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Transcript
A Chemical Look at Proteins: Workhorses of the Cell RNA RNA Life Sciences 1a Lecture Notes Set 4 Spring 2006 Prof. Daniel Kahne Dr Lue told you one important fact about HIV: it cannot replicate unless it can infect and coopt the machinery of a cell. It has a genome - an information carrier molecule - RNA in this case. What does the virus not have? It cannot do metabolism on its own. That is to say, it can’t carry out chemical transformations to take one molecule and convert it into another molecule. The molecules that carry out chemical transformations in cells are proteins and we will spend the next three lectures learning about the basics of these workhorses of the cell. 1 Life requires chemistry… HO HO OH H2N O N H H N O OH O N H O amino acid monomer …and it is proteins that make the chemistry happen. DNA is the information carrier of the cell. Lipids divide the cell into compartments. But proteins do the work. Proteins have an astonishing range of different functions because they are capable of adopting an enormous range of structures with different properties. Proteins are made of amino acids, and before we can begin to learn about how proteins work, we need to learn about their structures. 2 Lectures 6-8: The Molecular Basis of Translation Proteins: The Workhorses of Biology a. b. c. A chemical look at proteins i. Introduction to proteins and amino acids ii. Conformational peculiarities of peptide bonds iii. Structures and properties of the twenty natural amino acids iv. A closer look at four special amino acids -- Gly, Pro, Cys, and His. v. Collaborations between amino acids in proteins Protein structure i. The four levels of structure ii. A closer look at secondary structure Protein folding: i. Anfinsen’s experiment ii. Thermodynamic forces involved in protein structures. iii. Thermodynamics of protein folding iv. The Levinthal paradox (the kinetics of protein folding) v. Molecular chaperones Lecture Readings Alberts pp. 55-56, 74-75; McMurry Chapter 18 This is the outline for what we will talk about in the first lecture on proteins. After a brief introduction to some of the functions of proteins, we will learn about the building blocks of proteins -- the properties of the individual amino acids that make up these biological polymers and the properties of the bonds that join them. You will be expected to know all of the amino acids and their personalities and to have some understanding of the structure of the polypeptide backbone. 3 A polymer is built of repeating monomer units. Biological (Natural) Polymers O H N H NH2 N O P O HO OH N O N H CH3 N N N O N CH3 N N O N O N O O HO nucleotide monomer O P O O O O O O P O P O O O O O P O O O nucleic acid polymer OH OH HO HO H O N O OH OH sugar monomer O HO OH O HO OH O OH OH O HO O OH polysaccharide • DNA is the information carrier of life; along with RNA it provides instructions to make proteins. • Sugars are important in energy storage and have other functions that are not well understood. Many of the molecules found in the cell are polymers, which are large molecules comprised of repeating monomer units. We began by talking about the structure and function of nucleic acid polymers of DNA and RNA. These polymers are comprised of only four different building blocks each and they are highly negatively charged. DNA has a single structure -- the double helix -- and a single function that is explained by its structure. Its function is to transmit information and it does so in two ways -- through replication (DNA copying itself, which is important in the generation of new cells) and through transcription (DNA making RNA, which is important for protein synthesis). RNA is single stranded and can fold into many different kinds of structures, and it plays several different kinds of roles in the cell. For example, messenger RNA encodes proteins, amino-acid linked tRNA molecules help decode messenger RNA, and ribosomal RNA forms part of the ribosomal machine that is involved in decoding messenger RNA. Cells also make polymers of sugars. These polymers are called oligosaccharides or polysaccharides depending on how many sugars they contain. On this slide we have only mentioned one role for sugars -- the storage of energy -- but they play many other roles. For example, oligosaccharides on cell surfaces bind to circulating proteins and to other cell surfaces, so they play roles in communication between cells. As you will see in a few lectures, they also act as receptors for viruses, bacteria, and bacterial toxins that have evolved to use cell surface carbohydrates to help gain entry into cells. Specifically, you will learn more about how HIV uses a glycoprotein called gp120 to enter T-cells and macrophages. We aren’t going to talk very much about oligosaccharides and polysaccharides in this course, in part because their roles -- apart from energy storage -- really aren’t that well understood yet. 4 Proteins: Amino acid polymers HO HO H N OH H2N O amino acid monomer N H O OH O N H O protein polymer • Proteins have the most diverse shapes of the biological polymers. • Proteins are comprised of a wider variety of monomers and has a more varied charge distribution. • The different shapes combined with different properties allow proteins to have an incredible range of different functions. Proteins are the most diverse biological polymer. They are made of a wider variety of monomer units than nucleic acid polymers -- twenty monomers rather than four -- and they adopt a wider variety of shapes and display a much wider variety of properties. Whereas all DNA molecules are polyanions, proteins may be positively or negatively charged, and many proteins contain some regions that are highly negatively charged and others that are highly positively charged. Because they can adopt so many different shapes with so many different properties, they can carry out an incredible range of different functions. 5 Some important functions of proteins Enzymatic Structural proteins: Hair, skin, eyes, muscle, silk DNA polymerase Tubulin - cytoskeletal Hemoglobin - 02 carrier Digestion, blood clotting, replication, transcription, translation Carriers: Regulatory: Respiration and metabolism Coordinate events within and between cells Bcr-Abl - signal transduction Proteins play many roles in the cell. For example, they can be structural. Your hair is made of proteins. The outer layers of your skin, the single most important protective organ of the body, are made entirely of protein. Your fingernails, your wool hats, your silk scarves, your leather boots -- those are all made of proteins that have evolved to withstand particular kinds of mechanical stresses. Depicted here is a protein called tubulin that is found inside cells and that helps form an internal scaffold, or cytoskeleton, in the cell. Unlike your hair, which is pretty set in its ways, cytoskeletal proteins like tubulin are made of individual proteins that come together (polymerize) and fall apart (depolymerize) on a rapid timescale, and as they polymerize and depolymerize, they cause other molecules inside the cell to move in particular ways. For example, cytoskeletal proteins are involved in chromosome movement during the process of cell division. You will learn more about this protein when we talk about cancer later in the course. Thus, even within the category of “structural” proteins, there are a wide range or different structures, functions, and behaviors. Furthermore, the mechanical properties of different structural proteins are pretty interesting. Another major function of proteins is to act as enzymes, molecules that catalyze chemical reactions in cells. All life involves chemistry -- for example, the chemistry involved in the breakdown of nutrients and the synthesis of macromolecules from nutrients. None of the reactions involved in these processes occurs spontaneously in water on a timescale that would be consistent with life. For example, the spontaneous breakdown of a protein from food into its individual components would take hundreds to thousands of years in sterile water. The notes continue on the next page for this slide. 6 Some important functions of proteins Enzymatic Structural proteins: Hair, skin, eyes, muscle, silk DNA polymerase Tubulin - cytoskeletal Hemoglobin - 02 carrier Digestion, blood clotting, replication, transcription, translation Carriers: Regulatory: Respiration and metabolism Coordinate events within and between cells Bcr-Abl - signal transduction Enzymes accelerate the biological reactions that are necessary for life to exist. Because enzymes are so important in all aspects of cell growth and division, we will examine throughout this course several enzymes and how they work. We will also focus some attention on how one might inhibit an enzyme that is responsible for deleterious effects. We have already seen one type of enzyme in the previous lecture, DNA polymerase. This enzyme strings together the nucleotides to make a nucleic acid polymer. Later we will look at enzymes that catalyze the breakdown of proteins. Proteins also function as regulatory molecules that affect the activity of other enzymes. We have already pointed out that life depends on chemical reactions occurring on a rapid timescale. However, it is also necessary for these chemical reactions to be precisely coordinated. Thus, the activities of various enzymes are regulated (turned on and off) by other proteins that respond to environmental conditions. Later in this course we will look at a specific type of regulatory protein, Bcr-Abl. This protein plays a regulatory role in normal cells, but it can malfunction and when it does, it causes a certain type of cancer. The example of Bcr-Abl illustrates that the proper regulation of chemical reactions in a cell is absolutely essential for normal growth and division. The notes continue on the next page for this slide 7 Some important functions of proteins Enzymatic Structural proteins: Hair, skin, eyes, muscle, silk DNA polymerase Tubulin - cytoskeletal Hemoglobin - 02 carrier Digestion, blood clotting, replication, transcription, translation Carriers: Regulatory: Respiration and metabolism Coordinate events within and between cells Bcr-Abl - signal transduction Proteins can function as molecular transporters, delivering molecules to different parts of an organism. Hemoglobin is a very important protein that delivers oxygen to all parts of the body. It is found in high concentrations in red blood cells and it picks up oxygen in the lungs and releases oxygen in other parts of the body. It also carries CO2 back to the lungs for exhalation. Cholesterol, which we will talk about, is carried to different parts of the body from the gut by protein carriers. Other proteins exist that are not as easy to classify. Ion channels, for example, which allow ions to pass across cell membranes and are critically important in muscle contraction and nerve stimulation, can be thought of as enzymes (because they catalyze the transport of ions across lipid bilayers) or as carriers (because they deliver ions from one side of a membrane to another). 8 Crystal structure of DNA with P53 protein bound • The structural variability of DNA is limited. • Proteins can adopt many structures; predicting what a protein will look like from its sequence is hard. This slide shows a crystal structure of DNA with a protein bound to it. The protein shown is called P53, which is a tumor suppressor protein. When DNA damage is sensed with in the cell, P53 binds to DNA and turns on the synthesis of other proteins/genes that tell the cell to stop dividing. What you can see from this slide is that the DNA looks as you all expect it to look -- the archetypal double helix. You don’t even have to know what the sequence of nucleotides is in a stretch of duplex DNA to know that the DNA will adopt a double helical structure. However, the structures of proteins that bind to DNA are highly variable and they depend on the specific sequence of amino acids. The problem is that predicting what a protein will look like from its sequence is a major challenge, as you will see. 9 Protein 3D structure depends on primary sequence NH3 N H H N O N H O H N O O OH Lys Ser Ala Phe Amino acid sequence Folded polypeptide chain Question: What happens if you change a single amino acid in the primary sequence? Proteins are comprised of amino acids strung together into polypeptide chains. The specific order of amino acids in the chain is called the primary sequence, and the chain folds into a shape that depends on this primary sequence. 10 Small changes at the amino acid level can affect structure: Sickle Cell Anemia O O HN NH N H H N OH O N H O O C NH O N O His Leu Thr Pro Glu Hemoglobin: Helical, globular structure Glutamate at 6 position Normally forms tetramer HN NH N H H N O OH O N H O C NH Normal Red Blood Cells O N O His Leu Thr Pro Val Sickle -Hemoglobin: Valine at 6 position Quaternary structure clumps together Sickle Cell Red Blood Cells You might imagine that in a protein comprised of a hundred or more amino acids a single amino acid change would have no effect. Often this is the case, but sometimes a single change makes a profound difference. This slide shows one example where a single amino acid change alters the activity of a protein significantly. Hemoglobin, the oxygen carrier in red blood cells, contains a negatively charged residue, glutamate, at position 6 in the chain. Hemoglobin folds into a helical, globular protein (you will learn shortly what helical means; globular simply means that it is roughly spherical -- i.e., it has approximately the same dimensions along all three axes), and this globular protein associates to form a tetramer, which is the active form of the molecule. People who have sickle cell anemia have a single amino acid change, or mutation, in their hemoglobin. The mutation involves a change from a negatively charged glutamate to valine, which is an uncharged, non-polar amino acid. This single amino acid change leads to a dramatic change in how the individual hemoglobin proteins interact, and you can see from the slide that the mutant hemoglobin tetramer has a significantly different shape from the normal hemoglobin tetramer. This change in the shape of the tetramer is actually reflected in the shape of the red blood cells, which in the case of the valine mutant have a distorted, sickle shape because the hemoglobin inside is clumped together. Sickle-shaped cells do not carry as much oxygen and therefore deliver less oxygen to the body's tissues. The cells are also fragile and can break into pieces causing painful “crises” because they disrupt blood flow. The sickle cell mutation is recessive and a single copy of the mutant allele somehow enables people to resist infection with malaria, which is why it was selected for in areas where malaria is endemic. 11 Pymol: A useful tool The lab this week involves a playing around with a program called Pymol that allows one to view proteins. The ribbon diagrams of hemoglobin on this slide were made using Pymol. Pymol is a graphics package that reads files containing information about molecular structure. The files contain a list of all of the atoms in the molecule along with their coordinates in cartesian space as determined by X-ray crystallography or NMR spectroscopy. The coordinates are read into the Pymol program, which displays them as a three dimensional structure. Because it is difficult to grasp all the details of a three dimensional structure containing hundreds of atoms from a single representation, the Pymol program allows the user to display the structure in different ways, to focus in on different parts of the molecule, to color particular segments of the molecule with user-designated colors, and to rotate the molecule or parts thereof in order to become familiar with features that would otherwise be obscured by the overall complexity. 12 The user can choose to display all of the atoms in the protein, all but the hydrogen atoms, or only the backbone atoms as desired. The molecule can be displayed in a “ball and stick” representation, with sticks for bonds and balls for atoms; in a space-filling representation (called CPK) in which the relative size of each atom reflects its van der Waals radius; or as a ribbon diagram. Each of these representations can be useful. The ribbon diagram representation allows the user to visualize the structure of the backbone so that elements of secondary structure -helix, beta strands, turns, etc. -- are clearly revealed. (We will talk about secondary structure in more detail in a few slides.) The CPK representation most realistically conveys how tightly packed the interior of the protein is and reveals channels, grooves, clefts, etc. that may be important for function. The ball and stick representation creates the false impression that there is a great deal of empty space throughout the protein, but it also allows the user to see details of torsion angles and interactions between atom types that are obscured in the CPK representation. In this lab, students will learn how to use the Pymol program to display molecules starting with single amino acids and progressing to the quaternary structure of protein. The students should become familiar with the different display options and the advantages and disadvantages of each with respect to the reality of the protein. If I were doing this lab, I would be interested in position 6. Where is it? Is it surface exposed? Are there other charges in the vicinity? Is it part of a loop or a helix? (Hemoglobin has virtually no beta strands). Can I understand how changing this amino acid could change the quaternary structure by examining the interactions between the individual proteins in the normal tetramer? 13 Parts of an amino acid RH H2N OH α O amino acid building block: amine (basic) carboxylic acid (acidic) α-carbon is tetrahedral R groups distinguish amino acids All amino acids are composed of three elements: an amino group, a carboxylic acid, and an intervening carbon atom. This carbon atom is called the alpha carbon because it is adjacent to the first carbon of the amino acid - the carboxyl carbon - and for nineteen of the twenty amino acids it contains a substituent, designated as an R group. The twentieth amino acid, glycine, contains two hydrogens rather than an R group and a hydrogen. The personality of each amino acid is determined by its R group (or lack thereof in the case of glycine). We will learn more about the R groups of individual amino acids presently. 14 Amino acids with ‘R’ groups are chiral HR RH OH H2N O L - enantiomer HO NH2 O D - enantiomer • The building blocks of proteins are chiral. • When we string them together the protein is chiral. First, however, it is important to note that the presence of an R group on the alpha carbon makes an amino acid chiral. Earlier we learned about chirality and we learned a quick test to determine if a molecule has a chiral center: if the central atom is bonded to four different groups, it is a chiral center. Nineteen of the twenty amino acids are bonded to four different groups, an R group (side chain), an amine, a carboxylic acid and hydrogen. If there are two hydrogens, as in glycine, then the amino acid is not chiral. In general, amino acids in nature are the L-enantiomer. 15 A review of chirality O O H H L - carvone D - carvone caraway spearmint Although chirality may seem like an abstract concept, it is not. You are surrounded by chiral objects and many macroscopic structures as well as most molecules in your body, large and small, are chiral. For example, your feet are chiral, which is why you can’t wear your left shoe on your right foot. Your hands are chiral, which is why if you are left-handed it is hard to use most scissors, which are designed for right-handed people simply because the majority of people happen to be right-handed. Chirality in molecules can have as profound an effect on function as chirality in hands or feet. Two small molecules are shown in the above slide. Both molecules have the same number and type of atoms and the same bond connections, and both are called by the chemical name of carvone. When Professor Liu talked about these molecules, he pointed out that carvone has one chiral center, however, and so the two molecules are actually different. The one on the left is L-carvone whereas the molecule on the right is D-carvone. Most of you have experienced both of these molecules whether you know it of not, and your experiences of the two are very different. The one on the left is the dominant odor molecule found in caraway, the seed used in rye bread and swedish cookies, among other things. The one on the right is the dominant odor found in spearmint. No one would confuse the smell of caraway and spearmint. The reason these molecules smell so different is that the receptors they bind to in your nose are chiral themselves. Just as your left shoe binds differently to your right foot than to your left foot, so do L- and D-carvone bind differently to the chiral receptors in your nose. In other words, they may look the same to you on paper, the same as a pair of scissors looks the same, but they fit very differently. The take home message is that the chirality of amino acids is important because chirality is a fundamental property of structure and it plays a key role in molecular interactions. Things would be very different in a racemic world. 16 Fluvastatin has two chiral centers F N F OR OH stereoselective reduction OH O + N F OR OH O O racemic starting material N OR OH OH O In lab, you are making fluvastatin. This drug is a member of the statin class of drugs that are used to lower cholesterol. Fluvastatin inhibits a liver enzyme, HMG CoA reductase, which controls cholesterol biosynthesis by controlling the flux of an intermediate along the biosynthetic pathway. Cholesterol is an important component of eukaryotic cell membranes and you need a certain amount, but excess cholesterol forms waxy deposits that accumulate along the lining of blood vessels and can occlude blood flow. Cholesterol is formed in the liver and is also obtained from animal products in the diet. There is great variability in the amount of cholesterol that people produce, and there is compelling evidence that keeping blood levels of cholesterol below a certain level prevents atherosclerosis, which is the leading cause of death in the United States. Fluvastatin is the first fully synthetic member of the class (the other statins are derived from natural products). Fluvastatin is produced as a racemic mixture and only one of the enantiomers has activity. The other enantiomer is inactive but has no off-target interactions, meaning that it does not interact with any other biological receptors. Therefore, the company that sells fluvastatin has decided that there is no need to spend the extra money to separate the enantiomers to provide a pure compound. Usually, however, enantiomers do have off-target effects, making it necessary to work out chiral syntheses or to separate the products. 17 A peptide bond connects two amino acids R OH + H2N O amino acid R O H N H O NH H2N OH O R' amino acid OH H2O R' peptide bond O A protein contains many peptide bonds (from 40 to well over 1000s). + N H NH O NH NH O O OH Peptide bonds play a role in the shape of a protein. Okay. Now that we have established that chirality is fundamental and not simply a boring detail, we need to talk about peptide bonds. As mentioned earlier, polypeptide chains are formed of strings of amino acids in which the bonds between amino acids are amide bonds. They are constructed by connecting the amino terminal end of one amino acid to the carbonyl of another amino acid. These amide bonds, or peptide bonds as they are called in the context of polypeptides, are very important in maintaining the shape of the peptide. Those of you who take more chemistry will learn a lot more about how the properties of these amide bonds impose constraints on the polypeptide chain. For now, what is important to know is that some of the bonds in the peptide chain are free to rotate but the peptide bonds can only adopt certain conformations. They can only adopt certain conformations because of the nature of the atoms in the bond. The nature of the atoms influences the bond that forms between the atoms, and to understand the nature of an amide bond, we need to talk a little bit about what bonding is. 18 Bonding in ethylene H H C C H H Ethylene contains one double bond. A double bond is made up of a σ and a π bond. π bonding orbitals of ethylene Recall that bonds form between atoms that share electrons. You have learned that atoms form bonds because they want to have eight valence electrons. Carbon has four valence electrons and so it needs to form four bonds with other atoms so that it can obtain four more electrons. Even though these electrons are shared between the two atoms, they complete the valence shell around carbon. Carbon can form single bonds with other atoms, in which case it needs to be bonded to four other atoms, or it can form double bonds, in which case it needs to be bonded to two other atoms, or it can form a combination of single and double bonds, as in the example of ethylene above. In ethylene, which consists of only hydrogen and carbon, the two carbons are joined to one another via a double bond. (Each carbon also has two other bonds to hydrogen.) Electrons around atoms are found in orbitals, which describe the probable location of the electrons. The orbitals are represented by some combination of “lobes”, which represent areas of high probability for the electrons to be found, and “nodes”, where the electrons are never found. Bonds form when orbital lobes overlap. The probable location of the electrons is now described by the bonding orbital that forms, which is a combination of the atomic orbitals on each atom that overlap. A single bond (which we call a sigma bond sometimes) forms when part of the sigma bonding orbitals on carbon overlap, forming a cylindrically symmetrical molecular orbital. A double bond consists of one sigma bond as well as a second bond called a pi bond. The pi bond forms when the p orbitals overlap. If you compare double and single bonds between carbon atoms, you find that the double bonds are shorter and harder to stretch than the single bonds, which makes intuitive sense since the atoms are now held together by two bonds rather than one. (Notes for this slide continues on next page.) 19 Bonding in ethylene H H C C H H Ethylene contains one double bond. A double bond is made up of a σ and a π bond. π bonding orbitals of ethylene When you take organic chemistry in the future, you will learn why in order to achieve a trigonal planar geometry in the case of ethylene, we need to have atomic orbitals pointing at the vertices of a triangle. As you know, the s orbital is spherically symmetric, and does not point in any specific direction. The three p orbitals are dumb bell shaped and are directed orthogonal (at right angles) to each other. So it seems that none of any combination of these four orbitals could allow carbon to bond to three other atoms in a trigonal planar fashion. Therefore, scientists came up with a mathematical manipulation known as hybridization that allow these orbitals to “mix” in a way to produce a similar number of hybrid orbitals. In the case of the ethylene carbons, the s and two p orbitals were mixed to form three sp2 orbitals which point at the three vertices of a triangle, and thus allow bonding to 2 hydrogen atoms and one other carbon atom as shown above. 20 Peptide bonds are planar like ethylene Ethylene contains a carbon-carbon double bond that is not free to rotate. “flat” “twist breaks one bond” The peptide bond is typically drawn as a single bond, implying that it is free to rotate. However, it is known that it can not. Why not? “flat” “twisted amide” In addition to being shorter than a single bond, the double bonds in ethylene don’t twist the way single bonds do. In other words, the other atoms attached to the carbons (hydrogens in this case) can no longer change their relative orientations by rotation because double bonds just don’t undergo bond rotations. The reason they don’t undergo bond rotations is that the pi bond has specific orientation requirements. In order for the p orbitals to overlap, they must be parallel. Rotation around sigma bonds can occur readily because it doesn’t affect the overlap of the sigma orbitals, but rotation changes the overlap between p orbitals. When the p orbitals are perpendicular, as shown above, there is no bonding at all. Because pi bonds form when it is energetically favorable to do so, you can infer that it is energetically unfavorable to break a pi bond by rotation. So: it doesn’t happen. One consequence of the need to align p orbitals to achieve and maintain overlap in a pi bond is that the atoms on the carbon atoms are all in the same plane. Thus, ethylene is flat. Okay. Now that we know about ethylene we are ready to talk about amide bonds. Amide bonds are bonds between amines and carbonyl groups (CO). We normally draw amide bonds with a single bond between the amine and the carbonyl group, which would seem to imply that the atoms are free to rotate past one another. However, amide bonds behave a lot like ethylene in that the atoms attached to the nitrogen and carbon groups are in the same plane and rotation is restricted. Furthermore, spectroscopic and crystallographic studies show that an amide bond is shorter than a typical N-C single bond. In order to understand this behavior better, we need to think about the bonding between nitrogen and the carbonyl carbon. 21 Peptide Bonds have “partial” double bonds N H R : R NH O 60% N H H N O 40% • We can draw more than one Lewis dot structure without changing the position of the atoms. • We call these structures resonance structures. • Resonance structures are drawn using DOUBLEHEADED arrows. This notation is reserved strictly for resonance! If we look at an amide bond, there is a nitrogen atom that is attached to a carbon atom, which is attached to an oxygen atom through a double bond. Earlier we explained that this kind of chemical structure is called a carbonyl. Due to the differences in electonegativity between carbon and oxygen, most of the electrons involved in the carbon-oxygen double bond spend more time around the oxygen atom, making the carbon atom slightly electro-positive. There is a tendency for the nitrogen to want to share its lone pair of electrons with the electropositive carbon atom of the carbonyl. The ability to be able to distribute the lone pair over two atoms creates a lower energy state. We call this situation resonance stabilization. Explaining in a rigorous way why delocalizing electrons lowers the energy of a molecule is complicated and requires a knowledge of quantum mechanics. For now it is sufficient to say that it is energetically favorable for electrons to be distributed over two or more atoms rather than concentrated on one atom. Resonance – electron sharing between the nitrogen and the carbonyl carbon -- gives the amide bond 40% double bond character and 60% single bond character. It is important to realize that the resonance forms shown on this slide do not exist as discrete entities. Rather, the amide bond is a combination of both of these resonance forms. You should also note that the positions of the atoms in different resonance forms are identical. Only the positions of the electrons differ. Resonance forms are thus crude representations of probable distributions of electrons. By examining the resonance form on the right, we can see that a peptide bond is somewhat like ethylene -- planar. Thus, the resonance stabilization of the amide bond restricts the shape of the polypeptide chain – the amide bonds are planar whereas the bonds on either side of the carbonyl and nitrogen are attached to tetrahedral carbons. Earlier we learned that double bonds are less free to rotate because doing so requires breaking the pi bond. Amide bonds can rotate, but it costs a lot of energy to break the partial pi bond, and so the rate of rotation is slow. The adjacent bonds have purely single bond character and are able to rotate readily. 22 A peptide bond is flat and polar H R H R N O N δ+ R' O R' δdipole (separated charge) • These resonance structures together represent the structure of a peptide bond. • One resonance form makes it easy to see that peptide bonds are flat and have strong dipoles. • Dipoles are important for the shape and function of a protein. We have talked about how an amide bond can be represented as a combination of two different resonance forms with the electrons localized on different atoms. You can see from the resonance structure on the right that electron donation from the nitrogen lone pair to the carbonyl leads to a structure in which the nitrogen has a partial positive charge and the oxygen has a partial negative charge. This charge separation means that the amide bond has a dipole, which we represent by an arrow pointing in the direction of the partial negative charge. 23 Geometric isomerism around amide bonds O O ! ! N ! O H Trans: The α-carbons are on opposite sides (strongly favored for all amino acids except one) H N ! O Cis: The α-carbons are on the same side There are actual two possible geometric isomers around the amide bond. Whether or not there are R groups attached to both alpha carbons flanking an amide bond, the peptide bond adopts what we call a “trans” conformation, where the alpha carbons are trans across the amide bond. This arrangement avoids the non-bonded repulsive interaction that exist in the corresponding cis isomer. Thus, amide bonds are flat and the preferred relative orientation of the larger substituents is “trans”. In a polypeptide chain, there are three different types of backbone bonds: the amide bond, which we have already talked about; plus the bond between the alpha carbon and the nitrogen, and the bond between the alpha carbon and the carbonyl. We will now look at the other two peptide backbone bonds. 24 Partial double bond character of the peptide bond constrains the polypeptide conformation but . . . R N H O H N O R' R'' N H H N O O R''' •‘R’ groups play a major role in the particular three dimensional structure that forms. The Calpha-nitrogen and Calpha-carbonyl bonds are single bonds and can rotate freely, at least in a short polypeptide. However, even short polypeptides have definite conformational preferences. That is, some combinations of angles around these bonds are preferred over others. The conformations that are high in energy are those that place side chains in close proximity. Thus, polypeptide chains have two kinds of “rigidity”. One kind is determined by the amide bond’s strong preference for planarity, which results from favorable orbital overlap and which leads to high barriers to rotation. The other kind is determined by the desire to avoid steric clashes between atoms in the main chain and the side chains. You can have a steric clash -- a non-bonded interaction -- when electrons involved in a bond between two atoms get too close to electrons involved in an adjacent bond. You will see in the next lecture that some of the common shapes that peptides adopt can be predicted by considering “non-bonded” interactions (i.e., steric clashes) between side chains of the various amino acids and with the polypeptide backbone. The amino acid side chains, which we are about to discuss in detail, play a big role in the conformations that polypeptide chains can adopt. This is evident because if the amide bonds were all that mattered, all polypeptides would have the same conformations. 25 Acidic Polar O H2N O CH C OH H2N CH C O OH H2N CH C CH C O OH H2N O CH C OH H2N O CH C OH H2N CH C CH2 CH2 CH2 CH OH CH2 CH2 C O OH CH2 C CH2 CH3 OH SH C NH2 O OH Glutamic Acid Glu E CH C OH H2N CH C O Glutamine Gln Q O OH H2N O H2N H2N CH C CH2 CH2 Basic CH CH3 CH CH3 CH2 H OH OH H2N H2N CH C OH OH CH2 HN Glycine Gly G Isoleucine Ile I 20 natural amino acids O H2N H2N CH C CH2 CH2 CH2 CH2 Cyclic OH CH2 HN Lysine Lys K CH C C NH NH2 Arginine Arg R O OH H2N CH C CH2 NH2 Histidine His H CH3 OH CH2 NH Methionine Met M CH C CH2 N Proline Pro P O O O S OH CH2 CH3 CH C CH C C Cysteine Cys C Serine Ser S O O OH Threonine Thr T Important for Peptide Shape CH3 Valine Val V O OH NH2 CH CH3 Alanine Ala A CH3 Leucine Leu L C O CH3 CH C O Aspargine Asn N Nonpolar O H2N H2N CH2 Aspartic Acid Asp D H2N O OH O OH H2N CH2 CH C OH CH2 HN Phenylalanine Phe F OH Tyrosine Tyr Y Tryptophan Trp W The individual amino acid building blocks all have different personalities. To understand the different personalities, we need to first classify the amino acids according to different descriptors, and then consider why there are multiple different amino acids in each category. The amino acids can be classified by size, charge, polarity, polarizablity or by the unusual conformational features that they impart on the polypeptide backbone (as we will see with glycine and proline). There are twenty natural amino acids and you should know the structures and designations (both three letter and one letter code) for each one. The natural amino acids all exist as the L-enantiomer in higher organisms but in bacterial systems D-versions of the amino acids are also observed. The nonpolar amino acids are those with aliphatic hydrocarbon side chains (one, methionine, also contains a sulfur). These amino acids are “hydrophobic” and have no dipoles, and are likely to be found in the interior of proteins (although alanine has such a small side chain that it is found both on the inside and on the outside). Even though we group these amino acids into a single category, you should be aware that they are all somewhat different. Methionine, for example, is much more flexible than isoleucine or valine, both of which have a methyl group on the side chain close to the peptide backbone (on the beta carbon - second carbon from carboxyl carbon). This methyl group leads to a greater restriction of conformations available to the peptide backbone because many more conformations would create unfavorable steric clashes. The notes continue on the next page . . . 26 Acidic Polar O H2N O CH C OH H2N CH C O OH H2N CH C CH C O OH H2N O CH C OH H2N O CH C OH H2N CH C CH2 CH2 CH2 CH OH CH2 CH2 C O OH CH2 C CH2 CH3 OH SH C NH2 O OH Glutamic Acid Glu E CH C OH H2N CH C O Glutamine Gln Q O OH H2N O H2N H2N CH C CH2 CH2 Basic CH CH3 CH CH3 CH2 H OH OH H2N H2N CH C OH OH CH2 HN Glycine Gly G Isoleucine Ile I 20 natural amino acids O H2N H2N CH C CH2 CH2 CH2 CH2 Cyclic OH CH2 HN Lysine Lys K CH C C NH NH2 Arginine Arg R O OH H2N CH C CH2 NH2 Histidine His H CH3 OH CH2 NH Methionine Met M CH C CH2 N Proline Pro P O O O S OH CH2 CH3 CH C CH C C Cysteine Cys C Serine Ser S O O OH Threonine Thr T Important for Peptide Shape CH3 Valine Val V O OH NH2 CH CH3 Alanine Ala A CH3 Leucine Leu L C O CH3 CH C O Aspargine Asn N Nonpolar O H2N H2N CH2 Aspartic Acid Asp D H2N O OH O OH H2N CH2 CH C OH CH2 HN Phenylalanine Phe F OH Tyrosine Tyr Y Tryptophan Trp W The polar amino acids are those that have polar groups, meaning they have groups with dipoles in their side chains. Remember that dipoles are the result of charge separation, which results from differences in electronegativity of bonded atoms. All the polar side chains can function as both hydrogen bond acceptors and donors because they all have available lone pairs and heteroatoms with attached hydrogens. There are other amino acids with polar side chains such as the acidic and basic amino acids, but we put these into separate categories because they contain full charges at physiological pH. (And you need enough categories so that you can remember all the side chains!) The charged side chains include the acidic amino acids, aspartic acid and glutamic acid, which are negatively charged at physiological pH, as well as the basic amino acid side chains, lysine, arginine, and histidine, which are positively charged at physiological pH. (You should be aware that cysteine has a pKa of 9.0 and so it is easily deprotonated near physiological pH. It is the only polar amino acid that is readily ionized. You should also be aware that histidine has a pKa of 6.5 and it is the only”charged” amino acid that contains a significant percentage of the neutral form at physiological pH. We will talk about both of these amino acids in more detail later.) The notes continue on the next page . . . 27 Acidic Polar O H2N O CH C OH H2N CH C O OH H2N CH C CH C O OH H2N O CH C OH H2N O CH C OH H2N CH C CH2 CH2 CH2 CH OH CH2 CH2 C O OH CH2 C CH2 CH3 OH SH C NH2 O OH Glutamic Acid Glu E CH C OH H2N CH C O Glutamine Gln Q O OH H2N O H2N H2N CH C CH2 CH2 Basic CH CH3 CH CH3 CH2 H OH Glycine Gly G OH H2N CH2 Proline Pro P OH H2N CH2 CH2 CH2 O CH C Lysine Lys K CH2 Cyclic CH C C NH NH2 Arginine Arg R O OH H2N OH CH2 HN NH2 Histidine His H CH C CH2 CH2 NH H2N CH C CH2 N CH3 20 natural amino acids OH HN Methionine Met M Isoleucine Ile I H2N CH C OH O O O S OH CH2 CH3 CH C CH C C Cysteine Cys C Serine Ser S O O OH Threonine Thr T Important for Peptide Shape CH3 Valine Val V O OH NH2 CH CH3 Alanine Ala A CH3 Leucine Leu L C O CH3 CH C O Aspargine Asn N Nonpolar O H2N H2N CH2 Aspartic Acid Asp D H2N O OH O OH H2N CH2 CH C OH CH2 HN Phenylalanine Phe F OH Tyrosine Tyr Y Tryptophan Trp W There is also a group of aromatic amino acids, phenylalanine, tyrosine, and tryptophan. These amino acids have both polar elements and hydrophobic surfaces, and they have important spectral properties. Tyrosine and tryptophan, for example, absorb UV light strongly between 274-280 nm and can be used to quantitate protein concentrations (because the amount of light that is absorbed at a particular wavelength depends on the amount of each of these two amino acid side chains in a particular protein). Tryptophan is also fluorescent, and because fluorescence is sensitive to environment, tryptophan can be used as a probe of changes in environment that occur near this amino acid. That makes it useful for studies of protein folding and protein-protein interactions. Finally, there are two amino acids that are very different from all the others with respect to their conformational properties. These amino acids are glycine and proline, which we will talk about in more detail in a minute. 28 pKa values for amino acids with ionizing side chains acid conjugate base Aspartic Acid Asp O 3.9 - 4.0 O OH O OH O 4.3 - 4.5 Glutamic Acid Glu H N H N Histidine His Cysteine Cys 6.0 - 7.0 N N H 9.0 - 9.5 S SH O OH 10.0 - 10.3 Tyrosine Tyr Lysine Lys Arginine Arg Serine Ser pKa O O NH2 NH3 NH2 N H OH 10.4 - 11.1 NH N H NH2 12.0 NH2 O 13.0 This table shows you pKa values for all of the amino acids with ionizing side chains. As you can see, some of the amino acids exist in an equilibrium between two forms. (Recall: pKa = -log(Ka), and Ka = [H+][A-]/[HA]). You have learnt from Professor Liu that when the pH of a solution is equal to the pKa of an acid, the concentration of the acid ([HA]) equals the concentration of the conjugate base ([A-]). For example, when the pH of a solution containing a protein is 4, 50% of all the aspartic acid side chains are protonated, and 50% are deprotonated at any one time. See if you can convince yourself that at physiological pH (pH 7), the conjugate base form of aspartic acid will dominate by a thousand fold over the free acid form. In general, if the pKa of an amino acid side chain is more than two log units from physiological pH, we assume that it exists almost entirely as either the charged or uncharged form (i.e., depending on what form it has at physiological pH). You will see later in the next lecture that histidine is a very special amino acid because the pKa of its side chain is very close to physiological pH. You should be aware, however, that the pKas of amino acids in the active sites of enzymes can be different from what they are in water. You will hear from Professor Liu in detail about an example where aspartic acids in the active site of a protein are found in both their charged and their uncharged forms. 29