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RNA
Life Sciences 1aLecture Notes Set 4Spring 2006Prof. Daniel Kahne
A Chemical Look at Proteins:Workhorses of the Cell
RNA
Dr Lue told you one important fact about HIV: it cannot replicate unless it caninfect and coopt the machinery of a cell. It has a genome - an informationcarrier molecule - RNA in this case. What does the virus not have? It cannot dometabolism on its own. That is to say, it can’t carry out chemicaltransformations to take one molecule and convert it into another molecule.
The molecules that carry out chemical transformations in cells are proteins andwe will spend the next three lectures learning about the basics of theseworkhorses of the cell.
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Life requires chemistry…
…and it is proteins that make the chemistry happen.
H2N
O
HO
OHNH
O
HN
NH
O
O
HO
OH
amino acidmonomer
DNA is the information carrier of the cell. Lipids divide the cell intocompartments. But proteins do the work.
Proteins have an astonishing range of different functions because they arecapable of adopting an enormous range of structures with different properties.
Proteins are made of amino acids, and before we can begin to learn about howproteins work, we need to learn about their structures.
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Lectures 6-8: The Molecular Basis of TranslationProteins: The Workhorses of Biology
a. A chemical look at proteinsi. Introduction to proteins and amino acidsii. Conformational peculiarities of peptide bondsiii. Structures and properties of the twenty natural amino acidsiv. A closer look at four special amino acids -- Gly, Pro, Cys, and His.v. Collaborations between amino acids in proteins
b. Protein structurei. The four levels of structureii. A closer look at secondary structure
c. Protein folding: i. Anfinsen’s experimentii. Thermodynamic forces involved in protein structures.iii. Thermodynamics of protein foldingiv. The Levinthal paradox (the kinetics of protein folding) v. Molecular chaperones
Alberts pp. 55-56, 74-75;McMurry Chapter 18
Lecture Readings
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 learnabout the building blocks of proteins -- the properties of the individual aminoacids that make up these biological polymers and the properties of the bondsthat join them. You will be expected to know all of the amino acids and theirpersonalities and to have some understanding of the structure of thepolypeptide backbone.
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A polymer is built of repeating monomer units.
OOPHO
OH
O
N
N
N
N
NH2
HO
Biological (Natural) Polymers
nucleotidemonomer
nucleic acid polymer
O
O
N
N
N
NN
O
PO
OO
O
PO
OO
O
N
N
O
H
O
CH3H
H
OP
O
O
O
PO
OO
O
N
N
O
H
O
CH3
• DNA is the information carrier of life; along with RNAit provides instructions to make proteins.
• Sugars are important in energy storage and haveother functions that are not well understood.
sugarmonomer
polysaccharide
OHOHO
OHOH
OH
O
HO
O
OH
OH
O
HO
O
OH
OH
O
HOOH
OH
Many of the molecules found in the cell are polymers, which are large molecules comprisedof repeating monomer units.We began by talking about the structure and function of nucleic acid polymers of DNA andRNA. These polymers are comprised of only four different building blocks each and they arehighly negatively charged. DNA has a single structure -- the double helix -- and a singlefunction that is explained by its structure. Its function is to transmit information and it doesso in two ways -- through replication (DNA copying itself, which is important in thegeneration of new cells) and through transcription (DNA making RNA, which is important forprotein synthesis).RNA is single stranded and can fold into many different kinds of structures, and it playsseveral 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 formspart of the ribosomal machine that is involved in decoding messenger RNA.Cells also make polymers of sugars. These polymers are called oligosaccharides orpolysaccharides depending on how many sugars they contain. On this slide we have onlymentioned one role for sugars -- the storage of energy -- but they play many other roles. Forexample, oligosaccharides on cell surfaces bind to circulating proteins and to other cellsurfaces, so they play roles in communication between cells. As you will see in a fewlectures, they also act as receptors for viruses, bacteria, and bacterial toxins that have evolvedto use cell surface carbohydrates to help gain entry into cells. Specifically, you will learnmore about how HIV uses a glycoprotein called gp120 to enter T-cells and macrophages. Wearen’t going to talk very much about oligosaccharides and polysaccharides in this course, inpart because their roles -- apart from energy storage -- really aren’t that well understood yet.
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H2N
O
HO
OH
NH
O
HN
NH
O
O
HO
OH
protein polymeramino acidmonomer
Proteins: Amino acid polymers
• Proteins have the most diverse shapes of the biologicalpolymers.
• Proteins are comprised of a wider variety of monomers andhas a more varied charge distribution.
• The different shapes combined with different propertiesallow proteins to have an incredible range of differentfunctions.
Proteins are the most diverse biological polymer. They are made of a widervariety of monomer units than nucleic acid polymers -- twenty monomersrather than four -- and they adopt a wider variety of shapes and display a muchwider variety of properties. Whereas all DNA molecules are polyanions,proteins may be positively or negatively charged, and many proteins containsome regions that are highly negatively charged and others that are highlypositively charged. Because they can adopt so many different shapes with somany different properties, they can carry out an incredible range of differentfunctions.
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Some important functions of proteinsEnzymaticDigestion,blood clotting,replication,transcription,translation
Structuralproteins:Hair, skin, eyes,muscle, silk
Regulatory:Coordinateevents withinand betweencells
Carriers:Respiration andmetabolism
DNA polymerase
Tubulin - cytoskeletal
Bcr-Abl - signal transductionHemoglobin - 02 carrier
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 importantprotective organ of the body, are made entirely of protein. Your fingernails, your woolhats, your silk scarves, your leather boots -- those are all made of proteins that haveevolved to withstand particular kinds of mechanical stresses. Depicted here is a proteincalled tubulin that is found inside cells and that helps form an internal scaffold, orcytoskeleton, in the cell. Unlike your hair, which is pretty set in its ways, cytoskeletalproteins like tubulin are made of individual proteins that come together (polymerize)and fall apart (depolymerize) on a rapid timescale, and as they polymerize anddepolymerize, they cause other molecules inside the cell to move in particular ways.For example, cytoskeletal proteins are involved in chromosome movement during theprocess of cell division. You will learn more about this protein when we talk aboutcancer later in the course. Thus, even within the category of “structural” proteins, thereare a wide range or different structures, functions, and behaviors. Furthermore, themechanical properties of different structural proteins are pretty interesting.
Another major function of proteins is to act as enzymes, molecules that catalyzechemical reactions in cells. All life involves chemistry -- for example, the chemistryinvolved in the breakdown of nutrients and the synthesis of macromolecules fromnutrients. None of the reactions involved in these processes occurs spontaneously inwater on a timescale that would be consistent with life. For example, the spontaneousbreakdown of a protein from food into its individual components would take hundredsto thousands of years in sterile water.
The notes continue on the next page for this slide.
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Some important functions of proteinsEnzymaticDigestion,blood clotting,replication,transcription,translation
Structuralproteins:Hair, skin, eyes,muscle, silk
Regulatory:Coordinateevents withinand betweencells
Carriers:Respiration andmetabolism
DNA polymerase
Tubulin - cytoskeletal
Bcr-Abl - signal transductionHemoglobin - 02 carrier
Enzymes accelerate the biological reactions that are necessary for life to exist. Becauseenzymes are so important in all aspects of cell growth and division, we will examinethroughout this course several enzymes and how they work. We will also focus someattention on how one might inhibit an enzyme that is responsible for deleterious effects. Wehave already seen one type of enzyme in the previous lecture, DNA polymerase. This enzymestrings together the nucleotides to make a nucleic acid polymer. Later we will look atenzymes that catalyze the breakdown of proteins.
Proteins also function as regulatory molecules that affect the activity of other enzymes. Wehave already pointed out that life depends on chemical reactions occurring on a rapidtimescale. However, it is also necessary for these chemical reactions to be preciselycoordinated. Thus, the activities of various enzymes are regulated (turned on and off) byother proteins that respond to environmental conditions. Later in this course we will look at aspecific type of regulatory protein, Bcr-Abl. This protein plays a regulatory role in normalcells, but it can malfunction and when it does, it causes a certain type of cancer. The exampleof Bcr-Abl illustrates that the proper regulation of chemical reactions in a cell is absolutelyessential for normal growth and division.
The notes continue on the next page for this slide
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Some important functions of proteinsEnzymaticDigestion,blood clotting,replication,transcription,translation
Structuralproteins:Hair, skin, eyes,muscle, silk
Regulatory:Coordinateevents withinand betweencells
Carriers:Respiration andmetabolism
DNA polymerase
Tubulin - cytoskeletal
Bcr-Abl - signal transductionHemoglobin - 02 carrier
Proteins can function as molecular transporters, delivering molecules to different parts of anorganism. Hemoglobin is a very important protein that delivers oxygen to all parts of thebody. It is found in high concentrations in red blood cells and it picks up oxygen in the lungsand releases oxygen in other parts of the body. It also carries CO2 back to the lungs forexhalation. Cholesterol, which we will talk about, is carried to different parts of the bodyfrom the gut by protein carriers.
Other proteins exist that are not as easy to classify. Ion channels, for example, which allowions to pass across cell membranes and are critically important in muscle contraction andnerve stimulation, can be thought of as enzymes (because they catalyze the transport of ionsacross lipid bilayers) or as carriers (because they deliver ions from one side of a membrane toanother).
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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. Theprotein shown is called P53, which is a tumor suppressor protein. When DNAdamage is sensed with in the cell, P53 binds to DNA and turns on the synthesisof other proteins/genes that tell the cell to stop dividing. What you can seefrom this slide is that the DNA looks as you all expect it to look -- thearchetypal double helix. You don’t even have to know what the sequence ofnucleotides is in a stretch of duplex DNA to know that the DNA will adopt adouble helical structure. However, the structures of proteins that bind to DNAare 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 sequenceis a major challenge, as you will see.
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Folded polypeptide chainAmino acid sequence
Protein 3D structure depends onprimary sequence
Question: What happens if you change asingle amino acid in the primary sequence?
NH
O
HN
NH
O
O
HN
O
NH3
OH
Lys Ser Ala Phe
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.
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Small changes at the amino acid level canaffect structure: Sickle Cell Anemia
Hemoglobin: Helical, globular structure Normal Red Blood CellsGlutamate at 6 position Normally forms tetramer
Sickle -Hemoglobin:Valine at 6 position Quaternary structure clumps together Sickle Cell Red Blood Cells
NH
O
HN
NH
O
O
OH
NHHN
N
CO NH
O
His Leu Thr Pro Glu
O
O
NH
O
HN
NH
O
O
OH
NH
HN
N
CO NH
O
His Leu Thr Pro Val
You might imagine that in a protein comprised of a hundred or more aminoacids 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 showsone example where a single amino acid change alters the activity of a proteinsignificantly. Hemoglobin, the oxygen carrier in red blood cells, contains anegatively charged residue, glutamate, at position 6 in the chain. Hemoglobinfolds into a helical, globular protein (you will learn shortly what helical means;globular simply means that it is roughly spherical -- i.e., it has approximatelythe same dimensions along all three axes), and this globular protein associatesto form a tetramer, which is the active form of the molecule. People who havesickle cell anemia have a single amino acid change, or mutation, in theirhemoglobin. The mutation involves a change from a negatively chargedglutamate to valine, which is an uncharged, non-polar amino acid. This singleamino acid change leads to a dramatic change in how the individualhemoglobin proteins interact, and you can see from the slide that the mutanthemoglobin tetramer has a significantly different shape from the normalhemoglobin tetramer. This change in the shape of the tetramer is actuallyreflected in the shape of the red blood cells, which in the case of the valinemutant have a distorted, sickle shape because the hemoglobin inside isclumped together. Sickle-shaped cells do not carry as much oxygen andtherefore deliver less oxygen to the body's tissues. The cells are also fragileand can break into pieces causing painful “crises” because they disrupt bloodflow. The sickle cell mutation is recessive and a single copy of the mutantallele somehow enables people to resist infection with malaria, which is why itwas selected for in areas where malaria is endemic.
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Pymol: A useful tool
The lab this week involves a playing around with a program called Pymol thatallows one to view proteins. The ribbon diagrams of hemoglobin on this slidewere made using Pymol. Pymol is a graphics package that reads filescontaining information about molecular structure. The files contain a list of allof the atoms in the molecule along with their coordinates in cartesian space asdetermined by X-ray crystallography or NMR spectroscopy. The coordinatesare read into the Pymol program, which displays them as a three dimensionalstructure. Because it is difficult to grasp all the details of a three dimensionalstructure containing hundreds of atoms from a single representation, the Pymolprogram allows the user to display the structure in different ways, to focus inon different parts of the molecule, to color particular segments of the moleculewith user-designated colors, and to rotate the molecule or parts thereof in orderto become familiar with features that would otherwise be obscured by theoverall complexity.
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The user can choose to display all of the atoms in the protein, all but the hydrogen atoms, oronly 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 (calledCPK) in which the relative size of each atom reflects its van der Waals radius; or as a ribbondiagram. Each of these representations can be useful. The ribbon diagram representation allowsthe 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 inmore detail in a few slides.) The CPK representation most realistically conveys how tightlypacked the interior of the protein is and reveals channels, grooves, clefts, etc. that may beimportant for function. The ball and stick representation creates the false impression that thereis a great deal of empty space throughout the protein, but it also allows the user to see details oftorsion 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 withsingle amino acids and progressing to the quaternary structure of protein. The students shouldbecome familiar with the different display options and the advantages and disadvantages of eachwith respect to the reality of the protein. If I were doing this lab, I would be interested inposition 6. Where is it? Is it surface exposed? Are there other charges in the vicinity? Is it partof a loop or a helix? (Hemoglobin has virtually no beta strands). Can I understand howchanging this amino acid could change the quaternary structure by examining the interactionsbetween the individual proteins in the normal tetramer?
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H2N
O
RH
OH
Parts of an amino acid
amino acid building block:amine (basic)
carboxylic acid (acidic)α-carbon is tetrahedral
R groups distinguish amino acids
α
All amino acids are composed of three elements: an aminogroup, a carboxylic acid, and an intervening carbon atom. Thiscarbon atom is called the alpha carbon because it is adjacent tothe first carbon of the amino acid - the carboxyl carbon - and fornineteen 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 moreabout the R groups of individual amino acids presently.
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Amino acids with ‘R’ groups are chiral
L - enantiomer D - enantiomer
• The building blocks of proteins are chiral.• When we string them together the protein is
chiral.
NH2
RH
HO
O
H2NOH
O
RH
First, however, it is important to note that the presence of an R group on thealpha carbon makes an amino acid chiral. Earlier we learned about chiralityand we learned a quick test to determine if a molecule has a chiral center: ifthe 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 Rgroup (side chain), an amine, a carboxylic acid and hydrogen. If there are twohydrogens, as in glycine, then the amino acid is not chiral. In general, aminoacids in nature are the L-enantiomer.
16
A review of chirality
O
H
O
H
L - carvone D - carvone
caraway spearmint
Although chirality may seem like an abstract concept, it is not. You aresurrounded by chiral objects and many macroscopic structures as well as mostmolecules in your body, large and small, are chiral. For example, your feet arechiral, which is why you can’t wear your left shoe on your right foot. Yourhands are chiral, which is why if you are left-handed it is hard to use mostscissors, which are designed for right-handed people simply because themajority of people happen to be right-handed. Chirality in molecules can haveas profound an effect on function as chirality in hands or feet. Two smallmolecules are shown in the above slide. Both molecules have the samenumber and type of atoms and the same bond connections, and both are calledby the chemical name of carvone. When Professor Liu talked about thesemolecules, he pointed out that carvone has one chiral center, however, and sothe two molecules are actually different. The one on the left is L-carvonewhereas the molecule on the right is D-carvone. Most of you have experiencedboth of these molecules whether you know it of not, and your experiences ofthe two are very different. The one on the left is the dominant odor moleculefound in caraway, the seed used in rye bread and swedish cookies, among otherthings. The one on the right is the dominant odor found in spearmint. No onewould confuse the smell of caraway and spearmint. The reason thesemolecules smell so different is that the receptors they bind to in your nose arechiral themselves. Just as your left shoe binds differently to your right footthan to your left foot, so do L- and D-carvone bind differently to the chiralreceptors in your nose. In other words, they may look the same to you onpaper, the same as a pair of scissors looks the same, but they fit verydifferently. The take home message is that the chirality of amino acids isimportant because chirality is a fundamental property of structure and it plays akey role in molecular interactions. Things would be very different in a racemicworld.
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Fluvastatin has two chiral centers
N
O
OR
OH OH
F
N
O
OR
OH OH
FN
O
OR
OH O
F
stereoselective reduction
racemic starting material
+
In lab, you are making fluvastatin. This drug is a member of the statin class ofdrugs that are used to lower cholesterol. Fluvastatin inhibits a liver enzyme,HMG CoA reductase, which controls cholesterol biosynthesis by controllingthe flux of an intermediate along the biosynthetic pathway. Cholesterol is animportant component of eukaryotic cell membranes and you need a certainamount, but excess cholesterol forms waxy deposits that accumulate along thelining of blood vessels and can occlude blood flow. Cholesterol is formed inthe liver and is also obtained from animal products in the diet. There is greatvariability in the amount of cholesterol that people produce, and there iscompelling evidence that keeping blood levels of cholesterol below a certainlevel prevents atherosclerosis, which is the leading cause of death in the UnitedStates.
Fluvastatin is the first fully synthetic member of the class (the other statins arederived from natural products). Fluvastatin is produced as a racemic mixtureand only one of the enantiomers has activity. The other enantiomer is inactivebut has no off-target interactions, meaning that it does not interact with anyother biological receptors. Therefore, the company that sells fluvastatin hasdecided that there is no need to spend the extra money to separate theenantiomers to provide a pure compound. Usually, however, enantiomers dohave off-target effects, making it necessary to work out chiral syntheses or toseparate the products.
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A peptide bond connects two amino acids
H2NOH
O
R
+H2N
NH
O
R
R'
O
OH + H2ON
R'
O
OH
H
H
amino acid amino acid peptide bond
A protein contains manypeptide bonds (from 40 to well
over 1000s).NH
NH
O
O
NH
OH
O
NH
O
Peptide bonds play a role in the shape of a protein.
Okay. Now that we have established that chirality is fundamental and notsimply a boring detail, we need to talk about peptide bonds. As mentionedearlier, polypeptide chains are formed of strings of amino acids in which thebonds between amino acids are amide bonds. They are constructed byconnecting the amino terminal end of one amino acid to the carbonyl ofanother amino acid. These amide bonds, or peptide bonds as they are called inthe context of polypeptides, are very important in maintaining the shape of thepeptide. Those of you who take more chemistry will learn a lot more abouthow the properties of these amide bonds impose constraints on the polypeptidechain. For now, what is important to know is that some of the bonds in thepeptide chain are free to rotate but the peptide bonds can only adopt certainconformations. They can only adopt certain conformations because of thenature of the atoms in the bond. The nature of the atoms influences the bondthat forms between the atoms, and to understand the nature of an amide bond,we need to talk a little bit about what bonding is.
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Bonding in ethylene
C C
H
H
H
H
Ethylenecontains one
double bond. Adouble bond ismade up of a σand a π bond.
π bonding orbitals of ethylene
Recall that bonds form between atoms that share electrons. You have learned thatatoms form bonds because they want to have eight valence electrons. Carbon has fourvalence electrons and so it needs to form four bonds with other atoms so that it canobtain four more electrons. Even though these electrons are shared between the twoatoms, they complete the valence shell around carbon. Carbon can form single bondswith other atoms, in which case it needs to be bonded to four other atoms, or it can formdouble bonds, in which case it needs to be bonded to two other atoms, or it can form acombination of single and double bonds, as in the example of ethylene above. Inethylene, which consists of only hydrogen and carbon, the two carbons are joined toone 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 ofthe electrons. The orbitals are represented by some combination of “lobes”, whichrepresent areas of high probability for the electrons to be found, and “nodes”, where theelectrons are never found. Bonds form when orbital lobes overlap. The probablelocation of the electrons is now described by the bonding orbital that forms, which is acombination of the atomic orbitals on each atom that overlap. A single bond (which wecall a sigma bond sometimes) forms when part of the sigma bonding orbitals on carbonoverlap, forming a cylindrically symmetrical molecular orbital. A double bond consistsof one sigma bond as well as a second bond called a pi bond. The pi bond forms whenthe 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 ratherthan one.
(Notes for this slide continues on next page.)
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Bonding in ethylene
C C
H
H
H
H
Ethylenecontains one
double bond. Adouble bond ismade up of a σand a π bond.
π bonding orbitals of ethylene
When you take organic chemistry in the future, you will learn why in order toachieve a trigonal planar geometry in the case of ethylene, we need to haveatomic orbitals pointing at the vertices of a triangle. As you know, the s orbitalis spherically symmetric, and does not point in any specific direction. Thethree p orbitals are dumb bell shaped and are directed orthogonal (at rightangles) to each other. So it seems that none of any combination of these fourorbitals could allow carbon to bond to three other atoms in a trigonal planarfashion. Therefore, scientists came up with a mathematical manipulationknown as hybridization that allow these orbitals to “mix” in a way to produce asimilar number of hybrid orbitals. In the case of the ethylene carbons, the s andtwo p orbitals were mixed to form three sp2 orbitals which point at the threevertices of a triangle, and thus allow bonding to 2 hydrogen atoms and oneother carbon atom as shown above.
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Peptide bonds are planar like ethyleneEthylene contains a carbon-carbon double bond that isnot free to rotate.
The peptide bond is typically drawn as a single bond,implying that it is free to rotate. However, it is known thatit can not. Why not?
“flat” “twist breaks one bond”
“flat” “twisted amide”
In addition to being shorter than a single bond, the double bonds in ethylene don’t twist theway single bonds do. In other words, the other atoms attached to the carbons (hydrogens inthis case) can no longer change their relative orientations by rotation because double bondsjust don’t undergo bond rotations. The reason they don’t undergo bond rotations is that thepi bond has specific orientation requirements. In order for the p orbitals to overlap, theymust be parallel. Rotation around sigma bonds can occur readily because it doesn’t affectthe 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. Becausepi bonds form when it is energetically favorable to do so, you can infer that it isenergetically unfavorable to break a pi bond by rotation. So: it doesn’t happen. Oneconsequence of the need to align p orbitals to achieve and maintain overlap in a pi bond isthat 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. Amidebonds are bonds between amines and carbonyl groups (CO). We normally draw amidebonds with a single bond between the amine and the carbonyl group, which would seem toimply that the atoms are free to rotate past one another. However, amide bonds behave alot like ethylene in that the atoms attached to the nitrogen and carbon groups are in thesame plane and rotation is restricted. Furthermore, spectroscopic and crystallographicstudies show that an amide bond is shorter than a typical N-C single bond. In order tounderstand this behavior better, we need to think about the bonding between nitrogen andthe carbonyl carbon.
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Peptide Bondshave “partial” double bonds
• We can draw more than one Lewis dot structurewithout changing the position of the atoms.
• We call these structures resonance structures.
• Resonance structures are drawn using DOUBLE-HEADED arrows. This notation is reserved strictly forresonance!
NH
NH
O
R
NH
HN
O
R
60% 40%
:If we look at an amide bond, there is a nitrogen atom that is attached to a carbon atom, which isattached to an oxygen atom through a double bond. Earlier we explained that this kind ofchemical structure is called a carbonyl. Due to the differences in electonegativity betweencarbon and oxygen, most of the electrons involved in the carbon-oxygen double bond spendmore 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 electro-positive carbon atom of the carbonyl. The ability to be able to distribute the lone pair over twoatoms creates a lower energy state. We call this situation resonance stabilization. Explaining ina rigorous way why delocalizing electrons lowers the energy of a molecule is complicated andrequires a knowledge of quantum mechanics. For now it is sufficient to say that it isenergetically favorable for electrons to be distributed over two or more atoms rather thanconcentrated on one atom. Resonance – electron sharing between the nitrogen and thecarbonyl carbon -- gives the amide bond 40% double bond character and 60% single bondcharacter. It is important to realize that the resonance forms shown on this slide do not exist asdiscrete 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 ofprobable distributions of electrons. By examining the resonance form on the right, we can seethat a peptide bond is somewhat like ethylene -- planar. Thus, the resonance stabilization of theamide bond restricts the shape of the polypeptide chain – the amide bonds are planar whereasthe bonds on either side of the carbonyl and nitrogen are attached to tetrahedral carbons. Earlierwe learned that double bonds are less free to rotate because doing so requires breaking the pibond. Amide bonds can rotate, but it costs a lot of energy to break the partial pi bond, and so therate of rotation is slow. The adjacent bonds have purely single bond character and are able torotate readily.
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N
O
R
R'
H
N
O
R
R'
H
A peptide bond is flat and polar
δ+
δ-
dipole (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 combinationof two different resonance forms with the electrons localized on differentatoms. You can see from the resonance structure on the right that electrondonation from the nitrogen lone pair to the carbonyl leads to a structure inwhich the nitrogen has a partial positive charge and the oxygen has a partialnegative charge. This charge separation means that the amide bond has adipole, which we represent by an arrow pointing in the direction of the partialnegative charge.
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Geometric isomerism around amidebonds
Trans: The α-carbonsare on opposite sides(strongly favored for allamino acids except one)
Cis: The α-carbonsare on the same side
N
O
N
O
!
!
!!
O
H
HO
There are actual two possible geometric isomers around the amide bond.Whether or not there are R groups attached to both alpha carbons flanking anamide bond, the peptide bond adopts what we call a “trans” conformation,where the alpha carbons are trans across the amide bond. This arrangementavoids the non-bonded repulsive interaction that exist in the corresponding cisisomer. Thus, amide bonds are flat and the preferred relative orientation of thelarger substituents is “trans”.
In a polypeptide chain, there are three different types of backbone bonds: theamide bond, which we have already talked about; plus the bond between thealpha carbon and the nitrogen, and the bond between the alpha carbon and thecarbonyl. We will now look at the other two peptide backbone bonds.
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NH
N
O
R
R'
O
N
R''
O
N
R'''
OH
H
H
Partial double bond character of thepeptide bond constrains the polypeptide
conformation but . . .
•‘R’ groups play a major role in the particular threedimensional structure that forms.
The Calpha-nitrogen and Calpha-carbonyl bonds are single bonds and canrotate freely, at least in a short polypeptide. However, even short polypeptideshave definite conformational preferences. That is, some combinations ofangles around these bonds are preferred over others. The conformations thatare high in energy are those that place side chains in close proximity. Thus,polypeptide chains have two kinds of “rigidity”. One kind is determined bythe amide bond’s strong preference for planarity, which results from favorableorbital overlap and which leads to high barriers to rotation. The other kind isdetermined by the desire to avoid steric clashes between atoms in the mainchain and the side chains. You can have a steric clash -- a non-bondedinteraction -- when electrons involved in a bond between two atoms get tooclose to electrons involved in an adjacent bond. You will see in the nextlecture that some of the common shapes that peptides adopt can be predictedby considering “non-bonded” interactions (i.e., steric clashes) between sidechains 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 bigrole in the conformations that polypeptide chains can adopt. This is evidentbecause if the amide bonds were all that mattered, all polypeptides would havethe same conformations.
26
Acidic
H2N CH C
CH2
OH
O
C
OH
O
Aspartic AcidAsp
D
H2N CH C
CH2
OH
O
CH2
C
OH
O
Glutamic AcidGluE
H2N CH C
CH2
OH
O
N
NH
H2N CH C
CH2
OH
O
CH2
CH2
CH2
NH2
H2N CH C
CH2
OH
O
CH2
CH2
HNC
NH2
NH
HistidineHisH
LysineLysK
ArginineArgR
Basic
H2N CH C
CH3
OH
O
AlanineAlaA
H2N CH C
CH2
OH
O
CH CH3
CH3
LeucineLeu
L
H2N CH C
CH
OH
O
CH3
CH2
CH3
IsoleucineIleI
H2N CH C
CH
OH
O
CH3
CH3
ValineValV
H2N CH C
H
OH
O
HN
C OH
O
GlycineGlyG
ProlineProP
H2N CH C
CH2
OH
O
C
NH2
O
H2N CH C
CH2
OH
O
CH2
C
NH2
O
AspargineAsn
N
GlutamineGlnQ
H2N CH C
CH2
OH
O
CH2
S
CH3
H2N CH C
CH2
OH
O
SH
H2N CH C
CH2
OH
O
OH
H2N CH C
CH
OH
O
OH
CH3
MethionineMetM
ThreonineThrT
CysteineCysC
SerineSerS
Polar
Nonpolar Important for Peptide Shape
H2N CH C
CH2
OH
O
OH
H2N CH C
CH2
OH
O
HN
H2N CH C
CH2
OH
O
PhenylalaninePhe
F
TyrosineTyrY
TryptophanTrpW
Cyclic
20 naturalamino acids
The individual amino acid building blocks all have different personalities. Tounderstand the different personalities, we need to first classify the amino acidsaccording to different descriptors, and then consider why there are multipledifferent amino acids in each category. The amino acids can be classified bysize, charge, polarity, polarizablity or by the unusual conformational featuresthat they impart on the polypeptide backbone (as we will see with glycine andproline). There are twenty natural amino acids and you should know thestructures 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 butin 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 ofproteins (although alanine has such a small side chain that it is found both onthe inside and on the outside). Even though we group these amino acids into asingle category, you should be aware that they are all somewhat different.Methionine, for example, is much more flexible than isoleucine or valine, bothof 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 groupleads to a greater restriction of conformations available to the peptidebackbone because many more conformations would create unfavorable stericclashes.The notes continue on the next page . . .
27
Acidic
H2N CH C
CH2
OH
O
C
OH
O
Aspartic AcidAsp
D
H2N CH C
CH2
OH
O
CH2
C
OH
O
Glutamic AcidGluE
H2N CH C
CH2
OH
O
N
NH
H2N CH C
CH2
OH
O
CH2
CH2
CH2
NH2
H2N CH C
CH2
OH
O
CH2
CH2
HNC
NH2
NH
HistidineHisH
LysineLysK
ArginineArgR
Basic
H2N CH C
CH3
OH
O
AlanineAlaA
H2N CH C
CH2
OH
O
CH CH3
CH3
LeucineLeu
L
H2N CH C
CH
OH
O
CH3
CH2
CH3
IsoleucineIleI
H2N CH C
CH
OH
O
CH3
CH3
ValineValV
H2N CH C
H
OH
O
HN
C OH
O
GlycineGlyG
ProlineProP
H2N CH C
CH2
OH
O
C
NH2
O
H2N CH C
CH2
OH
O
CH2
C
NH2
O
AspargineAsn
N
GlutamineGlnQ
H2N CH C
CH2
OH
O
CH2
S
CH3
H2N CH C
CH2
OH
O
SH
H2N CH C
CH2
OH
O
OH
H2N CH C
CH
OH
O
OH
CH3
MethionineMetM
ThreonineThrT
CysteineCysC
SerineSerS
Polar
Nonpolar Important for Peptide Shape
H2N CH C
CH2
OH
O
OH
H2N CH C
CH2
OH
O
HN
H2N CH C
CH2
OH
O
PhenylalaninePhe
F
TyrosineTyrY
TryptophanTrpW
Cyclic
20 naturalamino acids
The polar amino acids are those that have polar groups, meaning they havegroups with dipoles in their side chains. Remember that dipoles are the resultof charge separation, which results from differences in electronegativity ofbonded atoms. All the polar side chains can function as both hydrogen bondacceptors and donors because they all have available lone pairs andheteroatoms with attached hydrogens.
There are other amino acids with polar side chains such as the acidic and basicamino acids, but we put these into separate categories because they contain fullcharges at physiological pH. (And you need enough categories so that you canremember all the side chains!)
The charged side chains include the acidic amino acids, aspartic acid andglutamic acid, which are negatively charged at physiological pH, as well as thebasic amino acid side chains, lysine, arginine, and histidine, which arepositively charged at physiological pH. (You should be aware that cysteinehas a pKa of 9.0 and so it is easily deprotonated near physiological pH. It isthe only polar amino acid that is readily ionized. You should also be awarethat histidine has a pKa of 6.5 and it is the only”charged” amino acid thatcontains a significant percentage of the neutral form at physiological pH. Wewill talk about both of these amino acids in more detail later.)The notes continue on the next page . . .
28
Acidic
H2N CH C
CH2
OH
O
C
OH
O
Aspartic AcidAsp
D
H2N CH C
CH2
OH
O
CH2
C
OH
O
Glutamic AcidGluE
H2N CH C
CH2
OH
O
N
NH
H2N CH C
CH2
OH
O
CH2
CH2
CH2
NH2
H2N CH C
CH2
OH
O
CH2
CH2
HNC
NH2
NH
HistidineHisH
LysineLysK
ArginineArgR
Basic
H2N CH C
CH3
OH
O
AlanineAlaA
H2N CH C
CH2
OH
O
CH CH3
CH3
LeucineLeu
L
H2N CH C
CH
OH
O
CH3
CH2
CH3
IsoleucineIleI
H2N CH C
CH
OH
O
CH3
CH3
ValineValV
H2N CH C
H
OH
O
HN
C OH
O
GlycineGlyG
ProlineProP
H2N CH C
CH2
OH
O
C
NH2
O
H2N CH C
CH2
OH
O
CH2
C
NH2
O
AspargineAsn
N
GlutamineGlnQ
H2N CH C
CH2
OH
O
CH2
S
CH3
H2N CH C
CH2
OH
O
SH
H2N CH C
CH2
OH
O
OH
H2N CH C
CH
OH
O
OH
CH3
MethionineMetM
ThreonineThrT
CysteineCysC
SerineSerS
Polar
Nonpolar Important for Peptide Shape
H2N CH C
CH2
OH
O
OH
H2N CH C
CH2
OH
O
HN
H2N CH C
CH2
OH
O
PhenylalaninePhe
F
TyrosineTyrY
TryptophanTrpW
Cyclic
20 naturalamino acids
There is also a group of aromatic amino acids, phenylalanine, tyrosine, andtryptophan. These amino acids have both polar elements and hydrophobicsurfaces, and they have important spectral properties. Tyrosine andtryptophan, for example, absorb UV light strongly between 274-280 nm andcan be used to quantitate protein concentrations (because the amount of lightthat is absorbed at a particular wavelength depends on the amount of each ofthese two amino acid side chains in a particular protein). Tryptophan is alsofluorescent, and because fluorescence is sensitive to environment, tryptophancan be used as a probe of changes in environment that occur near this aminoacid. That makes it useful for studies of protein folding and protein-proteininteractions.
Finally, there are two amino acids that are very different from all the otherswith respect to their conformational properties. These amino acids are glycineand proline, which we will talk about in more detail in a minute.
29
acid conjugate base pKa
pKa values for amino acids with ionizing side chains
S
Cysteine Cys
OH
O O
Aspartic Acid Asp
Glutamic Acid Glu
NH
HN
Histidine His
Tyrosine Tyr
NH2
NH
NH
NH2
Lysine Lys
Arginine Arg
OH
O
O OH
N
HN
SH
O
NH3
NH
NH2
NH2
O
O
3.9 - 4.0
4.3 - 4.5
6.0 - 7.0
9.0 - 9.5
10.0 - 10.3
10.4 - 11.1
12.0
Serine Ser
OHO
13.0
This table shows you pKa values for all of the amino acids with ionizing sidechains. As you can see, some of the amino acids exist in an equilibriumbetween two forms. (Recall: pKa = -log(Ka), and Ka = [H+][A-]/[HA]). Youhave learnt from Professor Liu that when the pH of a solution is equal to thepKa of an acid, the concentration of the acid ([HA]) equals the concentrationof the conjugate base ([A-]). For example, when the pH of a solutioncontaining a protein is 4, 50% of all the aspartic acid side chains areprotonated, and 50% are deprotonated at any one time. See if you can convinceyourself that at physiological pH (pH 7), the conjugate base form of asparticacid will dominate by a thousand fold over the free acid form. In general, if thepKa of an amino acid side chain is more than two log units from physiologicalpH, we assume that it exists almost entirely as either the charged or unchargedform (i.e., depending on what form it has at physiological pH). You will seelater in the next lecture that histidine is a very special amino acid because thepKa 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 bedifferent from what they are in water. You will hear from Professor Liu indetail about an example where aspartic acids in the active site of a protein arefound in both their charged and their uncharged forms.
