General Biochemistry

Part 1: Proteins

Dr. Eyal Bengal

Roles of Proteins:

• Enzymatic catalysis
• Transport and storage
• Coordinated motion
• Mechanical support
• Immune protection
• Transmission of nerve impulses
• Control of growth and differentiation
 Lehninger:
Amino Pages 35-42
Acids
Alpha Carbon bound to four groups: Amino, Carboxyl, Hydrogen and R.

The Ionization state of an amino acid depends on pH in solution.

Zwitteri
on

Configuration of D and L isomers

of the amino acids.

In proteins amino acids are found

only in the L configuration.
Amino Acids Can be Classified by
.R Group
 Cysteine is interesting in two respects:
First, its side chain can ionize at moderately high pH:

ond, oxidation can occur between pairs of cysteine side chain to form disulfide

matic Amino Acids carry aromatic side chain that strongly absorb light in the
r ultraviolet region of the spectrum. This is used for analytical detection of pro
measuring absorption at 280 nm:
 Modified Amino Acids:
me amino can be chemically modified after they are assembled into proteins.
modified groups are shown in red.

Summary
 Lehninger
Titration curves Pages: 82-85
acids contain groups that can ionize. These groups can act as a
donor) or as a base (proton acceptor). The ionization of each group
at a typical pH.
H at which half of a chemical group is found in ionize state and the
her half in non ionize state.

The α COOH and α ΝΗ 3 Groups of each amino acid has their pK values,
and some of the side chains that ionize contain pK values (for example:
the amino groups of basic amino acids and the carboxylic groups of the
acidic amino acids.
mino acids have characretistic Titration curves.

Titration involves the gradual addition or removal of protons.

For example: If we add OH ions to Glycine that is found in a
solution of pH=0 and we measure at each step the pH of the
solution, we will get a typical curve that describes the change
in pH depending on the concentration of OH ions:

pK is a measure of the tendency of a group to give up a proton.

very low pH the carboxyl group is fully protonated. At a higher
his group start to lose its proton and at the at a point where half of
carboxyl group lost its proton (COOH=COO-) the pH equals the pKa
at group (pKcooh=2.34). As titration proceeds another pH point is reac
hich removal of the first proton is completed. At this point (pH=5.97
ne is present as a dipolar ion (pH=pI, or the isoelectric point)
electric point: A pH at which a molecule will not move in an electric
his point the net charge of the molecule is 0.
second stage of titration corresponds to the removal of a proton fro
up of glycine. The pH at the midpoint of this stage is 9.60, equal to th
he NH3 group. The titration is completed at a pH of about 12, at whic
amino group is at the NH2 form.

mino acids with ionizable R group:

Glutamate

Histidine
ectric charge of amino acids and peptides:

Glutamate

at is the net charge of the peptide: Lys-Gly-Ala-Glu at pH 6.0

eptide contains two positive charges and two negative charg

ore, the net charge is zero.
 Lehninger
Pages:85-86,
118-119
The Peptide Bond
 The peptide bond:

ATP

Oligopeptide:
Residues

The peptide bond is a rigid planar unit:

In proteins the alpha carbons

around the peptide bond are
always in trans state.
The carbon-nitrogen
bond has a partial
double-bond character.
he three dimensional structure of proteins:

Myoglobin is a helical protein

that carries an oxygen in the
muscles. The Heme group is
purple planar molecule that
binds the oxygen.
The protein contains alpha
helical regions and turns.

amid plans are formed around the rigid peptide bond. Rotation is allowed on
und the alpha carbons. Rotations of the C-N and C-C bonds are possible and
ponsible for the three dimensional structure of proteins.
Protein conformation (continued): Pages: 88-89
our levels of Architecture in Proteins:

Primary structure includes the covalent

bonds between amino acids. The sequence
of the amino acids and locations of
disulfide bonds.

Secondary structure refers to regular ,

recurring arrangements in space of
adjacent amino acid residues in a
polypeptide chain. Like alpha helix.

Tertiary structure refers to the spatial

relationship among all amino acids in
a polypeptide. The three dimensional
structure of a polypeptide.

Quaternary structure refers to the spatial

relationship of the polypeptides or subunits
within a protein.

A protein’s conformation is stabilized by weak interactions

#The native (active) conformation of a protein is only marginally stable. It nee
a very small energy to denature a protein. For example raising the temperatu
by several degrees.
#The stability is contributed mainly by weak chemical bonds like: Hydrogen
bonds, van der vals, hydrophobic bonds, ionic bonds. There are hundreds of s
bonds in a protein.
# The reason for this stability is a net increase in entropy of water molecules:
The hydrophobic bonds inside the molecule causes an increase in the free mo
of water molecules (increase in randomness).
 Pages: 120-122
Alpha helix

Hydrogen bond between NH and CO groups of the main chain. CO is bonded

the NH group of an amino acid found 4 residues ahead in the linear sequence
the main chain NH and CO groups are hydrogen bonded.
Each residue is related to the next one by a rotation of 100 degrees, which g
3.6 amino acids per turn of helix.

3) The R groups (side chains) are on

the outside of the helix.

4) Alpha helix can have a right or a

Left turn. In globular proteins the
helix is always with a right turn.
 mino acid sequence affects α helix stability

acent Glu or Arg residues (with charge) repel each other and destabil
ix.

hape and size of residues like Asn, Thr, Leu prevent helix formation.

ractions between amino acid side chains every three

r residues that are found adjacent (one above the other)
It could be ionic interaction between positively charged
egatively charged side groups, or hydrophobic interactions.

line cannot be within the helix. In proline the nitrogene

s part of a rigid ring and rotation about the N-C bond
possible.

entity of amino acids near the end of the helix is important. A small e
sts in each peptide bond. The four amino acids at either sides of the
rticipate in hydrogen bonds. This creates partial positive and negati
ear the amino terminal and carboxyl ends, respectively. Therefore, a
ds stabilize the amino terminal end, and basic amino acids stabilize
xylic end.
 Page: 123
The β conformation (pleated sheet)

More extended than the helix.

gen bonds between NH and CO groups in different polypeptides or w

me polypeptide that zigzag.

nt chains can run in the same direction (parallel) or in opposite direc

rallel).

roups of adjacent amino acids protrude in opposite directions of the

The β conformation (continued)
 The β turn or β bend structure. Page: 124

curs where the polypeptide chain

verses direction. This can happen
tween two anti parallel beta structures,
between two alpha helixes.
e structure is a tight turn involving
ur amino acids. The peptide group
nking the first residue bonded to the
ptide groups flanking the fourth.

ycines and prolines are often occur in

ta turns. Glycine is small and flexible.
e peptide bond involving the imino nitrogen
proline can assume a cis conformation,
at makes a tight turn.
rotein Tertiary Structure (Globular proteins)
Pages: 129-135

Globular proteins have several types of secondary

structure in the same molecule. Globular proteins include
enzymes, transport proteins, peptide
hormones, immunoglobulins. They are compact due to the
turns in between
the regular structures of alpha helix and beta sheets.
e dimensional structure of proteins is stabilized by various interactio
ain backbone and of side chains:

s or thousands of interactions stabilize the three dimensional struct

 Myoglobin:

en carrier in muscle. One polypeptide chain of 153 a.a. Binds oxygen

roup (prosthetic group with a central iron atom).

e dimensional structure was determined by X ray crystallography.

alpha helixes (eight), 70% of the main chain. The rest are turns betw
Four of the turns contain prolines.
 Myoglobin (continued)

terior of the protein consists almost entirely of non polar residues (L

e. The only polar residues are two His that play a role in binding oxy
he outer shell is rich in polar residues

erior of the protein is very compact and stabilized mainly by hydroph

ions. The stability is also contributed by van der Waals interactions
intimate contacts and by many hydrogen bonds.

Polar residues are in blue

Hydrophobic in yellow
Heme group in red

The interior of the protein

is hidden from exposure to
water. The heme group is not exposed
to solvent. This is important to its
ability to bind and to release oxygen.
 Pages: 147-153
The amino acid sequence specifies the three dimensional
structure of a protein
tian Anfinsen studied this relation in the Ribonuclease protein.

ease is a single polypeptide of 124 a.a. It has four disulfide bonds tha
eversible by reducing them with reagents like beta mercaptoethanol

Agents like urea or guanidine hydro-

chloride disrupt noncovalent
nteractions in an unknown fashion.

eatment of Ribonuclease with

eta mercaptoethanol in 8M urea
aused the loss of enzymatic activity.
he protein was denatured by this
eatment.
enatured protein freed of urea and beta mercaptoethanol by dialysis,
zymatic activity (became native).

cluded that the information needed to specify the three dimensional

nuclease is contained in its amino acid sequence.

nt result was obtained when reduced ribonuclease was reoxidized wh

. This protein gained back only 1% of its activity.

e 105 possibilities of pairing 8 cysteins (7x5x3x1). 104 wrong pairing

prtoein.

inactive (Scrambled ribonuclease could gain

k its activity after 10 hours when trace amounts
beta mercaptoethanol were added in aqueous
ution of the protein.

explanation was that the native form of ribonuclease

ermodinamically more stable than the other
ctures.
 The folding pathway

One model:
A stepwise process
Of folding:
First, secondary structures,
Then adjacent regions,
Domains etc.
econd model:
ollapse into compact states
ediated by hydrophobic interactions
etween non polar amino acids.
Molten Globule)

Free energy funnel:

Unfolded states: high degree
of conformational entropy. As
Folding proceeds, narrowing of
The funnel represents reduced
Number of conformational
Species.
Small depressions represent
semi stable intermediates.
 Assisted folding of proteins

1. Aggregation is prevented and folding is

assisted by such as Hsp70 proteins, DnaJ
and DnaK that coat the unfolded protein and
prevent aggregation.
2. Chaperonins, GroEL/GroES actively fold
proteins by repeated cycles of binding and
movements
3. Protein disulfide isomerases (PDI) help to
form the correct s-s bridges.
4. Peptidyl prolyl cis-trans isomerase (PPI)
help to return the prolines to the correct
trans (in most cases) form.
 Chaperones in
protein folding
The Hsp70 family bind regions of unfolded proteins
and preventing aggregation.
Under stress conditions causing denaturing of
proteins.
binding to and releasing from the polypeptides involving ATP hydrol
 Chperonins (GroEL/ES)
in protein folding
king care of proteins that do not fold spontaneously.
GroEL/GroES structure
Protein Quaternary Structure
that contain more than one polypeptide chain (Hemoglobin, RNA pol

ngement of protein subunits in three dimensional complexes constitu

ary structure.
actions between subunits are stabilized by the same forces that stab
tructure: multiple non covalent structures
Hemoglobin

ypeptide chains and four heme prosthetic groups (Fe is part of the gr
ains and two beta chains.

alysis was resolved in 1959 by Perutz and Kendrew. The tertiary stru
bunit is very similar to myoglobin.

are many contact points between the alpha and beta subunits mostl
phobic nature.
 aturally occurring changes in amino acids of hemoglobin are known.
00 genetic variants are known. Most of them have only minor effects
re and function of the protein. An exception is a substitution of valin
ate at position 6 of the beta subunit. This residue is on the surface o
le and produce a sticky hydrophobic spot that cause abnormal assoc
oglobin. When oxygen concentration is low subunits polymerize into
of fibers that distort the cell shape. The result is sickling of erythrocy
se of sickle cell anemia. This erythrocytes are fragile, and their bre
nemia.

Comparing the saturation curve of myoglobin to

hemoglobin shows that myoglobin has high affinity to
oxygen, and binding behave like a simple
hyperbolic curve. In contrast, the affinity of
deoxyhemoglobin to oxygen is much lower and the
curve is sigmoid. This indicates that the affinity for
the first oxygen is low, but the second, third and
fourth oxygen molecules are bound at higher affinity.
The conversion of
deoxyhemoglobin to oxyhemoglobin
require the disruption of ionic interactions
between the subunits. The conformation change
cause increase in the affinity to oxygen
(positive cooperatively).
Deoxyhemoglobin

Oxyhemoglo
bin
 Lehninger
Exploring Proteins Pages: 89-101
ntain thousands of proteins. A pure preparation of a given protein is
l before its properties, amino acid composition and sequence is dete
fy a new protein, one has to use several methods to completely purif
of a method is empirical and many methods can be used before the m
e is determined.
one does not have any idea about the size and physical properties o
Therefore we have to have a biological assay. The catalytic activity o
can be measured if we know: (1) the equation of the reaction catalyz
cedure to analyze the disappearance of the substrates or appearanc
. (3) The conditions of the reaction (salt, pH, temperature…)
of enzyme activity is defined as the amount of enzyme causing trans
omole of substrate per minute at 25 degrees.
ty, refers to the total units of an enzyme in a solution
c Activity is the number of enzyme units per milligram of protein.
purification step, the activity of a preparation is assayed and the tot
n is determined, and their ratio gives the specific activity.

Differences between activity and specific

activity. In the two jars there are the same number
of red marbles (same activity). In the second jar
however, the red marbles represent a much
higher fraction of the total (Specific activity).
teins can be characterized by electrophoresis.
ation of proteins over an electric field is known as electrophoresis.

parations carried out in gels. Polyacrylamide

e mostly used because they are chemically inert,
d form by polymerizarion of acrylamide, and the
re size can be controlled.

rophoretic method used for estimation of purity and molecular weigh

he detergent sodium dodecyl sulfate (SDS).
ds to most proteins in an amount that
rtional to the molecular weight of the
(one SDS per two amino acids). SDS
nearly all noncovalent interactions.
rcaptoethanol is added to the sample to
sulfide bonds

The proteins in the ge

can be visualized by
staining with silver or
Coomassie. If proteins
are radioactive labeled
the gel is exposed to
X ray film
(autoradiography).
 The electrophoretic mobility
of a protein on an SDS
polyacrylamide gel is related
its molecular weight.
Standard proteins with known
MW are separated near the
unknown protein. A plot of the
log MW of the marker protein
versus the relative migration
allows to determine the MW o
the unknown according to its
relative migration.

Isoelectric focusing

dient is established by allowing a mixture of low molecular weight or

bases (ampholytes) to distribute across a gel. The proteins migrate
pH that match their pI. Each protein therefore, migrate to a differen
g to its pI.
Isoelectric focusing (continued)

Separation by pI

Separation by size
uantitative separation of proteins

1) Gel Filtration chromatography:

Big molecules pass between

the beads

Small molecules pass within

the beads
2) Ion exchange chromatography:

Proteins with negative charge at a certain pHProteins with positive charge at a certain p
will bind the positively charged beads will bind the negatively charged beads

Raising salt concentration that will

compete with the positively charged
groups of the protein.

Proteins with a weak positive charge

will be the first to release from the
beads.
 3) Affinity Chromatography:

This chromatography method

takes advantage of the high
affinity and specificity of
binding of proteins to chemicals
or enzymes to their substrates
or antibodies to their ligands.

For example: The plant protein concavalin A

binds glucose with high affinity. Glucose that
s covalently linked to beads can be used to separate
he protein from a protein extract.
Determination of amino acid sequence:

y should one determine amino acid sequence of proteins?

unction of a protein depends on its amino acid sequence

changes in amino acid sequence are possible. 20-30% of proteins in

ymorphic- having amino acid variants in the population; changes tha
ect the function of a protein.
changes in amino acids can be critical to the function of a protein.
etic diseases that are caused by a single amino acid change:

2) Comparative research between proteins with

the same function
within the same organisms.
within different organisms.
 etermination of protein sequences:

3 Frederich Sanger worked out the sequence of amino acids of the

ptides of the insulin hormone (Nobel prize).

al steps in the determination of the sequence of a peptide:

1) Hydrolysis and
determination of
composition.

2) Identification of
amino terminal
esidue. Use of the
hemical reagent
FDNB or Dabsyl
hloride.

3) Edman
Degradation:
Phenylisothiocyanate
A chemical that binds
only the with terminal
amino group. Labels
he amino terminal
esidue. Under
acidic conditions, the
erminal residue is
emoved from the rest
of peptide.
) Determination of amino acid composition:

. hydrolysis: 110 degrees, 6N HCl

paration of amino acids over sulfonated polystyrene resin.

Sulfonated
polystyrene

omposition of peptide is: Ala, Arg, Asp, Gly2, Phe

ification of the releasing amino acids with ninhydrin that when react
o groups give a blue color.
dentification of the amino terminal residue of the protein.

identified by labeling it with compaunds like Fluorodinitrobenzen (F

chloride, Dansyl chloride.

Identified by its chromatographic

characteristics

3) Edman degradation
Sequentially removes one residue at a time from the am
end of the peptide.

sothiocyanate (PTH) with amino group of the peptide and under mild
ns, a cyclic derivative of the terminal amino acid is liberated and can
d by chromatographic procedures.
procedure is done automatically: Sequenators.

es of about 50-60 a.a can be sequenced in this method.

oteins can be cleaved into small peptides

A. Chemicals

Cyanogen
bromide

B. Proteolytic enzymes

Trypsin

Determination of peptide order:

mary: Determination of the composition and sequence of proteins

Determination
of a.a
composition.

N-terminal
amino acid

Cleavage
to peptides

Sequencing
of peptides

Order of
peptides.

no acid sequences can be deduced from DNA sequences.

lity to sequence DNA in a very simple way and the knowledge of the
n simplify the determination of protein sequences.