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7.013: Introductory Biology, Spring 2015: MIT.

Dr. Sinha & Prof. Sive 1

7.013 Recitation 2 Spring 2015 Summary of Lectures 2 & 3: Major elements of the biological macromolecules: All matter is composed of atoms, each of which contains a positively charged nucleus around which one or more negatively charged electrons move. An element is a pure substance that is comprised of only one kind of atoms. Of the different elements shown in periodic table, Carbon (C), Hydrogen (H), Oxygen (O), Nitrogen (N), Phosphorous (P) and Sulphur (S) are the six major elements found in biological macromolecules. The number of electrons within an atom determines how it will interact/ bond with the atoms of the same or different element i.e. number of electrons regulate the chemical bonding and geometry of an atom. Bonding: A chemical bond is an attractive force that links two electrons together in a molecule. There are many types of bonds that hold molecules together. a) Covalent bonds: These are the strongest of all the types of bonds. This type of bond results when two atoms attain stable electron numbers in their outermost shells by sharing one or more pairs of electrons between them, such as the bonds between C and H in methane. These can be represented as lines between the chemical symbols for linked atoms. For example, C-H represents one covalent bond between carbon (C) and hydrogen (H) whereas O=O represents two nonpolar covalent bonds formed by equal sharing of electrons between two oxygen atoms. The disulfide bond (S-S) is another example of covalent bond that is formed between the side-chains of two cysteine amino acids. Hydrogen likes to form a single bond with another atom (as shown by a line), Carbon prefers to form four bonds (shown by four lines), Nitrogen likes to make three bonds (shown by three lines) in this form the nitrogen is neutral, Nitrogen can also form four bonds in this form the nitrogen is positively charged Oxygen likes to make two bonds (shown by two lines) in this form the oxygen is neutral. Oxygen can also form a single bond in this form the oxygen will be negatively charged The most common bonding to sulfur in biomolecules is similar to oxygen (although sulfur can adopt higher valence states) Phosphorous likes to form 5 bonds (shown by 5 lines). The most common occurrence of phosphorus is in phosphate and phosphate esters. Electronegativity & Polar covalent bonds: When two atoms sharing the pair(s) of electron(s) are of different elements, the sharing is not necessarily equal. The nucleus of one atom may have a stronger affinity (higher electronegativity) for the shared pair of electrons compared to the other, so that the shared pair of electron(s) tends to be closer to that atom resulting in the formation of polar covalent bond i.e. as observed in water molecules where oxygen is more electronegative and hence has a higher affinity for the shared pair of electrons compared to the two hydrogen atoms. Of the six major elements in the biological macromolecules, Oxygen and nitrogen have a much higher electronegativity compared to others and therefore covalent bonds, for example with carbon and hydrogen and these elements tend to be polarized.

H3C C

OH

HH C EN = 2.5O EN = 3.5

So C-O bond polarized with partial negative on O and partial positive on C

+-

H3C C

O

HH H EN = 2.1

O EN = 3.5So O-H bond polarized with partial negative on O and partial positive on C

+-

H

b) Ionic bonds: occur between atoms with a very high difference in electronegativity. This results in a complete transfer of one or more electrons from one atom to another that has a higher electronegativity. For example, in table salt (NaCl), the sodium acquires a positive change (Na+) by donating its electron to chlorine, which now acquires a negative charge (Cl-).

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c) Hydrogen bonds: occur between polar molecules, such as molecules of water, because of the partial negative charge on the O and the partial positive charge on the H i.e. difference in the electronegativity of O and H atoms. In biological systems you most often see the formation of hydrogen bonds if the Hydrogen is between two O atoms or two N atoms or an O and N.

RO

HCO

N

R

RH

OH

H

= a hydrogen bond

d) Hydrophobic interactions or Van Der Waals forces (VDW): Molecules that are water- loving are called hydrophilic and polar (they can be either charged or uncharged). In comparison, molecules that are water- repelling are called hydrophobic. In aqueous environment, the hydrophobic molecules tend to aggregate with one another rather than interact with polar water molecules. This interaction between two hydrophobic molecules in an aqueous environment is called a hydrophobic interaction. Hydrophobic effects promote the association of hydrophobic molecules together in order for them to avoid water and thereby increase entropy. The interactions between nonpolar molecules are enhanced by VDW, which occurs when atoms of two molecules are in close proximity. These brief interactions result from the random variations in the electron distribution in one molecule, which creates opposite charge distribution in the adjacent molecule. Thus there is a weak, temporary + and - attraction. Although a single VDW is brief and weak, the sum of many such interactions over the entire span of a large nonpolar molecule can result in substantial attraction. Biological macromolecules: All the cells, whether prokaryotic or eukaryotic, have same major classes of biological macromolecules: lipids, carbohydrates, nucleic acids and proteins. With the exceptions of lipids, the biological macromolecules are polymers made by the covalent bonding small monomers through dehydration/condensation reactions. The polymers may be hydrolyzed to individual monomers with the release of water molecule(s) as a reaction byproduct. Functional groups define the properties of macromolecules: Certain small groups of atoms called functional group, occur frequently in biological macromolecules. Each functional group has specific properties and when attached to a large molecule, it confers its properties to the large molecule. Since macromolecules are so large, they may contain diverse functional groups some of which are polar and uncharged, others are polar and charged whereas some as nonpolar and hydrophobic. So the attached functional groups different specific properties to the local sites within the macromolecules, allows them to interact with each other and regulate the overall 3D-structure, chemical properties and interaction of the macromolecules with other reactants. a) Lipids: Lipids or fats are predominately hydrocarbon chains that are used as energy storage and insulation. Lipids are hydrophobic in nature. Modified lipids form phospholipids, steroid hormones, cholesterol and some vitamins. Fats and oils are triglycerides also known as simple lipids. Triglycerides that are solid at room temperature are fats whereas those that exist as liquids at room temperature are oils. Triglycerides are formed by condensation reaction that covalently attaches three fatty acids chains with three hydroxyl groups (-OH group) of a glycerol molecule by forming three covalent bonds/ ester linkages. In saturated fatty acids, all the carbon atoms within a fatty acid chain are saturated by hydrogen atoms i.e. there are only C-H and C-C bonds. In comparison, in unsaturated fatty acids the hydrocarbon chain contains one or more double bonds i.e. C=C. The saturated fatty acids have fewer kinks and are more closely packed and have higher melting points compared to the unsaturated fatty acids. Cis-fatty acids have hydrogen atoms on the same side of the C=C bond compared to the trans-fatty acids where they are on the opposite planes of the C=C bond. Trans- saturated fatty acids are bad for

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health and often increases the chances of a person developing cardiovascular problems. Cholesterol is another example of lipids which is transported in the circulation with the help of lipoproteins i.e. LDL, HDL etc). Increase in blood cholesterol and LDL is strong risk factor for cardiovascular disorders. Cell membranes are composed of lipid bilayers, which separate the aqueous inside of the cell (the cytoplasm) from the aqueous outside of the cell (the extra cellular environment). One specific type of lipid, called phospholipids, has polar phosphate groups as heads and two long hydrocarbon tails that are hydrophobic. These are amphipathic and form lipid bilayers in aqueous solutions that expose the phosphate heads on each side of the layer and hide the hydrophobic tails in between the two rows of head groups. Cell membrane is selectively semi-permeable. Only small hydrophobic molecules can pass through cell membranes. All large or polar or charged molecules must cross the membrane through protein pores in the membrane. b) Carbohydrates: Carbohydrates include monosaccharides (such as glucose, fructose and galactose) and disaccharides (such as sucrose and lactose), trisaccharide or polysaccharides. A monosaccharide is composed of carbons flanked by H atoms or -OH groups, is hydrophilic and can exist in a linear or cyclic structure. Monosaccharides are linked together by glycosidic bonds to form polysaccharides including starch, chitin and cellulose. Carbohydrates are used as an energy source, as a source for carbon, and can be attached to other macromolecules, including lipids and proteins, to serve as recognition markers for the cell. Glycoproteins: These are the proteins that contain oligosaccharide chains (glycans) covalently attached to polypeptide side-chains. We have different blood groups due to the variation in the glycans that are attached to cell surface protein on mature red blood cells. c) Nucleic acids: Nucleic acids [Deoxyribonucleic acids (DNA)/Ribonucleic acids (RNA)] are biological macromolecules specialized for the storage, transfer and use of genetic information. They are built from basic building blocks called nucleotide triphosphates (deoxy-ribonucleotide or dNTP in DNA and ribonucleotides or rNTPs in RNA). The nucleotide triphosphates are of five major types: Adenosine triphosphate (ATP), Thymidine triphosphate (TTP), Guanosine triphosphate (GTP), Cytosine triphosphate (CTP) and Uridine triphosphate (UTP). Each nucleotide has a triphosphate group, a pentose sugar (ribose in RNA and deoxy-ribose in DNA) and a nitrogenous base (A, T, G and C in DNA and A, U, G, C in RNA). The A and G are purine bases (with two aromatic rings) whereas the T, C and U are pyrimidine bases (with one aromatic ring). These are the names of the nitrogenous bases: A = Adenine, T = Thymine,C = Cytidine, G Guanine, U = Uracil. The 5 carbons of pentose sugars are labeled as 1C (attached to the nucleotide base), 2C (attached to a OH group in the ribose of rNTP and to an H in deoxy-ribose of dNTP), 3C (attached to a OH group), 4C (attached to an H atom) and a 5C (attached to the triphosphates). The -OH group at the 2C position of each rNTP makes RNA more reactive and less stable compared to DNA. Polymers of nucleic acid are formed by condensation or dehydration reaction that leads to the formation of a covalent phosphodiester bond, which links the 5 phosphate group of incoming nucleotide to the 3 hydroxyl group on the pentose sugar of last nucleotide in the growing nucleic acid strand. Thus the nucleic acids are always synthesized in a 5->3 direction with the 3end of the growing nucleic acid chain being the receiving end for the incoming base. Each condensation reaction produces a water molecule and a pyrophosphate (two phosphates) as the byproducts. It is worth noting that each condensation reaction is an energy requiring or endergonic reaction, which utilizes the energy that is released by the breaking of the two phosphates from the incoming nucleotide. DNA in the cell is the hereditary material and is usually double-stranded. The two strands of DNA are complementary i.e. A (a purine) forms two hydrogen bonds with T (a pyrimidine) and G (a purine) forms three hydrogen bonds with C (a pyrimidine). Thus the ratio of pyrimidine : purine within a DNA duplex is always 1:1 (Chargaffs rule). The two strands of DNA duplex are also anti- parallel (the 5 end

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of one DNA strand faces the 3 end of its complementary strand to make a DNA duplex). Within a cell, DNA exists in the form of chromatin (a complex of DNA and associated basic proteins such as histones which compact the DNA and make the chromosomes). The modifications of histones play a crucial role in folding and unfolding of chromosomes and regulation of gene expression. The RNA is usually single-stranded and is of three major types: ribosomal (rRNA), transfer (tRNA) and messenger (mRNA). The mRNA gets translated to proteins. In comparison, the tRNA and rRNA are involved in the synthesis of proteins. Recent research has also revealed new classes of RNAs known as siRNA (small interfering) and miRNA (micro), which play important regulatory roles. Nucleotides also serve as energy sources (ATP and GTP) and second messengers (cAMP) during intracellular or cell-cell signaling. d) Proteins: Proteins are linear chains of amino acids of which there are 20 distinct types. The order of the amino acids in the chain dictates the shape that the protein will take and therefore the function of the protein in the cell. There are 4 levels of protein structure: primary (1st order or a linear chain of amino acids that are attached by covalent peptide bonds), secondary (2nd order of protein structure) tertiary (3rd and highest order of protein structure for those proteins that are comprised of single polypeptide chain) quaternary (4th order of protein structure). The polypeptide chain of a protein is always WRITTEN in an N-> C direction and as we shall see later it is also biosynthesized in an N-> C direction. A proteins secondary structure (-helix and -pleated) is comprised of regular repeated spatial patterns in different region of polypeptide chains and is predominantly stabilized by hydrogen bonds between the backbone peptide bonds acting as H-bond donors and acceptors. The different interactions between the side chain groups of the amino acids determine the 3-dimensional tertiary structure of proteins. Quaternary structure results when two or more polypeptide chains in a protein bind to and interact with each other. Proteins perform all possible functions within a cell except storage of hereditary information. Proteins can be structural, can be used to transport materials, can be used for locomotion of cells and be used as enzymes or biological catalysts. Proteins perform all possible functions within a cell except storage of hereditary information. Proteins can be structural (e.g. collagen and elastin), can be used to transport molecules (hemoglobin transports O2 and CO2 and lipoproteins transport cholesterol esters in the blood), can be used for locomotion of cells (e.g. actin and tubulin proteins) and be used as enzymes or biological catalysts. Each protein has a unique 3D-conformation, which is maintained under optimal conditions i.e. specific pH, salt concentration and temperature conditions. The protein 3D structure is crucial for function. There are many examples of diseases that are related to the alteration in synthesis or functioning of one or multiple proteins. Some are included below. For example a substitution of Glutamic acid -> valine at amino acid position 6 in the beta globin chain of hemoglobin results in sickle cell anemia. Similarly, a single amino acid substitution from arginine -> tryptophan at the active site of phenylalanine hydroxylase enzyme results in phenylketonuria (PKU), which is an inborn error of metabolism.

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Questions: 1. Of the five structures given below

OO

OO O

N

N N

N

OO

N CO2

NH

O

O

OO

a) Circle the structure(s) that can serve as a building block of proteins. Underline the central carbon atom of this structure and classify the side- chain as polar/ nonpolar, charged/ uncharged, hydrophobic/ hydrophilic. b) Box the structure(s) that can serve as a building block of carbohydrates. c) Shade the structure(s) that can serve as a building block of nucleotide.

2. Patients with a high blood cholesterol levels do not benefit very significantly if they follow a cholesterol free dietary regimen. Briefly explain why is this so. 3. The following diagram represents a substrate molecule bound to the active site of a protein. The R groups from the amino acids in the proteins substrate-binding region are shown. Each of the four R groups from the protein that interacts with the substrate is numbered on the figure below. For each side chain, state the strongest type of interaction it could have with the substrate in the configuration shown below. Your choices are: Covalent, Hydrophobic/ VDW, Ionic and Hydrogen. Also classify each R group as hydrophobic, polar or charged.

CC C C

CCC O

O

H H

HH

NH

H

HC C

O

OC

H

H

NH

HCC

O

CHH

CC

CHH H

HHH H

C

SCH HC

H

Protein

Solution

Substrate

1

2

3

4

This is the simplest correct bonding arrangement of the atoms and charges.



a) Identify the amino acids (1), (3) and (4) in the schematic above. b) Classify the amino acids that you have identified above as polar or nonpolar, charged or uncharged, and hydrophilic/hydrophobic. c) Complete the following table.

R Group Interaction(s) of R Group with Substrate

Classification of R Group

(1)

(3)

(4)

4. Pepsin is a digestive enzyme that acts on proteins in stomach. This requires the presence of hydrochloric acid (HCl) for its normal function. a) What may be the type of reaction catalyzed by pepsin? b) Pepsin functions well in the stomach but it ceases to function in the intestine. Explain why pepsin does not function in the intestine? c) Propose a mechanism that explains how the cell degrades a misfolded protein. 5. The following schematic represents an ATP molecule that is used to build RNA and also serves as the energy currency of the cell.

N

NN

N

NH2

O

OHOH

OPO

O

O

PO

O

O

PO

O

O

a) Circle the group(s) that you will alter so that this molecule could serve as a monomer for DNA. b) Draw an arrow to the atom that forms a covalent bond with the terminal nucleotide of a growing nucleic acid polymer. c) Name the molecule(s) that are produced from ATP if it is used as an energy source. d) Draw an asterisk by the atom(s) that form hydrogen bond(s) with a nitrogenous base on the complementary strand of DNA or RNA. Identify the base in DNA and in RNA. e) If a double stranded DNA has 30% A, give the percentages of G, C, T in this molecule.



6. The following diagram represents a complex three-dimensional conformation of a RNA molecule.

a) Within Region 2, what bonds/interactions are primarily involved in stabilizing the RNA structure? b) In Region 1, if the sequence of the top region is AUUUGUAA, can you predict the % of bases i.e. %A, %U %C and %G of the bottom region? Explain. c) In the Region 2, if the sequence of the top region is AUUUGUAA, can you predict the % of bases i.e. %A, %U %C and %G of the bottom region? Explain.

Region 1

Top region Region 2 Bottom region

5 3



Solution key: 1. Of the five structures given below

a) Circle the structure(s) that can serve as a building block of proteins. Underline the central carbon atom of this structure and classify the side- chain as polar/ nonpolar, charged/ uncharged, hydrophobic/ hydrophilic. The amino acid and the central carbon atom is circle. Based on the side-chain this is a nonpolar/ uncharged/ hydrophobic amino acid. This amino acid is tryptophan. b) Box the structure(s) that can serve as a building block of carbohydrates. c) Shade the structure(s) that can serve as a building block of nucleotide. (2nd structure in the top row). 2. Patients with a high blood cholesterol levels do not benefit very significantly if they follow a cholesterol free dietary regimen. Briefly explain why is this so. Cholesterol is a part of membranes in all cells. It also serves as a precursor for vitamin D, bile acids, steroid hormones clearly reflecting that this is an important molecule that is made my our cells. Since the body makes cholesterol, you dont see a significant decrease in the blood cholesterol level even if you strictly follow a cholesterol free dietary regimen.



3. The following diagram represents a substrate molecule bound to the active site of a protein. The R groups from the amino acids in the proteins substrate-binding region are shown. Each of the four R groups from the protein that interacts with the substrate is numbered on the figure below. For each side chain, state the strongest type of interaction it could have with the substrate in the configuration shown below. Your choices are: Covalent, Hydrophobic/ VDW, Ionic and Hydrogen. Also classify each R group as hydrophobic, polar or charged.

a) Identify the amino acids (1), (3) and (4) in the schematic above. b) Classify the amino acids that you have identified above as polar or nonpolar, charged or uncharged, and hydrophilic/hydrophobic. c) Complete the following table.

R Group Interaction(s) of R Group with Substrate

Classification of R Group

(1) Ionic, (Hydrogen bonding also possible)

Charged, polar, hydrophilic

(3) Hydrophobic / Van der Waals

Nonpolar, hydrophobic

(4) Hydrogen bonding

Polar, hydrophilic

4. Pepsin is a digestive enzyme that acts on proteins in stomach. This requires the presence of hydrochloric acid (HCl) for its normal function. a) What may be the type of reaction catalyzed by pepsin? Pepsin digests the proteins to small polypeptides at acidic pH. b) Pepsin functions well in the stomach but it ceases to function in the intestine. Explain why pepsin does not function in the intestine? Each enzyme maintains its active three-dimensional conformation at a specific pH. The acidic pH in the stomach is the required pH for pepsin activity whereas the alkaline pH of the intestine is not.

CC C C

CCC O

O

H H

HH

NH

H

HC C

O

OC

H

H

NH

HCC

O

CHH

CC

CHH H

HHH H

C

SCH HC

H

Protein

Solution

Substrate

1

2

3

4

This is the simplest correct bonding arrangement of the atoms and charges.



c) Propose a mechanism that explains how the cell degrades a misfolded protein. The misfolded proteins can be degraded by the lysosomal enzymes ( autophagy). We briefly talked about this in the first lecture. 5. The following schematic represents an ATP molecule that is used to build RNA and also serves as the energy currency of the cell.

a) Circle the group(s) that you will alter so that this molecule could serve as a monomer for DNA. b) Draw an arrow to the atom that forms a covalent bond with the terminal nucleotide of a growing nucleic acid polymer. c) Name the molecule(s) that are produced from ATP if it is used as an energy source. ADP, AMP, Phosphate and Pyrophosphate d) Draw an asterisk by the atom(s) that form hydrogen bond(s) with a nitrogenous base on the complementary strand of DNA or RNA. Identify the base in DNA and in RNA. You can put an asterisk next to any electronegative atom i.e. O or N. e) If a double stranded DNA has 30% A, give the percentages of G, C, T in this molecule. T= 30%. G= 20% or C= 20% 6. The following diagram represents a complex three-dimensional conformation of a RNA molecule.

a) Within Region 2, what bonds/interactions are primarily involved in stabilizing the RNA structure? Hydrogen bonds between complementary bases and phosphodiester bonds between the adjacent bases are involved in stabilizing the structure. b) In Region 1, if the sequence of the top region is AUUUGUAA, can you predict the % of bases i.e. %A, %U %C and %G of the bottom region? Explain. No, since it is existing as single stranded RNA in this region with no hydrogen bonding between complementary bases. c) In the Region 2, if the sequence of the top region is 5AUUUGUAA3, can you predict the % of bases i.e. %A, %U %C and %G of the bottom region? Explain. Yes, since it is existing as double stranded RNA in this region with hydrogen bonding between complementary bases. There will 3Us, 2As, 2Cs and 1G to give the sequence 3UAAACAUU5 which will be complementary to the sequence given above.

Region 1

Top region Region 2 Bottom region

5 3

N

NN

N

NH2

O

OHOH

OPO

O

O

PO

O

O

PO

O

O

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