Topics 1-6: Dr. Bonnie Deroo Rev. 2013
Topic 1: The Central Dogma of Biology
The term “dogma” describes a doctrine or code of beliefs accepted as authoritative. The central dogma of
biology refers to the way that genetic information is stored and retrieved in living cells. The classic
relationship is DNA → RNA → Protein. Thus DNA functions as the information storage molecule, and
this information is "read out" into RNA molecules. Some of these RNAs are intermediates and carry the
information used to produce proteins. It is the proteins (and some RNAs) that are the "active" workers in
the cell — catalyzing reactions, moving things around, creating structures, etc. Thus the information
stored in DNA is the genotype (the sum of inheritable potential) and when this information is translated
into RNA and protein, a phenotype (the sum of observable characteristics) is produced.
The discovery of the structure of DNA by
Watson and Crick in 1953 was a milestone
for biology, leading to a molecular
understanding of how the sequence of
nucleotides making up the DNA molecule
Historically, much of our knowledge of reactions occurring in cells has come from isolating and studying
individual types of protein molecules. This resulted in the delineation of various metabolic pathways,
signaling events, structural elements, etc. and eventually to tools for manipulating DNA itself. You will
learn about these later in the course.
These methods have now allowed access to vast stores of genetic information. The Human Genome
Project (begun in 1990, with a working draft completed 10 years later) led to the development of fast and
accurate DNA sequence determination techniques. Over the past 20 years a huge quantity of sequence
information has been generated. In 1996, scientists completed the total nucleotide sequence of DNA from
yeast: about 12 million base pairs of DNA representing over 6000 genes were identified. Since then, the
chromosomal DNA of many microbes has been sequenced (now over 400 organisms). A virtually
complete sequence of human DNA was completed in 2002. The human genome consists of approximately
3.2 billion base pairs and encodes approximately 25,000 genes. However, we do not yet know the
function of many of these genes. The power to manipulate DNA sequences gives us new ways to probe
these functions and to answer questions about how cells work.
Since proteins are the active molecules in the cell and some of the key reagents in the technology behind
molecular biology, we will begin the course with a discussion of their general properties. Proteins are
made from building blocks called amino acids, which are strung together in long polymer chains. The
chains fold and coil in three dimensions to achieve a structure with a biological function. Understanding
this critical process requires an in-depth discussion of the various forces that stabilize a protein into a
given conformation (shape).
Note: Chapter 2 of the textbook, "Essential Cell Biology" (3 edition) by Alberts et al., entitled
“Chemical Components of Cells” provides a review of material covered in OAC Chemistry, Chem 1050
and Bio 1222. This chapter deals with atoms, electron shells, chemical bonding (ionic, covalent, polar
covalent, H-bonding, etc.), major chemical components of cells, water, weak acid and bases, amino acids,
etc. You are expected to understand this basic chemistry because it is important to protein structure,
which will be discussed in the next few lectures.
Proteins are macromolecules with molecular weights ranging from about 5 kilodaltons (kDa) to several
thousand kDa. A simple cell such as yeast contains about 6,000 different proteins. Many of these proteins
are biological catalysts (enzymes), which catalyze a single chemical reaction in the cell but others serve a
range of functions, as you will see (see Panel 4-1, p. 120). In fact, proteins are the most diverse class of
macromolecules, with a huge range of sizes, shapes, copy number, solubility, etc. as well as function (Fig. 4-9, p. 127), but underlying this complexity is a very simple fundamental structure. All proteins are
synthesized from combinations of some or all 20 amino acids. Basically, proteins are linear polymers of
amino acids. To understand protein structure, we have to start with the amino acids.
Topic 2: Amino Acids
Readings: p. 72-73
More properly known as alpha-amino acids, their general structure is:
Nineteen of the twenty amino acids have the same arrangement around the central
alpha-carbon: a. an amino group, b. a carboxyl group, c. a hydrogen, and d. an R
group (called the "side chain") which differs for each amino acid. Recall that when
4 different groups are attached to a carbon atom, stereoisomers are possible.
Therefore, amino acids are designated as D- and L-amino acids. Not all the amino
acids have D- and L- isomers, but for those that do, only the L- forms are
incorporated into proteins.
An important property of amino acids is their net charge, derived from the ionization of weakly acidic or
basic groups. The net charge on a group changes as the hydrogen ion concentration (pH) changes because
of the association of hydrogen ions with the groups.
Recall that for a weak acid:
RCOOH <=> H + RCOO
This equilibrium is characterized by a constant Ka for each
This equilibrium shows that lowering t-e pH (increasing H ) will drive the equilibrium to the left, as
written, resulting in decreased RCOO and increased RCOOH. In other words, the fraction of the
molecules that are ionized will decrease. Thus the net charge on the group will decrease. For basic groups
such as amino groups, the effect is the opposite. That is, the fraction of the molecules that are ionized
increases with decreasing pH.
As we will see, proteins have multiple ionizable groups, so their net charge depends on the sum of the
charges from all groups.
Categories of Amino Acids
Every amino acid has a 3-letter abbreviation and a one-letter code. I don’t expect you to
memorize all the codes, but you will have to memorize a few (see below). Amino acids are
classified according to the properties of their side chains.
1. The largest group has non-polar side chains:
a. Some have only H or CH in side chains: glycine (gly, G), alanine (ala, A), valine (val, V),
leucine (leu, L) and isoleucine (ile, I)
b. Some contain a sulfur atom: cysteine (cys, C), methionine (met, M)
c. Two are aromatic: phenylalanine (phe, F), tryptophan (trp, W)
d. Finally, the one odd one is actually an imino acid meaning that its immediate synthetic
precursor was an imino acid (ie. it contained an imine, or C=NH group): proline (pro, P)
2. Charged side chains: basic or acidic
a. contain carboxyl groups: glutamic acid (glu, E), aspartic acid (asp, D)
b. contain basic groups: lysine (lys, K), arginine (arg, R), histidine (his, H)
3. Uncharged polar side chains: a. contain hydroxyl groups: Serine (ser, S), threonine (thr, T), tyrosine (tyr, Y)
b. contain amide groups: glutamine (gln, Q), asparagine (asn, N)
Note: You will be expected to know the structure of the following 8 amino acids: glycine (G), alanine
(A), cysteine (C), serine (S), proline (P), lysine (K), aspartic acid (D), phenylalanine (F).
While only these 20 amino acids are used to make proteins, other amino acids can be found in proteins
due to modifications that happen after the protein is made. This allows the introduction of specialized
groups for specific purposes, and often changes the properties of the protein. A common example is
phosphorylation of the hydroxyl-containing amino acids ser, thr and tyr. These phosphoamino acids have
a phosphate esterified on the hydroxyl group of their side chain. You will come across a variety of other
modifications as you study Biochemistry.
NOTE: Topic review questions are available on the Biochem2280_2288 Forum. Please join in the
discussion of this material.
Topic 3: Protein Structure
Readings: p. 121-140
Proteins are the largest and most varied class of biological molecules, and they show the greatest
variety of structures. Many have intricate three-dimensional folding patterns that result in a
compact form, but others do not fold up at all ("natively unstructured proteins") and exist in
random conformations. The function of proteins depends on their structure, and defining the
structure of individual proteins is a large part of modern Biochemistry and Molecular Biology.
To understand how proteins fold, we will start with the basics of structure, and progress through
to structures of increasing complexity.
To make a protein, amino acids are connected together by a type of amide bond called a "peptide
bond". This bond is formed between the alpha amino group of one amino acid and the carboxyl
group of another in a condensation reaction. When two amino acids join, the result is called a
dipeptide, three gives a tripeptide, etc. Multiple amino acids result in a polypeptide (often
shortened to "peptide"). Because water is lost in the course of creating the peptide bond,
individual amino acids are referred to as "amino acid residues" once they are incorporated.
Another property of peptides is polarity: the two ends are different. One end has a free amino
group (called the "N-terminal") and the other has a free carboxyl group ("C-terminal").
In the natural course of making a protein, polypeptides are elongated by the addition of amino
acids to the C-terminal end of the growing chain. Conventionally, peptides are written N-
terminal first; therefore gly-ser is not the same as ser-gly or GS is not the same as SG. The
connection gives rise to a repeating pattern of "NCC-NCC-NCC…" atoms along the length of
the molecule. This is referred to as the "backbone" of the peptide. If stretched out, the side chains
of the individual residues project outwards from this backbone.
The peptide bond is written as a single bond, but it actually has
some characteristics of a double bond because of the resonance
between the C-O and C-N bonds:
This means that the six atoms involved are coplanar, and that
there is not free rotation around the C–N axis. This constrains the
flexibility of the chain and prevents some folding patterns.
Primary Structure of Proteins It is convenient to discuss protein structure in terms of four levels (primary to quaternary) of increasing
complexity. Primary structure is simply the sequence of residues making up the protein. Thus primary
structure involves only the covalent bonds linking residues together.
The minimum size of a protein is defined as about 50 residues; smaller chains are referred to simply as
peptides. So the primary structure of a small protein would consist of a sequence of 50 or so residues.
Even such small proteins contain hundreds of atoms and have molecular weights of over 5000 Daltons
(Da). There is no theoretical maximum size, but the largest protein so far discovered has about 30,000
residues. Since the average molecular weight of a residue is about 110 Da, that single chain has a
molecular weight of over 3 million Daltons.
This level of structure describes the local folding pattern of the polypeptide backbone and is
stabilized by hydrogen bonds between N-H and C=O groups. Various types of secondary
structure have been discovered, but by far the most common are the orderly repeating forms
known as the α helix and the β sheet.
An α helix, as the name implies, is a helical arrangement of a single polypeptide chain, like a
coiled spring (see Fig. 4-10, p. 130). In this conformation, the carbonyl and N-H groups are
oriented parallel to the axis. Each carbonyl is linked by a hydrogen bond to the N-H of a residue
located 4 residues further on in the sequence within the same chain. All C=O and N-H groups are
involved in hydrogen bonds, making a fairly rigid cylinder. The
alpha helix has precise dimensions: 3.6 residues per turn, 0.54
nm per turn. The side chains project outward and contact any
solvent, producing a structure something like a bottle brush or a
round hair brush. An example of a protein with many α helical
structures is the keratin that makes up human hair.
The structure of a β sheet is very different from the structure of an α
helix. In a β sheet, the polypeptide chain folds back on itself so that
polypeptide strands lie side by side, and are held together by hydrogen
bonds (see Fig. 4-10, p. 130), forming a very rigid structure. Again,
the polypeptide N-H and C=O groups form hydrogen bonds to
stabilize the structure, but unlike the α helix, these bonds are formed
between neighbouring polypeptide (β) strands. Generally the primary
structure folds back on itself in either a parallel or antiparallel
arrangement, producing a parallel or antiparallel β sheet (see Fig. 4-
14, p. 132). In this arrangement, side chains project alternately upward
and downward from the sheet (Fig. 4-10D, p. 130). The major constituent of silk (silk fibroin) consists
mainly of layers of β sheet stacked on top of each another.
Other types of secondary structure. While the α helix and β sheet are by far the most common
types of structure, many others are possible. These include various loops, helices and irregular
conformations. A single polypeptide chain may have different regions that take on different
secondary structures. In fact, many proteins have a mixture of α helices, β sheets, and other
types of folding patterns to form various overall shapes (Fig. 4-16, p. 133).
What determines whether a particular part of a sequence will fold into one or the other of these
structures? A major determinant is the interactions between side chains of the residues in the
polypeptide. Several factors come into play: steric hindrance between nearby large side chains,
charge repulsion between nearby similarly-charged side chains, and the presence of proline. Proline contains a ring that constrains bond angles so that it will not fit exactly into an α helix or
β sheet. Further, there is no H on one peptide bond when proline is present, so a hydrogen bond
cannot form. Another major factor is the presence of other chemical groups that interact with
each other. This contributes to the next level of protein structure, the tertiary structure.
This level of structure describes how regions of secondary
structure fold together - that is, the 3D arrangement of a
polypeptide chain, including α helices, β sheets, and any other
loops and folds. Tertiary structure results from interactions
between side chains, or between side chains and the
polypeptide backbone, which are often distant in sequence.
Every protein has a particular pattern of folding and these can
be quite complex (e.g. Panel 4-2, p. 128, right).
Whereas secondary structure is stabilized by H-bonding, all four “weak” forces contribute to
tertiary structure (p. 122). Usually, the most important force is hydrophobic interaction (or
hydrophobic bonds). Polypeptide chains generally contain both hydrophobic and hydrophilic
residues. Much like detergent micelles, proteins are most stable when their hydrophobic parts are
buried, while hydrophilic parts are on the surface, exposed to water. Thus, more hydrophobic
residues such as trp are often surrounded by other parts of the protein, excluding water, while
charged residues such as asp are more often on the surface (Fig. 4-5, p. 124).
Other forces that contribute to tertiary structure are ionic bonds between side chains, hydrogen bonds, and
van der Waals forces (Fig. 4-4, p. 123). These bonds are far weaker than covalent bonds, and it takes
multiple interactions to stabilize a structure.
There is one covalent bond that is also involved in tertiary structure, and that is the disulfide bond that can
form between cysteine residues. This bond is important only in non-cytoplasmic proteins since there are
enzyme systems present in the cytoplasm to remove disulfide bonds.
Visualization of protein structures Because the 3D structures of proteins involve thousands of atoms in
complex arrangements, various ways of depicting them so they are understood visually have been
developed, each emphasizing a different property of the protein. Panel 4-2 (p. 128) illustrates a few of
these different ways, from a simple backbone to a space-filling representation. Software tools have been
written to depict proteins in many different ways, and have become essential to understanding protein
structure and function.
Structural Domains of Proteins
Protein structure can also be described by a level of organization that is distinct from the ones we
have just discussed. This organizational unit is the protein “domain,” and the concept of domains
is extremely important for understanding tertiary structure. A domain is a distinct region
(sequence of amino acids) of a protein, while a structural domain is an independently-folded part
of a protein that folds into a stable structure. A protein may have many domains, or consist only
of a single domain. Larger proteins generally consist of connected structural domains. Domains
are often separated by a loosely folded region and may create clefts between them. Structural
domains are often functional units as well. Examples of structural domains are illustrated in Fig.
4-16, p. 133 and 4-17, p134.
Quaternary Structure Some proteins are composed of more than one polypeptide chain. In such proteins, quaternary structure
refers to the number and arrangement of the individual polypeptide chains. Each polypeptide is referred to
as a subunit of the protein. The same forces and bonds that create tertiary structure also hold subunits
together in a stable complex to form the complete protein.
Individual chains may be identical, somewhat similar, or totally different. As examples, CAP
protein (Fig. 4-19, p. 136) is a dimer with two identical subunits, whereas hemoglobin (Fig. 4-20,
p. 136) is a tetramer containing two pairs of non-identical (but similar) subunits. It has 2 α
subunits and 2 β subunits. Secreted proteins often have subunits that are held together by
disulfide bonds. Examples include tetrameric antibody molecules that commonly have two larger
subunits and two smaller subunits (“heavy chains” and “light chains”) connected by disulfide
bonds and noncovalent forces (Panel 4-3, p. 144, top left).
In some proteins, intertwined α helices hold subunits together; these are called coiled-coils (Fig.
4-13, p. 132). This structure is stabilized by a hydrophobic surface on each α helix that is created
by a heptameric repeat pattern of hydrophilic/hydrophobic residues. The sequence of the protein
can be represented as “abcdefgabcdefgabcdefg...” with positions “a” and “d” filled with
hydrophobic residues such as A, V, L etc. Each α helix has a hydrophobic surface that therefore
matches the other. When the two helices coil around each other, those surfaces come together,
burying the hydrophobic side chains and forming a stable structure. An example of such a
protein is myosin, the motor protein found in muscle that allows contraction.
How and why do proteins naturally form secondary, tertiary and quaternary structures? This
question is a very active area of research and is certainly not completely understood. A folded,
biologically-active protein is considered to be in its “native” state, which is generally thought to
be the conformation with least free energy.
Proteins can be unfolded or “denatured” by treatment with solvents that disrupt weak bonds.
Thus organic solvents that disrupt hydrophobic interactions, high concentrations of urea or
guanidine that interfere with H-bonding, extreme pH or even high temperatures, will all cause
proteins to unfold. Denatured proteins have a random, flexible conformation and usually lack
biological activity. Because of exposed hydrophobic groups, they often aggregate and
precipitate. This is what happens when you fry an egg.
If the denaturing condition is removed, some proteins will re-fold and regain activity. This
process is called “renaturation.” Therefore, all the information necessary for folding is present in
the primary structure (sequence) of the protein. During renaturation, the polypeptide chain is
thought to fold up into a loose globule by hydrophobic effects, after which small regions of
secondary structure form into especially favorable sequences. These sequences then interact with
each other to stabilize intermediate structures before the final conformation is attained.
Many proteins have great difficulty renaturing, and proteins that assist other proteins to fold are
called “molecular chaperones.” They are thought to act by reversibly masking exposed
hydrophobic regions to prevent aggregation during the multi-step folding process. Proteins that
must cross membranes (eg. mitochondrial proteins) must stay unfolded until they reach their
destination, and molecular chaperones may protect and assist during this process.
Protein families/Types of proteins
Proteins are classified in a number of ways, according to structure, function, location and/or
properties. For example, many proteins combine tightly with other substances such as carbohydrates (“glycoproteins”), lipids (“lipoproteins”), or metal ions (“metalloproteins”). The
diversity of proteins that form from the 20 amino acids is greatly increased by associations such
as these. Proteins that are tightly bound to membranes ar