Amino acids & Protein Structure Reading: Chapter 12: p 413-428 Problems: Ch 12.1, 2, 11,17,21,23, 25,33,35, 37,39,41,45,47,49, 50 1. 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: 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?
amino group
H
side chain
carboxyl group H
H
N
C
O C
R
O
H
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.
L-Alanine
D-Alanine
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C. Amino acids can be grouped based on similarities in the side chains (R group) 1. Non-polar 2. Polar but uncharged at normal pH 3. Negatively charged (Remember, we are referring to the side chain.) 4. Positively charged
Ala
Gly
H2N
H
O
C
C
OH
H2N
Val
H
O
C
C
OH
H2N
H
O
C
C
OH H
H
H
C
H
H
H
H
C
C
H H
C
H
H
Lys
Asp
Cys H
O
H2N
C
C
H
C
H
OH
H
O
H2N
C
C
H
C
H
H2N
OH
H
H
O
O
C
C
OH
(CH2)4 N
C
S
H
H
O
H
D. Why should we care 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. Importance of amino acid side chain structure in human health:
Wild type human Hb has glutamic acid (Glu) at position 6 of its β-chain. Sickle-cell Hb has Val at position 6. The remaining 145 amino acid residues are identical! Val
Glu H
H
O
H
H
O
N
C6
C
N
C6
C
CH2
CH
CH3
CH2
CH3
C O-
O
2
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-.....-An-2-An-1-An O
O
H
O
C
N
+
C
H
H
N H
2. In proteins, aa are the monomers. They are linked together by peptide bonds. N-C-C-N-C-C- repeating pattern (see next page).
Note -N-C-C-
(Which C is the carbonyl-C, and which is the α-C?)
Gly-Ala CH3
H O H H
C
N+ H
C
C H
N
OC O
H
Atoms shown in red are all in the same plane. Comment re. nature of peptide bond.
H
peptide bond
4. 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 5. Peptides can be named by listing their aa sequence, starting from the amino terminal end. What is the amino terminal end?
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A peptide containing 8 amino acid residues (an octomer) Full name: Valyl-Histidyl-Leucyl-Threonyl-Prolyl-Glutamyl-Glutamyl-Lysine
In 3 letter abbrev: Val-His-Leu-Thr-Pro-Glu-Glu-Lys
In 1 letter abbrev: V-H-L-T-P-E-E-Y O +H3N
CH
C
CH
CH3
O H N
CH
C
CH2
CH3
O H N
CH
C
CH2 CH
CH3
O H N
CH
C
CH
OH
Atoms in the peptide backbone are shown in blue.
O N
H C
C
O H N
CH3
CH
C
O H N
CH
C
O H N
CH
CH2
CH2
CH2
CH2
CH2
CH2
C
C
C
O-
N CH3 NH
-O
O
O
CH2 -
O
CH2
Can you locate each residue's side chain and determine whether it is non-polar, polar but not charged, acidic, or basic?
H
N+ H
Can you make predictions about the solubility in water of each residue's side chain?
H
4
6. The peptide bond exhibits resonance. What is it? (Darth Vader’s voice.) a) Resonance occurs when there is more than one stable way to arrange the electrons in a molecule or ion. See below. b) Structures with resonance often behave like something in between the different resonance forms. c) Structures that exhibit resonance tend to be more stable than you would otherwise think. Ca
H C
O
Ca
H C
N Ca
-O
N+ Ca
Peptide bond resonance. 1. All six atoms shown are in the same plane. 2. Both the central C and N atoms behave like they are trigonal planar, sp2 hybridized. (righthand structure). 3. Note that neither of the structures above provides a perfect model for all aspects of peptide bond structure/function 4. The bond between C and N cannot rotate.
IV. Primary (1̊) 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. (Heterozygosity?) B. This sequence is called the primary structure of the protein. (Polymorphism?)
V. Secondary (2̊) Structure of Proteins: repetitive A. Secondary structure involves the bending, folding and twisting of the peptide backbond. It is held in place by intrachain Hydrogen Bonding and comes in two main forms: α-helix and β-sheet. 1. α-helix The backbone is twisted in a coil like a spring or slinky.
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Part of the A 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 αhelical 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]
Drawing of a β-sheet
H
O
N
R
C
H
CH
O
N
R
C
H
CH
O
N
C
CH
N
C
CH
N
C
CH
R
H
O
R
H
O
R
R
O
CH
H
C
R
N
H
O
CH
C
R
N
CH
C
N
CH
C
N
CH
C
N
O
H
R
O
H
R
O
H
indicates Hydrogen Bond
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Can you: 1) Trace the peptide backbones? 2) Find the amino terminal ends of the backbones? 3) Is this a parallel or anti-parallel structure? 4) Are these Hydrogen Bonds perfectly aligned? B. Some generalizations: 1. Most proteins have obvious 2o structure. 2. Many proteins have more than 50% of their aa involved in 2̊ structure. C. Specialized 2o structures exist. Example: Collagen triple helix. (proline hydroxylation and scurvy knaves.) Collagen amounts to almost 1/3 of the total protein in the human. It ia a structural protein found in tendons, cartilage, bone, blood vessels, and teeth. Collagen proteins twist into helices and three wrap around each other to form a triple helix. Every third aa is glycine. Gly-Pro-Hyp Conversion of Pro to Hyp requires vitamin C. Hydroxyproline deficient collagen is relatively weak and can be pulled apart. Scurvy produces skin lesions, fragile blood vessels and bleeding gums.
VI. Tertiary (3̊) Structure of Proteins A. Tertiary (3̊) 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 3o structure; this is a very non-random, highly organized situation (so unfavorable entropy). An exception: Protein involved with “Mad Cow”. If something is unfavorable in entropy terms (∆S), there must be a significant amount of bonding (∆H) holding it in place. What forces maintain the very non-random structures? Remember: ∆G = ∆H − T∆S ?
1. Peptide bonds (covalent) maintain 1o structure. 2. Hydrogen bonds maintain 2o 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 (??? re. H bonds to solvent) c) ionic bonds (salt bridges) d) hydrophobic interactions (keep the inside on the inside) (actually, mostly a system ∆S term)
Hydrophobic Collapse & protein folding e) London Forces Essentially all proteins do b-e in various amounts. 7
VII. Quaternary (4̊) 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. Space-filling model
Ribbon model (backbone)
2o 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.
Hemoglobin: four subunits, each with a heme. B. Hemoglobin (Hb) and O2 & CO2 transport. 8
1. Consider the job of hemoglobin: it must ______________________________________ and yet ________________________________________________ 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. 1. One way to look at the problem: Human Hb is really good at binding O2, but not as good at releasing it. 2. What consequences does this have for the fetus? 3. How do we deal with this problem? a) When you were a fetus you didn’t make much Hb β-chain. b) You made a variant of the β-chain called γ, so fetal Hb is α2γ2. c) 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 oxygen conditions.) d) This allows O2 to flow from mom to fetus. D. Sickle Cell Anemia. (__________= sickle cell hemoglobin) Thoughts from the group. 1. Small change in structure = big change in function.
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Normal (wt) human β-hemoglobin subunit sequence:
1 6 10 Val-His-Leu-Thr-Pro-Glu-Glu-Lys-Ser-Ala-.......-His146 Sickle-cell human β-Hb subunit sequence:
1 6 10 Val-His-Leu-Thr-Pro-Val-Glu-Lys-Ser-Ala-.......-His146 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 (∆S). 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: http://www.dnalc.org/resources/3d/17-sickle-cell.html 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 fetal hemoglobin γ-chain, reducing the amount of the mutated β-chain. Seems to reduce the number and severity of crisis episodes.
5. How different are human and chimpanzee Hb? Hemoglobin, myoglobin and related proteins have been studied in many organisms. Scientists have looked at the amino acid sequence of many proteins from different organisms. In the last few years, they have also sequenced DNA from many organisms currently alive (and some from fossils). 10
They use this data to determine the relatedness of organisms and to investigate the origins of humans. Chimpanzees are considered our closest living relative.
X. Denaturation of Proteins Heat, -S-S- reductants, and detergents cause lost of 3o, which causes loss of function. What kinds of bonds are broken when a protein is denatured?
XI. Dietary Protein & Digestion We (no longer) have the ability to make all of the necessary amino acids from scratch. We must obtain some from our diet. These aa’s are called A. They are: isoleucine leucine lysine methionine phenylalanine threonine tryptophan valine histidine Comment on high lysine corn. B. Example of an amino acid we can make: serine 3-phosphoglycerate ➔ 3-phosphohydroxypyruvate ➔ 3-phosphoserine ➔ serine Where in our metabolic processes is 3-phosphoglycerate produced?
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