Download A Chemical Look at Proteins: Workhorses of the Cell

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.
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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.
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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.
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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).
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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. 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.
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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