Any questions on the material from the previous lecture?
A Little More Nomenclature for Amino Acids
In the previous lecture, we looked at the general structure of an amino acid, including the α carbon,
the α-amino and α-carboxylate groups and the side chain. When referring to the atoms of the side chain, greek letters
are traditionally used, following in sequence from the α carbon:
Side Chain Hydrophobicity
An important chemical property of amino acid side chains is their degree of hydrophobicity
Some amino acids, such as the aliphatics and aromatics, can have side chains that are quite hydrophobic.
Others, such the basics and the acid/amides, have side chains that are very hydrophilic. Others have side
chains with a hydropathy that is between these two extremes
The hydropathy of an amino acid is a measure of its relative hydrophobicity or hydrophilicity
There are a number of ways to measure hydropathy. One common scale, shown in Table 3.1 in the text,
measures hydropathy values in terms of the change in free energy of an amino acid residue as it moves
from a hydrophobic to a hydrophilic environment
Hydropathy Scale
Section 3.3: Non-standard Amino Acids
Although the 20 'standard' are the ones used in proteins, there are many other types of amino acids
produced in the cell for other purposes
Some of these appear as intermediates in various metabolic reactions. One example is homocysteine,
used in the biosynthesis of methionine and cysteine (Chapter 17). Another is ornithine, an intermediate
for producing arginine and eliminating excess nitrogen in the urea cycle
Other non-standard amino acids are used as components for signaling molecules such as neurotransmitters
and hormones. Examples include the modification of glutamate to GABA and conversion of histidine
to the regulatory molecule histamine:
Many of these modifications come about through decarboxylation and deamination reactions,
which involve removal of the carboxyl and amino groups of an amino acid, respectively
Non-standard Amino Acids in Proteins
The 20 canonical amino acids are the ones used for building proteins. After being assembled
into the polypeptide chain, some amino acid residues are subsequently modified
One example of such a post-translational modification is the conversion of proline
into hydroxyproline in collagen:
The addition of a hydroxyl group allows for more hydrogen bonding between the chains of a
collagen fiber, increasing its strength (Section 4.11)
The 21st and 22nd Amino Acids
In most proteins, just the 20 standard amino acids are incorporated during translation,
as specified by one of the 64 codon sequences of the genetic code.
However, in the late 1980's, a startling discovery was made that
selenocysteine (Sec) was sometimes used in translation. Selenocysteine is a variant of cysteine, with
a selenium atom in place of the sulfur:
Because of this, selenocysteine is considered the 21st amino acid, although it is encoded differently than the
others, relying on some contextual mRNA information and an alternate interpretation of the UGA 'stop' codon
Even more recently, a 22nd amino acid was identified, pyrrolysine, a modified form of lysine.
Its occurrence appears to be much rarer than selenocysteine, as it is only found in certain species of archaebacteria
Section 3.4: Ionization of Amino Acids
In previous lectures, we have seen how acids and bases can become ionized by a change in pH. For individual
amino acids, we can observe such ionizations at least twice: once for the α-carboxyl group and once for the
α-amino group
The pKa value of an acid determines the pH value at which ionization occurs. For an amino acid such as alanine,
we can identify the two pKa values from its titration curve:
Amino Acids are Zwitterions at Physiological pH
The titration curve shows that alanine has both a negative and a positive charge in the pH
interval from 6.8 to 7.4 (physiological pH). This dual charge means that it is a zwitterion
(dipolar ion) in this range
At the lower pKa (pH 2.4), the net charge is +0.5. At the upper pKa (pH 9.9), the net charge is -0.5.
Consequently, at the halfway point of pH 6.15 the net charge will be 0. This pH value is
called the isoelectric point (pI) for alanine
Amino Acids with Two Ionizable Groups
The diagram below summarizes the ionization state of amino acids with only
two ionizable groups (the α-amino and α-carboxyl groups):
As shown in Table 3.2 in the text, all of the 20 standard amino acids have α-carboxyl pKa values
less than 3.0 and α-amino pKa values less than 11
Consequently, the 13 amino acids without an ionizable side chain will be positively charged
below pH 3, largely neutral around pH 7 and negatively charged above pH 11
Amino Acid pKa Values
Table 3.2 summarizes the pKa values for the α-carboxyl, α-amino and side chain groups
of the 20 standard amino acids
Only 7 of the 20 amino acids have ionizable side chains
They are:
The two acids
The three basics
Cysteine and Tyrosine
Ionization of Side Chains
The titration curve for histidine shows three inflection points: with pKa values of 1.8, 6.0 and 9.3.
Here, the side chain contributes an additional positively-charged species below pH 6, giving the amino
acid a net positive charge at pH values between pKa1 and pKa2. Consequently,
the isoelectric point for histidine occurs between the second and third pKa values:
pI = (pK2 + pK3) / 2 = (6.0 + 9.3) / 2 = 7.65
Section 3.5: Peptide Bonds
The linkage between the amino acid residues in a protein is called a peptide bond. This
bond results from a condensation reaction between the α-carboxyl group of one amino acid and the
α-amino group of another:
The resulting peptide has a charged amino group called the N-terminus at one end and
a charged carboxyl group or C-terminus at the other end
This gives a direction to the resulting polypeptide chain, from N terminus to C terminus. This
direction also reflects the temporal order in which the amino acids are added to a growing chain by the ribosome
Protein Sequence and Direction
The direction of a linear protein chain, from N to C terminus, is shown below:
By convention, the sequence of amino acids (its primary structure) is also written from N to C
For example, the peptide with sequence 'LEGGY' represents:
A living cell can sometimes be likened to a bewildering, complex and invisible molecular jungle.
We can try to make some sense of it by isolating and analyzing individual components (ultimately, we must
also synthesize what we find to produce a deeper understanding)
Purification techniques are used to isolate a protein of interest from the complex stew of starting materials.
A variety of techniques exist, and different ones are usually applied in a sequential way
that moves from an initial crude separation to progressively more refined fractions
Some commonly used techniques include Salting Out, Dialysis,
Chromatography and Ultracentrifugation
Salting Out
What is 'salting out'?
Salting out is a technique for precipitating a protein from solution by increasing the salt concentration
The solubility of a protein depends on a variety of factors specific to that protein, including surface
charge, size and polarization. Consequently, different proteins will precipitate at different salt concentrations
Ammonium sulfate, (NH4)2SO4, is commonly used for salting out because of
its high solubility
Some examples:
fibrinogen precipitates at 0.8M ammonium sulfate
serum albumin precipitates at 2.4M ammonium sulfate
Dialysis
What is dialysis?
Dialysis is a method for removing small molecules from a solution by diffusion through a semipermeable membrane
Large molecules such as proteins are retained within the dialysis bag while small molecules and ions pass through:
Gel-Filtration Chromatography
Gel-filtration chromatography separates molecules on the basis of size
It involves passing a solution through a column (long tube) that is packed with beads of a hydrated insoluble
material such as dextran, agarose or polyacrylamide
The smaller molecules in the solution spend more time interacting with the beads while larger molecules pass by
Consequently, the larger molecules leave the column first
Ion-Exchange Chromatography
Ion-exchange chromatography separates molecules based on differences in net charge
Proteins with a net positive charge will be retained on negatively-charged columns such as carboxymethyl-cellulose
Proteins with a net negative charge will be retained on positively-charged columns such as diethylaminoethyl-cellulose
Affinity Chromatography
Affinity chromatography can be used to purify proteins that have a high specific affinity to some chemical group
For example, concanavalin A, shown above, is a sugar-binding protein with a high affinity for glucose.
A column with glucose residues attached will retain concanavalin A, which can then be later released by adding a
solution of free glucose
This technique is not always be applicable because the specific high-affinity groups may not always be obtainable
However, for certain categories of proteins such as transcription factors, the high affinity group can be
a specific sequence of DNA, which is easily prepared
HPLC
HPLC is a very powerful technique for separation and purification of biomolecules.
What is HPLC?
HPLC is an abbreviation for High Pressure Liquid Chromatography
This technique employs high-pressure pumps that can force solutions through columns to produce
better separation of purification fractions
It can be applied to all of the previous chromatography techniques mentioned above
Ultracentrifugation
Ultricentrifugation creates huge forces on biomolecules in order separate them by mass and shape.
An ultracentrifuge is typically the size of a washing machine, with rotational speeds of 70,000 - 100,000 rpm
The movement of a particle in the centrifuge is quantified by the Svedberg equation, which takes into
account the size, shape and density of a particle.
In general, larger particles move faster than smaller ones, more compact particles move faster than more extended ones,
and denser particles move faster than less dense ones. These properties, in conjunction with the
measured speed, are combined to give a Svedberg coefficient for a particular biomolecule
The Svedberg coefficient is often used when referring to different kinds of biomolecules that vary in size.
For example, the ribosomal subunits are referred to by their Svedberg coefficents of 30S for the small subunit,
50S for the large subunit and 70S for the complex
Questions
Questions about the material covered today?
References
A Forgotten Debate: Is Selenocysteine the 21st Amino Acid?, Robert Longtin,
Journal of the National Cancer Institute, Vol 96, No. 7, April 7, 2004
Brief review of selenocysteine as the 21st amino acid
Aminoacyl-tRNAs: setting the limits of the genetic code, Ibba and Soll,
Genes and Development, 18:731-738, 2004
Discusses selenocysteine and pyrrolysine in the larger context of aminoacyl-tRNA structure and function
