COMMONWEALTH OF AUSTRALIA Copyright Regulation

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1 Page1 University of Sydney Library Electronic Item CURSE: MBLG1001 Lecturer: Dale Hancock Title of Lecture: Molecules of Life: Proteins CMMNWEALTH F AUSTRALIA Copyright Regulation WARNING This material has been reproduced and communicated to you by or on behalf of the University of Sydney pursuant to Part VB of the Copyright Act 1968 (the Act). The material in this communication may be subject to copyright under the Act. Any further reproduction or communication of this material by you may be the subject of copyright protection under the Act. Do not remove this notice.

2 Page2 MBLG Lecture 2: Molecules of Life: Proteins I am also assuming that you are all well versed in the nature of chemical bonding so I won t bore you by going over it too much. Let s consider the extremes: At one end of the spectrum you have the perfect coupling where each atom shares its electron with the other. This perfectly equal, harmonious relationship is, like couples in real life, quite rare. The quintessential example given is H 2. Both atoms are equal and very small. They both share their only electron equally so the charge is evenly distributed. The other examples of even sharing are the carbon carbon bond and the carbon-hydrogen bond. Note that both carbon and hydrogen have a half-full (or half-empty depending on your state of mind) outer or valence shell of electrons. At the other end of the spectrum is the completely ionic bond, characterized by NaCl. This pair of atoms is such a dysfunctional couple that one atom (Cl) grabs the electron from the other atom (Na) and hogs it. There is no sharing in this relationship; one partner completely takes the electron from the other. Note that the Cl has a full outer shell bar one electron and the Na has only one electron in its outer shell. This makes both these atoms incredibly reactive; one wants to lose its only valence electron and the other wants to take it. In relationship terms it is completely one sided but the analogy falls down here because both atoms are satisfied with the arrangement; two very reactive elements form a compound, common table salt, which is very stable. In solution this compound exists as Na + and Cl - ions. These two ions are in atom heaven; they have full outer shells! It is the in-between coupling (sometimes termed polar covalent) that is often interesting to molecular biologists and biochemists; bonds which show some covalent and some ionic character. The two atoms in the partnership share the bonding electron(s), but not evenly. ne of the atoms involved in the coupling has a stronger affinity for the electron(s), hence we generate a partial charge or dipole, denoted as delta positive (δ+) or delta negative (δ-). It is not a complete swap and no true ion forms. But it gives the compound a polar character. The best and most important example from a life scientist s point of view is water. The oxygen is very electronegative (that means it has a great affinity for electrons in any bonding. It has 6 electrons in its outer shell; of which 2 pairs never participate in bonding. It only needs another 2 for a complete outer shell: nirvana or heaven for atoms). The hydrogen-oxygen bond has a dipole so the water has a delta negative charge at the oxygen and a delta positive charge at the hydrogen. Water is a very polar solvent and this property has immense significance for the workings of the cell. Molecules which also have a dipole will be attracted to the water and will be soluble in it. They are termed hydrophilic (or water loving) or polar. Molecules with no dipole are not attracted to the water and

3 Page3 are much less soluble in aqueous solutions. They are termed hydrophobic (or water hating) or non-polar. Two common compounds found in everyday life display these properties; fat (hydrophobic) and polysaccharides (hydrophilic). Both these compounds are very important polymers (VIPs) for life. Let s briefly consider these two polymers to see what gives them their different solubility properties. Fats or more scientifically lipid has the general formula (- -) n. Examples are fatty acids such as palmitic acid (where n=15) with a CH group attached at one end. Three of these fatty acids esterify to a glycerol molecule to form triglycerides. Lipids are very hydrophobic, the long carbon chains are very non-polar. They consist of C-C and C-H bonds; both of which are evenly sharing covalent bonds. The long chains are known as aliphatic chains; the longer this chain the more hydrophobic. This polymer is made up of the same monomer, it has repeating units and hence we can give a general formula. Carbohydrate or hydrated carbon has the general formula (H-C-H) n. Polymer examples are cellulose, starch and glycogen (fuel for your body). The polymer has the general name polysaccharide (many saccharides), the monomer is a saccharide or sugar. Sugars with 5 or 6 carbons readily cyclise forming ring structures, the most common being glucose C 6 H Sugars are also components of nucleic acids, hence I would like to mention one property of sugars that impacts on the properties of DNA and RNA. Sugars are very water soluble or hydrophilic. The H groups (remember the -H bond in water) are responsible for this property. Carbohydrate polymers are also made up of repeating units.

4 Page4 Now let s introduce the next 2 classes of biopolymer: nucleic acids and protein. These polymers differ from the others in that a number of different types of the monomer are joined to make them up AND THE RDER IS IMPRTANT. When we refer to genetic information transfer this is the information that is transferred; the order of the monomer. To have a sequence dependent polymer you must have a template. There must be some way of copying that template and ensuring that the template copy is accurate. The cell goes to extraordinary lengths to ensure the accuracy of the synthesis of DNA, RNA and protein, particularly DNA. Let s see why we need to be so fussy about order or is the cell just anal!! I will briefly consider DNA and RNA now and return to these polymers later. Both DNA and RNA are nucleic acids, named thus as they were first isolated from the nucleus and they were acidic. They are composed of four monomers (I am sure you know this from high school biology!!). These monomers, however, are made up of more than just the base. Each monomer contains a sugar moiety, either ribose or deoxyribose, and a phosphate. These groups confer on nucleic acids some of their familiar chemical properties; water solubility and acidity. Both nucleic acids are composed of a repeating sugar phosphate backbone. The variation, and hence information, comes about from the order of the 4 bases. These bases are attached to the sugar and hang off the backbone. Proteins are polymers made up of amino acids. There are 20 different amino acids; they differ in the side chain. We have side chains that are hydrophobic, aromatic, polar, acidic and basic. The type of side chains and their sequence determines many of the properties of the protein. Some side chains are attracted to each other; some are repelled. Some form weak bonds with each other. Proteins are found everywhere in your cell. They function to hold the shape of the cell (cytoskeleton), receptors, transporters carrying molecules in and out of the cell and most importantly enzymes. They make up whole classes of hormones and growth factors, toxins and antibodies. These molecules are the doing molecules, definitely the molecules of life. Some proteins are hydrophobic and are synthesized embedded in membranes; some are water soluble, located in the cytosol. The incredible diversity of protein structures and hence their function is made possible by having 20 different amino acids and by maintaining the order of these amino acids each time a new protein molecule is synthesized. How is the order determined and maintained? In eukaryotic cells transcription and replication are carried out in the nucleus and translation is carried out in the cytoplasm. In prokaryotes, which have no organelles to compartmentalize the processes, transcription is tightly coupled to translation. ne follows the other almost

5 Page5 simultaneously. Replication is carried out by DNA polymerases, transcription by RNA polymerases and translation is performed on ribosomes. How do amino acids combine to form proteins? To understand what s going on lets look at your typical amino acid. It has the following general structure: alpha carbon Amino group R - Carboxyl group Sidechain or R group; there are 20 different ones! Two amino acids combine, by condensation polymerization to form a dipeptide R 1 R 2 H 2 N CH C - R 1 H R 2 Peptide bond The peptide bond is a strong covalent bond with some unique properties. Amino acids are added in a sequential process to the growing peptide chain, at the carboxyl (or C-terminal) end. The incorporated portion of the amino acid is termed an amino acid residue (some of the monomer has been lost in the polymerization). The mrna is the template for translation; this is copied from the

6 Page6 DNA. Notice as the polypeptide grows that you have a common backbone, irrespective of the sequence; it is only the side chain hanging off the backbone where the variation occurs. It has a defined beginning, termed the N terminal and a chemically different end, the C terminal. The protein then must be folded, often modified (glycosylated, phosphorylated etc) then transported to the correct location before we have a fully functional protein. All the enzymes you will meet in the up-coming lectures will have undergone this process. The peptide bond: The double bond between the oxygen and the carbon resonates between the C= and the C=N. This resonance gives the C-N bond a partial double bond character. It is considered to exist as C= for 60% of the time and C=N for the remaining 40%. nce the peptide bond is formed the carboxyl group of amino acid 1 loses its charge as does the amino group of the second amino acid. The bond formed is an amide bond, having the dipole properties of an amide ie. the N has a net positive charge (δ+ve) and the carbonyl C (C=) has a net negative charge (δve). The partial double bond character of the peptide bond restricts rotation and has a big impact on the 3-D conformations the protein can exist as. The partial charge or dipole of the peptide bond allows for H-bonding between different portions of the backbone. - N N+ Let s now consider the different side chains available for protein synthesis. Recapping..There are 20 amino acids found in all naturally occurring proteins. These vary in the side chain or R group attached. We will divide them up by their chemical properties. alpha carbon Amino group R - Carboxyl group Sidechain or R group; there are 20 different ones!

7 Page7 Some of the amino acid side chains can be modified post translationally. The types of modification which can occur include: phosphorylation, hydroxylation, glycosylation, addition of lipid moiety. These modifications can be carried out for regulation, solubility, anchoring or for structural reasons. Amino acid side chains are grouped by their chemical properties as follows: Hydrophobic, including aliphatic and aromatic side chains Polar non-ionic Acidic Basic Rather than go through all 20 side chains I will show you a representative from each group. The structures of each of the 20 side chains are presented in the lecture supplement in the front of your resource manual. The 3 letter and 1 letter code is also quoted. You need to be familiar with the 3 letter code. Hydrophobic aliphatic amino acids e.g. Leucine, (Leu, L). Leucine has a branched aliphatic (- -) chain which has no dipole (C-C and C-H bonds share their electrons very evenly) making it quite hydrophobic (insoluble in water). It does not participate in H-bonding or ionic interactions. It does, however, interact with other hydrophobic side chains and is often found buried in the interior of water soluble proteins or exposed on the outside of membrane embedded portions of proteins. CH CH 3 CH 3 H H Aromatic amino acids e.g. Phenylalanine (Phe, F). Phenylalanine contains an aromatic ring in its side chain which makes it, not only hydrophobic but also confers UV absorption properties on the side chain. Proteins absorb UV light at ~280 nm as a result of the aromatic side chains: tyrosine, tryptophan and phenylalanine (as well as cysteine and histidine to a lesser extent). Phenylalanine and tryptophan are quite hydrophobic. Tyrosine, although it contains the aromatic ring, has a hydroxyl attached. This H is polar and actually dissociates at high phs.

8 Page8 Polar non-ionic amino acids e.g. Serine (Ser, S). The side chain of serine contains an H which gives the side chain its polar properties. Except in very rare circumstances (active site of serine proteases) the proton does not dissociate from but its polarity facilitates H-bonding. The serine side chain acts as an H-bond donor. It can also be phosphorylated post translationally; a common mechanism for enzyme regulation. H H H Acidic amino acids e.g. Glutamate (Glu, E). These side chains contain a carboxylic acid which dissociates with a pka of ~4. At neutral ph these side chains carry a negative charge, enabling ionic interactions with basic (positively charged) side chains. They are hydrophilic and often found on the outside of water soluble proteins. The dissociation follows the general formula: HA H + + A - C H Basic amino acids e.g. Lysine (Lys, K). This group is characterized by side chains with a group containing a protonated N. The dissociation follows the general formula: BH + B + H +. At physiological ph these side chains carry a positive charge and are often found on DNA binding proteins, interacting with the sugar phosphate backbone. H NH 2 Amino acids (19 of the 20) have a chiral carbon (the alpha carbon), having both a D and an L isomer. Some have more than one chiral carbon. Which amino acid has no chiral carbon? Which have 2 chiral carbons? Check the structures in the supplement. All naturally occurring proteins contain only L amino acids, except for a few nasty toxins e.g. actinomycin D. How to distinguish L from D?

9 Page9 H H 3 N + C R H C C- C- R + NH 3 L isomer C R N spelt in a clockwise D isomer C R N spelt in an anti-clockwise Important properties of amino acids. UV absorption. Certain side chains containing aromatic rings eg tyrosine, tryptophan and to a lesser extent phenylalanine absorb UV light strongly, with an absorption maximum of ~280 nm. This property is often exploited when detecting proteins experimentally, providing a quick and relatively inexpensive detection method which doesn t destroy the sample. Charge. All amino acids contain charged groups as part of their basic structure. The amino group and the carboxyl group attached to the alpha carbon are charged at certain phs. Let s consider the charge pattern of a standard simple amino acid, glycine.

Housekeeping. Housekeeping. Molecules of Life: Biopolymers

Housekeeping. Housekeeping. Molecules of Life: Biopolymers Molecules of Life: Biopolymers Dr. Dale Hancock D.Hancock@mmb.usyd.edu.au Room 377 Biochemistry building Housekeeping Answers to the practise calculations and a narration are on WebT. Access these through