. O2 transport and the unit. There is a barrier between the maternal and fetal bloodstreams. The oxygen concentration is low in the location that fetal hemoglobin picks it up. 1. When you were a fetus you didn t make much Hb β-chain. 2. You made a variant of the β-chain called γ, so fetal Hb is α2γ2. 3. Fetal Hb has higher affinity for O2 than does adult Hb, because fetal Hb does not bind BPG very well. (So fetal Hb stays in the higher affinity form under lower O2 conditions.) 4. This allows the fetal hemoglobin to be able to better bind oxygen.

9 D. Sickle Cell Anemia. ( = sickle cell hemoglobin) Thoughts from the group. 1. Small change in structure = big change in function. 9 Normal (wt) human β-hemoglobin subunit sequence: Val-His-Leu-Thr-Pro-Glu-Glu-Lys-Ser-Ala-...-His 146 Sickle-cell human β-hb subunit sequence: Val-His-Leu-Thr-Pro-Val-Glu-Lys-Ser-Ala-...-His 146 Only one difference out of 146 aa residues!!! 2. How does this change alter Hb and rbc function? a) The Glu side chain is charged. It is hydrophilic (water loving). b) The Val side chain is non-polar. Does water like to be by it? Recall the ordered H2O cages are unfavorable (ΔS). c) RBC are normally biconcave discs in shape and flexible. d) RBC from people with sickle cell, can form rigid elongated or crescent shaped cells. e) In low oxygen conditions, HbS molecules stick together to minimize Val6 contact with water. f) The HbS forms insoluble polymer chains that form stiff rods that distort the cell shape. g) Leads to many problems, including blocking of blood vessels, anemia, pain, infection, etc 1.5 min movie about Sickle Cell: 3. Why has HbS trait persisted in some populations? Individuals that have just one gene for sickle cell (sickle-cell trait) are resistant to malaria, increasing their survival rate. People who live in areas where malaria is endemic (or who are descended from people who lived in such areas) are more likely to carry a gene for sickle-cell. 4. One treatment: hydroxyurea. Hydroxyurea induces the expression of reducing the amount of the mutated -chain. Seems to reduce the number and severity of crisis episodes.

10 10 5. Genetic studies of hemoglobin sequences was one of the first systems scientists used to determine the evolutionary relatedness of organisms. Since then, scientists have looked at the aa sequence of many proteins from different organisms, and in the last few years, they have also sequenced DNA from many organisms currently alive (and some from fossils). It has been determined that Chimpanzees are our closest living relative. This data is also used to determine the origins of humans. X. Denaturation of Proteins Heat, -S-S- reductants, and detergents cause lost of 3 o, which causes loss of function. What kinds of bonds are broken when a protein is denatured? Bonds that hold 3 o and 4 o structure together? Bonds that hold 2 o structure together? Bonds that hold 1 o structure together? Draw a picture of what happens. What happened to the enzyme activity when you boiled it?