Amino Acids, Peptides and Proteins 2018

BIOCHEMISTRY I
Amino Acids, Peptides

and Proteins
Amino acids, peptides and proteins
• Proteins
• Proteins are biological macromolecules.
• Macromolecules have large (molecular weights ranging from
about 104 to 1010) include carbohydrates, nucleic acids, and lipids
in addition to proteins.
• Proteins are made up of building blocks call amino acids.
• There are 20 amino acids that a commonly found in proteins;
these are called protein amino acids or canonical amino acids.
• Proteins play a wide range of roles in organisms.
• Some examples are shown on the next slide.
2
• Some functions of proteins.
• Enzymes—Biocatalysts which facilitate thousands of chemical
reactions in organisms.
• Membrane Proteins—Proteins and lipids form the major structural
components of cell membrane.
• Hormones—Signaling molecules that play an important role in the
regulation of metabolic reactions.
• Transport Protein—Examples are hemoglobin and albumins that carry
oxygen and other chemicals in blood.
• Structural Proteins—Examples are keratins in skin and hair, collagens in
connective tissues, actin and myosin in muscles, microtubules in many
cells.
• Antibodies—They bind to specific foreign particles, such as viruses and
bacteria, to help protect the body. 3
• The protein amino acids have
common structural features.
• They are all α-amino acids, i.e.
they have both a carboxyl group
and an amino group bonded to the
α−carbon atom.
• The amino acids differ in their R
groups which vary in the size,
structure and charge.
4
• All the protein amino acids are
chiral except one, glycine.
• They are L-amino acids.
• D-amino acids are found in cells
but they are not incorporated into
proteins at the ribosome.
• Two canonical amino acids have
two chiral centers, isoleucine and
threonine (more later).
5
• Amino acids are often organized by R group.
• Although there are several ways they might be grouped, the text
places them in five groups depending on their R
substituents:
• Nonpolar, aliphatic (7)
• Aromatic (3)
• Polar, uncharged (5)
• Positively charged (3)
• Negatively charged (2)
• See next few slides. 6
7
8
9
• Tryptophan and tyrosine
absorb UV light with a peak of
absorbance near 280 nm.
• So a common way of both
identifying the presence of
proteins and of quantifying
them is to measure the
absorbance of biological
extracts at 280 nm.
10
Amino acids, peptides
and proteins
• Proteins are made of amino acids
linked to each other by peptide bonds.
• In this case the α amino group of one
amino acid reacts with the α carboxyl
group of a second amino acid and are
called polypeptides. peptide bonds
• All of the α amino and an a carboxyl
groups can be linked in the chain
except one α amino group will be free
called the amino-terminal end or N-
terminus and one α carboxyl with be
free called the carboxyl-terminal end polypeptide
or C-terminus. 11
• Proteins are synthesized (i.e.
amino acids are linked together)
by ribosomes (more on this
later).
• After the proteins are
synthesized additional covalent
links can form.
• One type is the reactions of
cysteine side chains with each
other to form disulfide bridges.
• This can be important in
stabilizing the three dimensional The pronunciation can Is pronounced,
structures of proteins as well as be a bit confusing as it
linking two or more separate sis-teen but also
polypeptide chains together. is sis-tay-een but may sis-tyne.
sound like sis-teen. 12
• In addition to
disulfide bond
formation, other
modifications can
be made to the
amino acids in
proteins.
• However, note that
these result from
modifications to
the 20 canonical
amino acids.
13
• Some other covalent
modifications to
protein amino acids
are reversible.
• For example,
phosphorylation and
dephosphorylation
of hydroxyl amino
acids are important
in regulation of both
enzymes and gene
expression.
14
• Many other non-
protein amino acids
are found in cells.
• These amino acids
have other
important
biological functions
such as being
neurotransmitters,
antibiotics,
metabolic
intermediates, etc.
15
• Amino acids are given both a 3 letter abbreviation (e.g. Gly for glycine)
and a 1 letter symbol (e.g. G for glycine).
• The reason for the single letter code is because in the very early days of
bioinformatics, the very fastest computers were, in fact, pretty slow.
• Dr. Margaret Oakley Dayhoff, a founder of the field of bioinformatics,
shortened the code from three letters to a single letter to reduce the
size of the data files needed to describe the sequence of amino acids in
a protein.
• Since there are 20 amino acids commonly found in proteins and 26
letters in the alphabet, most of the letters are used.
• Dr. Dayhoff tried to make the 1 letter code easy to remember.
• See link :
http://www.biology.arizona.edu/biochemistry/problem_sets/aa/Dayhof
f.html
16
• The molecular weights for the common amino acids range
from 75 for the smallest, glycine, to 204 for the largest,
tryptophan. The average for all 20 is 136.
• The tables on the following two slides shows some of the
properties and characteristics of the common protein amino
acids.
• These include three and one letter abbreviations, the
molecular weights, the pKa values for the α amino and α
carboxyl groups as well as the R groups that have extra acid-
base functions, the values for the pI or isoelectric points of
the amino acids, the hydropathy index and finally the relative
abundance of the individual amino acids in proteins.
• So let’s review some of the characteristics. 17
18
19
• Amino acids can act as Brønsted acids and
bases, both as free amino acids and within
proteins.
• The titration curve for the simple amino acid
glycine is shown here.
• Remember from the earlier discussion, the
midpoint of the titration can be used to
determine the pKa values.
• As we can see there are two buffering
regions: the first, estimating pK1 to be 2.34, is
for the carboxyl group and the second,
estimating pK2 as 9.60, is for the amino group.
20
• In cells at neutral pH (around pH 7) the amino acids exists as

zwitterions. Why?
• Recall from the previous slide, for glycine the pK1 is 2.34 (pKa for
the α carboxyl group) and pK2 is 9.60 (pKa for the α amino group) is
9.60.
• See calculations on next slide.
21
pK1 for α carboxyl group.
pK 2 for α amino group.
-
[-COO ] [-NH 2 ]
pH = pK1 + log pH = pK 2 + log
[-COOH] [-NH 3+ ]
[-COO - ] [-NH 2 ]
7 = 2.34 + log 7 = 9.60 + log
[-COOH] [-NH 3+ ]
[-COO - ] [-NH 2 ] • At pH 7, the overall charge is -
4.66 = log
[-COOH]
- 2.60 = log
[-NH 3+ ]
0.999978 + 0.9975 = -0.00248
and very slightly negative.
Taking antilogs of both [-NH 3+ ]
sides of the equation :
2.60 = log
[-NH 2 ] • So at pH 7, the carboxyl is
almost all in the –COOH- form
[-COO - ] Taking antilogs of both
10 4.66
= = 45709 and the amino group is almost
[-COOH] sides of the equation :
all in the –NH3+ form or
Let [-COOH] = 1, then [-COO - ] = 45709
10 2.60
=
[-NH 3+ ]
= 398 zwitterion form shown below.
Fraction in base or - COO form, - [-NH 2 ]
- [-COO - ] Let [-NH 2 ] = 1, then [-NH 3+ ] = 398

[-COO ] =
[-COOH] + [-COO - ] Fraction in acid or - H 3+ form,
45709 + [-NH 3+ ]
= = 0.999978 [NH ] =
[NH 3+ ] + [NH 2 ]
3
1 + 45709
So the charge is -0.999978 398
= = +0.9975
1 + 398 22 22
• Do you recall the pKa estimate for carboxyl group of acetic acid
from before? It was 4.76.
• So why is the pKa for the glycine carboxyl group lower than for
acetic acid carboxyl?
• The perturbed pKa of glycine is caused by nearby positively
charged amino group on the α-carbon atom; the opposite
charges lower the pKa by stabilizing the zwitterion.
• Another factor is that in acetic acid the carboxyl is bound to a
methyl group which is an electron donating group. In glycine an
H atom is replaced by amino group which is electron
withdrawing. So the pKa of the carboxyl in glycine is lower than
the carboxyl of acetic acid.
• See next slide.
23
24
• What about the amino group?
• As can be seen on the previous slide the pKa for the amino group
of methylamine is 10.6 but the pKa for the amino group of glycine
is 9.6.
• The pKa of the amino group in glycine is perturbed downward
relative to the average pKa of an amino group.
• So why is the pKa for the glycine amino group lower than for the
methylamine amino group?
• This effect is due partly to the electronegative oxygen atoms in
the carboxyl groups, which tend to pull electrons toward them,
increasing the tendency of the amino group to give up a proton.
• Hence, the α-amino group has a pKa that is lower than that of an
aliphatic amine such as methylamine.
• See previous slide. 25
• What is the isolectric point or pI?
• For chemicals like amino acids and proteins that have
multiple functional groups capable of taking both positive
and negative charges, the pI is the pH where the overall
charge on the molecule is zero.
• At the isolectric point a molecule like glycine has both
positive and negative charges. But the plus and minus
charges are equal and balance each other so the net
charge is zero.
• For an amino acid like glycine with one carboxyl group
and one amino group, the pI can be estimated by
averaging the pK1 and pK2 values (see below).
• pI =
( pK1 + pK 2 )
=
( 2.34 + 9.60 ) = 5.97
2 2 26
• What about amino acids with R-groups having extra carboxyl or amino
groups?
• Titration curves for
glutamic acid with an
extra carboxyl group
and for histidine which
has an extra amino
group are shown here.
• Note the extra pKR (the
pKa for the function in
the R group) in each
case.
27
• Some additional information about amino acids with R-groups
having extra carboxyl or amino groups.
• Can we estimate the pI for the amino acids with extra acid/base
groups? Yes.
• We don’t average all 3 pKs, we average the pKs for the groups
that are similar, i.e. for glutamic acid the pKs for the 2 carboxyl
groups. From the previous slide pK1 = 2.19, and pK2 = 9.67 and
pKR = 4.25.
 pI =
( pK1 + pK R )
=
( 2.19 + 4.25 )
= 3.22
2 2
• Why does this make sense (see next slide)?
28
• We estimated the pI to
be 3.22.
• Let’s use the
Henderson-Hasselbalch
equation to estimate
the charges on the
various carboxyl and
amino groups, when
pK1 = 2.19, pK2 = 9.67
and pKR = 4.25 and the Glutamic acid
pH = 3.22.
29
Amino acids, peptides and proteins 30

pK1 for α carboxyl group. pK R for γ carboxyl group.
[-NH 2 ]
[-COO - ] -
[-COO ] pH = pK 2 + log
pH = pK1 + log pH = pK R + log [-NH 3+ ]
[-COOH] [-COOH] [-NH 2 ]
-
[-COO ] - 3.22 = 9.67 + log
3.22 = 2.19 + log 3.22 = 4.25 + log
[-COO ] [-NH 3+ ]
[-COOH] [-COOH] [-NH 2 ]
[-COO ] - - 6.45 = log
[-NH 3+ ]
-
1.03 = log [-COO ]
-1.03 = log
[-COOH] [-COOH] [-NH 3+ ]
Taking antilogs of both Taking antilogs of both 6.45 = log
[-NH 2 ]
sides of the equation : sides of the equation : Taking antilogs of both
-
sides of the equation :
-
[-COO ] [-COO ]
101.03 = = 10.71 10-1.03 = = 0.0933
[-COOH] [-COOH] [-NH 3+ ]
6.45
10 =
Let [-COOH] = 1, then [-COO - ] = 10.71 Let [-COOH] = 1, then [-COO - ] = 0.0933 [-NH 2 ]
Fraction in base or - COO - form, Fraction in base or - COO - form, Let [-NH 2 ] = 1, then [-NH 3+ ] = 2818383
- [-COO - ] - [-COO - ] Fraction in acid or - H 3+ form,
[-COO ] = [COO ] =
[-COOH] + [-COO - ] [-COOH] + [-COO - ] [-NH 3+ ]
+
10.71 0.0933 [NH ] =
[NH 3+ ] + [NH 2 ]
3
= = 0.915 = = 0.085
1 + 10.71 1 + 0.0933 2818383
So the charge is -0.915 So the charge is -0.085 = = + 0.9999996 ≈ + 1
1 + 2818383
So the charge is essentially + 1
• Thus, the overall charge on the α
carboxyl group is -0.915.
• The overall charge on the γ
carboxyl group is -0.085.
• The overall charge on the two
carboxyl groups is -0.915 + -0.085
= -1.
• The charge on the α amino group
is ≈ +1.
• So finally the net charge on
glutamic acid at pH 3.22 is zero. Glutamic acid
31
• Proteins and peptides vary widely in
size and function.
• Both peptides and proteins are comprised
of amino acids linked together by peptide
bonds as noted earlier.
• Although there is not clear distinction
between them, polypeptides with
molecular weights above 10,000 daltons
are usually characterized as proteins while
those smaller are designated as peptides.
• The average size of a human protein is
about 53,000 molecular weight and has
480 amino acids. 32
• Some functional proteins may be composed of a single
polypeptide chain.
• By contrast, a functional protein may be made of several
polypeptide chains linked together.
• The peptide chains are often linked noncovalently.
• The peptide chains can be linked covalently via disulfide
bridges.
• A multiple subunit protein my be composed of more than
one identical subunits or different subunits.
• Some protein contain other chemical species in addition to
amino acids; these are called conjugated proteins. 33
• A tightly bound non-protein
species is called a prosthetic
group. It may be linked
covalently or non-covalently.
• Some examples are the
following:
• Small organic chemicals.
• Lipids (liporpoteins).
• Carbohydrates
(glycoproteins).
• Metal ions (metalloproteins).
34
• Proteins can be separated and purified.
• The first step in isolating and purification of a protein is breaking
the cell to release the cellular components.
• Some cell disruption methods are listed below.
• Autolysis, freezing and thawing.
• Air, vacuum and freeze drying.
• Pressure disruption (French press, etc).
• Tissue slicing (razor blades, bacon cutter, microtome, etc).
• Grinding in a mortar and pestle.
• Hand and mechanical homogenizers.
• Homogenization with blender.
• Sonic disruptors (sonicators, high-frequency oscillators)
• Use of detergents Tween 20, Triton X-100, SDS, etc).
35
• Proteins may be separated by a number of methods
based on their characteristics; some example are the
following:
• Solubility Characteristics
• Charge Characteristics
• Size
• Biological or Chemical Affinity
• Organelle Association
36
• Proteins and biomolecules may be separated from
each other by chromatography.
• All chromatographic systems have some common features:
• mobile phase
• stationary phase
• component mixture to be separated
• What is the basis of separation of chemical species by
chromatography?
• Chemical species partition between a mobile phase and a
stationary phase.
• What is the basis for partitioning?
• It can be related to solubility, charge, size, biological
affinity and others. 37
• One example is differences in solubility.
• Consider two liquid phases in contact that do not
mix, such as hexane (organic) and water
(aqueous).
• If a third component, a solute, dissolves to some
extent in both phases, the Partition Coefficient (K)
is the ratio of the amounts of the solute material
that is dissolved in the two phases at equilibrium.
• The partition coefficient is a constant for a given
pair of solvents with a particular solute.
• By convention when relating aqueous/organic systems, the organic
phase concentration is in the numerator and the aqueous phase
concentration in the denominator.
• In most cases the upper phase is an organic solvent and the lower
phase is water (but not always as it depends on the relative
densities). 38
• In chromatography, differences in the partition coefficient
between the mobile phase and the stationary phase can
provide the foundation for separation (although the basis of
partitioning is not always due to solubility as we will see
below).
• What type chemical species would you expect to “partition”
mainly into the organic phase? More hydrophobic species.
• What chemical species would partition into the aqueous
phase? More hydrophilic chemicals.
• So back to chromatography.
39
• The stationary phase might be cellulose
which, similar to paper, has a tightly bound Thin-Layer Chromatography: A
Two-Component Mixture
layer of water covering it.
• The mobile phase might be acetone: water. solvent front
• If a chemical mixture is spotted on a cellulose component B Less polar!
thin layer plate and developed (i.e. the

solvent front
component B
solvent is allowed to migrate up the plate), component A

component A More polar!
what might we expect? origin mixture

solvent front
origin origin
• A neutral (less polar component B) compound Increasing Development Time
should be more soluble in acetone than water

and thus travel further while a more polar
(component B) should be more soluble in
water bound to the cellulose layer and
therefore not move as far (see figure). 40
• There are several types of chromatography (for example):
• Paper
• Thin-layer
• Gas
• Column (liquid).
• In all cases there is a mobile phase, a stationary phase, and a
third component or a component mixture that partitions
between the two phases.
• Obviously, the mobile and stationary phases may vary.
• What are the typical mobile and stationary phases for the
following types of chromatography?
• Paper
• Mobile phase is organic solvent; stationary phase is water.
41
• Thin-layer
• Could be cellulose which is similar to paper but
depends on thin-layer material and solvent system.
• Gas
• Mobile phase is carrier gas; stationary phase can vary.
Examples?
• Liquid
• Mobile phase and stationary phase can vary.
• Normal phase chromatography?
• Reverse phase chromatography?
42
• Proteins may be separated by different types of column (liquid)
chromatography which is based on different characteristics of the
protein (see next few slides).
• One is ion exchange chromatography which exploits differences in charges
on various proteins.
• In ion-exchange chromatography a column is packed with insoluble beads
with charged functional groups.
• If the beads have a negative charge what type chemicals will bind to the
matrix? Positively charged species or cations!
• So separation of positive species from each other by binding to a negatively
charged matrix is called cation-exchange chromatography.
• Note the charged groups on the column are negatively charged and are
thus anionic, but the species that bind are cations, hence cation-exchange.
43
• If the beads have a positive charge what type chemicals
will bind to the matrix? Negatively charged species or
anions! This is called anion exchange chromatography.
• Recall there is no charge on zwiterionic chemical species
when the pH is equal to the pI.
• But what is the charge when the pH is above or below the
pI?
• Recall for glycine the pI was calculated to be 5.97 so lets
consider the cases where the pH is below the pI and where
the pH is above the pI.
• See next slides. 44
• Let’s pK1 for α carboxyl group. • The overall
[-NH 2 ]
consider pH = pK + log
charge is -0.977
-
[-COO ]
[-NH 3+ ]
2
pH = pK + log
the case
1
[-COOH]
3.97 = 9.60 + log
[-NH ] plus 0.999998 =
2
where the
-
+0.023.
[-COO ] [-NH ] +
3.97 = 2.34 + log 3
pH is two
[-COOH] [-NH ]
• So when the pH
2
- - 5.63 = log +
units below
[-COO ] [-NH ]
1.62 = log 3
the pI for
[-COOH]
5.63 = log
[-NH ] +
3 is below the pI
Taking antilogs of both
glycine. sides of the equation :
[-NH ] 2 the zwitterionic
Taking antilogs of both
molecule will
• Since the pI sides of the equation :
-
[-COO ]
have an overall
1.63
10 = = 42.6
is 5.97 what [-COOH] +
[-NH ]
positive charge
5.63 3
10 = = 426579
would be
-
Let [-COOH] = 1, then [-COO ] = 42.6 [-NH ]
Let [-NH ] = 1, then [-NH ] = 426579 and should bind
2
+
the charge
-
Fraction in base or - COO form, 2 3
to the negatively
+
on the
-
- [-COO ] Fraction in acid or - H form, 3
[-COO ] =
[-COOH] + [-COO ] - +
charged cation-
protein at + [-NH ] 3
[NH ] =
exchange
3 +
42.6 [NH ] + [NH ]
pH 3.97? = = 0.977 3 2
column. 45
1 + 42.6 426579
= 0.999998
So the charge is -0.977 1 + 426579
Amino acids, peptides and proteins • The overall charge is
-0.999998 plus 0.977 =
pK for α carboxyl group.
• Now let’s 1 pK for α amino group.
2 -0.023.
consider
-
[-COO ] [-NH ]
• So when the pH is
2
pH = pK + log pH = pK + log
the case
1 2 +
[-COOH] [-NH ]
above the pI the
3
where the
-
[-COO ] [-NH ]
zwitterionic molecule
2
7.97 = 2.34 + log 7.97 = 9.60 + log +
pH is two [-COOH] [-NH ] 3
will have an overall

units [-COO ] - [-NH ] 2
1 .63 = log
negative charge and
5.63 = log [-NH ] +
above the [-COOH] 3
pI. Taking antilogs of both 1.63 = log

[-NH ] +
3 should not bind to the
sides of the equation : [-NH ] 2 negatively charged
• Since the [-COO ]- Taking antilogs of both
cation-exchange
pI is 5.97
5.63
10 = = sides of the equation :
[-COOH] column.
what -
Let [-COOH] = 1, then [-COO ] = 426579 10 = [-N
1.63 H ]+
3
• So one could bind

= 42.6
would be Fraction in base or - COO form, - [-NH ] 2
the charge - Let [-NH ] = 1, then [-NH ] = 42.6 glycine to a cation

2
+
3
[-COO ]
on the [-COO ] = -
[-COOH] + [-COO ]
Fraction in acid or - H form,
-
+
3 exchange column at pH
protein at 426579 [NH ] +
=
[-NH ] +
3 3.97 and then raise the
pH 7.97? =
1 + 426579
= 0.999998 3 +
[NH ] + [NH ]
3 2 pH to 5.97 to release or
So the charge is -0.999998 =
42.6
= +0.977 elute glycine from the
1 + 42.6
column. 46
• Ion Exchange Chromatography
• Depending on the isoelectric point of the protein and the
pH, a protein (or other zwiterionic molecule) should bind
to the column if it is positively charged.
• It will be positively charged when the pH is below the pI.
• Again how can we elute (wash off) the bound species?
• As noted on the previous page, one way is to raise the pH
above the pI leading to a negative overall charge on the
molecule; in this case it will no longer bind to the
negatively charged beads and can be eluted.
• There is another way to elute the molecule: increase the salt concentration. A salt like KCl
with have both positive K+ and Cl- ions in solution. The K+ will compete with the positively
charged protein for binding sites on the beads and therefore can displace and in turn
elute the positively charged protein.
• What about anion exchange column and proteins? 47
and proteins
• Size-exclusion chromatography (also called
molecular-exclusion or gel-filtration
chromatography).
• This methods exploits differences in size of various
proteins.
• The column is packed with insoluble beads that
have pores of specific sizes.
• What is the basis for interacting with the column?
• The smaller molecules can enter the pores and are
retarded in moving through the matrix.
• By contrast the larger molecules are too big to enter
the pores and thus simply pass around the beads
and are not slowed down as much as they pass
around the beads. 48
• Size-exclusion chromatography can be used to
separate proteins of different sizes from each
other and also to estimate the molecular weight
of proteins and other molecules.
• The elution volume of proteins of known sizes
can be plotted as elution volume (i.e. volume of
solvent needed to wash the protein through the
column) versus the log of their molecular
weights.
• This gives a linear plot over a range of molecular
weights and can be used to generate a standard
curve. log Mr = 4.05
taking antilogs of both sides
• Thus by comparison to the standard curve, the Mr = 104.05 ≈ 11,200
elution volume of an unknown protein may be
used to estimate its molecular weight.
49
• Still another method is affinity
chromatography
• This methods exploits the fact than many proteins have
evolved to bind specific molecules.
• An example would be a hormone (chemicals like
hormones that bind to proteins are often called ligands)
and the protein could be a receptor for the hormone.
• The column is packed with insoluble beads to which a
ligand is covalently attached.
• So as the protein passes over the column, it specifically binds, while other proteins pass
trough.
• How is the bound protein eluted?
• One way is by adding free ligand. Why would this work?
• Another way might be adding a denaturing agent. Why would this work? 50
• Dialysis is a method for partially purifying
proteins.
• Dialysis is typically used to remove small molecules, such
as sugars, amino acids, or salts, from the protein
preparation rather than for separating proteins from each
other (although protein separation could occur under the
appropriate conditions).
• This method usually employs a membrane with pores too
small for a protein to pass through but large enough for
small molecules and ions to pass.
• A sample of proteins and small chemicals in placed in a
membrane bag and immersed in a buffer (usually for
several hours or more).
• The small chemicals diffuse into the medium while the
protein is retained within the dialysis bag.
• Note from the diagram, after dialysis the concentration of
small chemical species in the bag is greatly reduced. 51
• Proteins may also be separated and analyzed by electrophoresis?
• Electrically charged biomolecules placed in an electric field will move towards
the electrode of opposite charge.
• The velocity of movement of a charged molecule in an electric field may be
described in part by the following equation:
Ez
V =
f
 Where: V = velocity of movement of a charged molecule in an electric field
E = electric field strength
z = charge on the molecule
f = frictional coefficient 52
𝐸𝐸𝐸𝐸
V=
𝑓𝑓
• Examining the equation above suggests molecules will move faster as z (the
net charge on the molecule) and E (the electric field strength) increase and
slower as f (the frictional coefficient) goes up.
• In general f is related to the size (molecular mass) and shape of the particle,
the porosity of the supporting matrix, and a few other factors.
• Larger molecules and those with less charge move slower while smaller one
and those with greater charge migrate faster.
• Also if the supporting matrix (typically a gel) is more porous the molecules
migrate faster and if it is less porous they move slower.
• Since most proteins differ is size and charge they may be separated. 53
• The migration not only reflect size but also charge to mass ratio; also, depending on
the isoelectric points different proteins may migrate toward the negative pole and
some toward the positive pole.
• Actually, the most common type of protein electrophoresis is SDS-PAGE (sodium
dodecyl sulfate-polyacrylamide gel electrophoresis). Na
+
• SDS is a common household detergent.

• Interestingly, SDS binds to every protein in roughly the same proportion, which is
about one molecule for every two amino acid residues, and also denatures proteins.
• What do we mean by denatures?
• SDS carries with it a negative charge, and
the cumulative negative charge renders
the intrinsic net charge of the protein
insignificant (i.e. SDS coats proteins with
negative charges) so all move to plus pole. 54
• Therefore, every protein will have
approximately the same charge to mass - Polyacrylamide
ratio, which will cause all proteins to gel matrix.
migrate towards the positive pole with a
velocity that is mainly dependent on their Denatured
sizes. proteins
separated by
• The smaller molecules migrate faster than size.
+
larger molecules (see figure here).
• This technique is not commonly used to
purify proteins for activity assays because,
as noted above, SDS denatures proteins.
• SDS-PAGE is commonly used to analyze the
purity of proteins or estimate protein
molecular weights. 55
• The migration of proteins in a
SDS gel is nearly linearly related
to the log of the molecular
weight.
• So protein standards of known
molecular weight may be
separated along with unknown
proteins.
• After staining to visualize the
proteins (see previous slide) a
plot of migration distance versus
log molecular weight may be
used to estimate the size of
unknown proteins. 56
• Larger numbers of proteins may be
analyzed simultaneously by two-
dimensional electrophoresis?
• In this case a protein mixture is separated at first by
isoelectric focusing.
• With this method separation is based on
differences in isoelectric points of proteins.
• Samples are applied to gel strips which chemical
attached to a matrix that establishes a pH gradient.
• When a current is applied, proteins will migrate
toward the positive or negative pole until reaching
the point in the strip where the pH is equal to the
isoelectric point when there is no charge and thus
no further migration.
57
and proteins
• After isoelectric focusing the gel
strip can be placed on top of an SDS
gel and the samples separated in
the second dimension by denaturing
electrophoresis.
• So proteins are separated based on
two different characters: first by pI
and second by molecular weight.
• This allows hundreds of proteins to
be analyzed at the same time.
58
• Structure of proteins?
• Primary structure—the amino
acid sequences.
• Second structure—stable
arrangement of recurring
structural patterns like α helices
or β sheets.
• Tertiary structure—all aspects of
the three-dimensional folding of a
polypeptides.
• Quaternary structure—
arrangement of two or more
polypeptides into a final three-
dimensional structure. 59
• The three-dimensional structure and function of proteins
ultimately depends on the primary structure, i.e. the amino acid
sequence (more on this later).
• How are primary structures determined?
• There are several ways including the following:
• Traditional Edman degradation method.
• Mass spectrometer methods.
• Deduced sequences from DNA gene sequences.
• DNA sequencing is much simpler and less expensive than
protein sequencing.
• If you know the DNA sequence for a gene the protein
sequence can be deduced. 60
• The traditional method for protein sequencing is
called Edman degradation.
• It was first used in 1950 and automated in 1967.
• The first step is to break any disulfide bridges that link
polypeptide chains in a protein that has quaternary
structure so that they may be separated into
individual polypeptides.
• Why is this important?
61
• There are two common ways to
break disulfides and prevent
reformation of disulfide bridges.
• One is oxidation with performic
acid to convert the cysteine sulfur
atoms to cysteic acid.
• A second is to reduce the disulfide
with DTT or another reducing
agent (e.g. β-mercaptoethanol,
abbreviated BME) to form the
thiols followed by
carboxylmethylation by
iodoacetate.
• Why is the last step needed? 62
• Identification of the N-terminal
amino acid.
• The reagent 1-Fluoro-2,4-
dinitrobenzene (FDNB) reacts with
the α-amino group to form a 2,4-
Dinitrophenyl derivative of the
polypeptide.
• Heating in 6 N HCL leads to
hydrolysis of the protein freeing all
amino acids in the protein.
• Only one, the N-terminal amino
acid, will be a 2,4-dinitrophenyl
derivative which may be
determined by chromatographic
method.
• See diagram here.
63
• The Edman degradation method is
illustrated here.
• The reagent phenylisothiocyanate (PITC) reacts
with the α-amino group to form a
phenylthiocarbamyl derivative of the
polypeptide.
• Under acidic conditions an anilinothiozalone
derivate of the N-terminal amino acid is liberated
which in turn is converted to the
phenylthiohydantoin derivative of the free amino
acid which is analyzed to determine its identity.
• That generates a polypeptide with a new N-terminus which can then be purified and subjected
to a new round of reactions leading to the identification of the second amino acid.
• Additional rounds allow determination of still more amino acids in the protein sequence.
• A snag with the Edman method is that normally only about 40 amino acids may be sequenced
at a time so other steps are needed.
• See next slide. 64
and proteins
• Traditional sequencing process using
Edman method.
• Isolate and purify the protein.
• Determine the amino acids distribution.
Why?
• Reduce disulfide bridges.
• Cleave the protein into smaller fragments?
This is done by using enzymes such as
trypsin and chymotrypsin or chemical
reagents like cyanogen bromide which all
cleave the protein chain at particular sites.
Why?
• Purify the fragments.
• Carry out Edman degradation.
• Knowing the N-terminal assemble the
fragments using sequence overlap derived
from the different smaller peptides. 65
• In the last decade or two mass spectrometry has become a
second method for protein sequencing.
• Mass spectrometry had been used to analyze smaller
molecules for many years but proteins were too large and
not volatile enough for past method.
• Two more recent techniques have enabled mass
spectroscopy to be used with proteins.
• One method is matrix-assisted laser desorption-ionization
mass spectrometry (MALDI MS) and a second is electrospray
ionization mass spectrometry (ESI MS).
66
• In mass spectrometry the samples may
be ionized by several different methods.
• In ESI MS a protein solution is dispersed
through a needle in a high-voltage field
and passes into a vacuum chamber
where the droplets evaporate leaving a
charge on the protein which then enters
the mass spectrometer where mass to
charge ratio (m/z) is determined.
• The spectrum is a family of peaks, with
each successive one corresponding to a
charged species increased by 1 in both
mass and charge.
• From this a computer generated
transformation gives the mass of the
protein. 67
• OK so basic mass spectrometry can provide information about
the molecular weights of proteins. But what about sequencing?
• As in conventional sequencing the protein is purified and then broken
into shorter fragments.
• Sequencing is then carried out by a method called tandem MS or
MS/MS.
• In this case two mass spectrometers are linked together.
• The sample is injected and sorted by the first MS so that only one of the
fragments emerges.
• Between the two mass spectrometers is a collision cell where the
fragment is broken into smaller pieces.
• Next, a second MS measures the m/z rations. 68
• Each successive peak has one fewer amino acid than the peak before, so
the difference in the mass from peak to peak identifies the amino acid
sequence. Leu and Ile have the same mass so require additional methods.
69
and proteins
• Peptides and proteins can
be synthesized.
• Methods for chemical synthesis
were developed in the early
1960s by Bruce Merrifield who
won the Nobel Prize in Chemistry
in 1984 for his work.
• The key innovation was to
synthesize the peptide on a
insoluble polymer that allowed
easy purification from one round
of synthesis to the next.
• A summary of the steps is shown
here.
70

Amino Acids, Peptides and Proteins 2018

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Amino Acids, Peptides and Proteins 2018

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BIOCHEMISTRY I

Amino Acids, Peptides

• In cells at neutral pH (around pH 7) the amino acids exists as

- [-COO - ] Let [-NH 2 ] = 1, then [-NH 3+ ] = 398

pK 2 for α amino group.

• If a chemical mixture is spotted on a cellulose component B Less polar!

thin layer plate and developed (i.e. the

solvent is allowed to migrate up the plate), component A

what might we expect? origin mixture

• A neutral (less polar component B) compound Increasing Development Time

should be more soluble in acetone than water

pH is two [-COOH] [-NH ] 3

will have an overall

above the [-COOH] 3

pI. Taking antilogs of both 1.63 = log

• So one could bind

the charge - Let [-NH ] = 1, then [-NH ] = 42.6 glycine to a cation

• SDS is a common household detergent.

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