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Why should we study proteins?

Thousands of different proteins are present in the human body with
thousands of different functions. Their structure determines the function.
Proteomics have a renaissance for the determination of protein structure,
function and interactions.


The function of a protein can be structural or dynamic.

Structural proteins provide matrix for bone and connective tissue, giving
structure and form to the human organism. E.g. collagens, elastin, keratin,
histone proteins forming chromatin with DNA.

Dynamic proteins provide transport, metabolic control, contraction, and

chemical transformation.
Important classes of dynamic proteins:
o Enzymes to catalyze chemical reactions, e.g. trypsin, glucokinase,
thrombin, etc.
o Transport proteins e.g. hemoglobin, albumin, transferrin, ion
channels, etc.
o Protective proteins to fight bacterial or viral infection, e.g.
immunoglobulins, interferon; or blood coagulation proteins, e.g.
o Movement proteins muscle proteins, myosin and actin
o Regulatory proteins in metabolism:
Hormones to regulate metabolism, e.g. insulin, glucagon, thyrotopin
Receptors to transduce the signal
Regulatory proteins to bind to enzymes for changing their activity
o Gene regulatory proteins to control transcription and translation


Proteins are polymers of -amino acids
There are 20 common amino acids, with at least one codon for each in the
genetic code.
Derived amino acids are post-translationally modified and have specific
roles in proteins.
o desmosine and isodesmosine in elastin
o hydroxyproline and hydroxylysine in collagen
o -carboxyglutamate in prothrombin
General structure of the common amino acids
Amino acids contain a central alpha carbon to which a carboxylic (COOH),
an amino group (NH2) and a hydrogen atom are attached as well as a
specific R group (side chain).
The alpha amino group can be protonated (positively charged ammonium
ion) or unprotonated (neutral NH2 form).
The carboxylic acid group can be unprotonated (negatively charged
carboxylate ion) or protonated (neutral COOH form).

Exception is proline,
which is heterocyclic,
i.e. the amino group
and the side chain
form a ring.

Figure 1.1. Lippincott. General Structure of the Common Amino Acids

Amino acids are asymmetric

The common amino acids have four different substituents around the carbon, except for glycine that has 2 hydrogens (the R-group is also a H).
Consequently, they are optically active, can rotate polarized light. They
can exist in two forms, the L and D optical isomers.
Only L amino acids are found in human proteins.
o L means the amino acid has its amino group to the left.

Figure 3.8, Devlin. Absolute Configuration of an Amino Acid

Side chains define chemical nature and structures of different amino acids
Major groups of amino acids according to their side chains:
1. Monoamino, monocarboxylic acids: they are neutral at pH 7.
Unsubstituted or alkyl amino acids: have alkyl group (hydrocarbon) side
chains and include glycine, alanine, valine, leucine and isoleucine.
(note: Gly has an H+ for an R group)
Proline is unique since it incorporates the alpha amino group in a five-member ring.
(note: proline causes a rigid structure when built into proteins)

Aromatic amino acids: have aromatic groups as side chains and include
phenylalanine, tyrosine and tryptophan.
Sulfur containing amino acids: are cysteine, with a mercapto, and
methionine, with a thioether group.
Two hydroxyl group (alcohol)-containing amino acids are serine and
Carboxamide group containing amino acids: have amides in place of the
OH of the carboxylic groups
o Glutamine is amidinated glutamic acid.
o Asparagine is amidinated aspartate acid.

2. Monoamino, dicarboxylic acids: contain a carboxylic group in the side chain.

They are negatively charged at pH 7.
Aspartate contains a carboxylic group separated from the alpha carbon by
one methylene group.
In glutamate there are two methylene groups.
3. Diamino, monocarboxylic acids: include a nitrogen-containing group in the
side chain. They are positively charged at pH 7.
Lysine side chain has a primary amino group.
Arginine side chain contains a guanidino group.
Histidine side chain contains a five-membered heterocyclic structure, the
imidazole ring.

FYI only Figure 3.3. Devlin: Structures of the Common Amino Acids. (Charged forms at pH 7.0.)

Abbreviations for the Amino Acids

Note: 1-letter abbreviations of amino acids are for your reference only.

Amino Acid

Three Letter One Letter

Asparagine or aspartate
Glutamine or glutamate



Amino acids are polymerized into peptides and proteins

The reaction of the primary amino group with the primary carboxyl group
of another amino acid yields a dipeptide. The chemical bond formed
between the amino acids is the peptide bond.
The peptide bond has a 50% double bond character.
This means that the electrons are shared among the O
the C and the N;
As a result, rotation does not occur around the peptide
bond (between the C (carbonyl carbon) and the
nitrogen of the peptide bond).
This results in the C=O and the N-H groups lying in a
common plane, while the bonds next to them, between
the C and C, can rotate. This phenomenon becomes
important in protein folding.

Figure 2.2. Lippincott. Formation (A) and isomer structure (B) of a peptide bond.

Repetition of this reaction creates a polypeptide.

In general, the term protein is used for molecules composed of over 50

amino acids and peptide is used for those of less than 50 amino acids.
Not only long proteins but also several biologically active peptides play
important biochemical and physiological roles in humans.

The sequence of the amino acids in peptides and proteins are listed from
the N-terminal to the C-terminal direction. N-terminal is the free aminogroup of the first amino acid; C-terminal is the free carboxyl group of the
last amino acid.

Some Examples of Biologically Active Peptides (FYI only)

Amino Acid Sequence



Thyrotropin- Secreted by hypothalamus;

causes anterior pituitary
gland to release thyrotropic
Vasopressin Secreted by posterior
(antidiuretic pituitary gland; causes
kidney to retain water from
Methionine Opiate-like peptide found in
brain that inhibits sense of
Little gastrin Hormone secreted by
mucosal cells in stomach;
causes parietal cells of
stomach to secrete acid

Pancreatic hormone
involved in regulating
glucose metabolism

II (horse)

Pressor or hypertensive
peptide; also stimulates
release of aldosterone from
adrenal cortex
Vasodilator peptide

Substance P


The NH2-terminal Glu is in the pyro form in which its -COOH is covalently joined to its -NH2 via
amide linkage; the COOH-terminal amino acid is amidated and thus also not free.
Cysteine-1 and cysteine-6 are joined to form a disulfide bond structure within the nonapeptide.
The Tyr 12 is sulfonated on its phenolic side chain OH.
The COOH-terminal amino acid is amidated.


Amino acids can undergo a variety of chemical reactions
Various reagents that are specific to certain side chains can be used to inactivate
amino acid side chains and probe function. (E.g. an enzyme inhibitor that
chemically binds to the active center of an enzyme will inactivate the enzyme)
Cystine is derived from two cysteines
This is derived post-translationally by oxidation of the -SH side chains of two Cys
residues to form a disulfide bond.
Disulfides are involved in stabilization of the protein structure and are formed and
dissipated in oxido-reductive reactions.

Figure 3.7, Devlin. Cystine bond formation

Glutathion is derived from 3 amino acids

Glutathione is the most important reductive agent that fight reactive oxygen
species. It plays a role in repairing oxidative damage to the cell membrane. The
composition is: gammaGlu-Cys-Gly. When membrane proteins are oxidized,
glutathione can reduce them back. In the meantime, glutathione is oxidized (S-S
bridge forms between two glutathiones). Glutathione reductase enzyme can
reduce back glutathione to its reduced form.

Ionizable groups of amino acids are critical for biological function of


Ionizable groups in a protein can carry charges and influence the

properties of the protein.

Ionizable groups in proteins are:

free N-terminal residue;
free C-terminal residue;
side chains of monoamino dicarboxylic and diamino monocarboxylic
amino acids as well as
cystein and tyrosine.

Depending on the pH of the solvent, these groups can be protonated or

de-protonated, a.k.a. charged or neutral.

Positively charged amino acid residues in proteins stabilize negatively

charged ones, thus play role in folding the protein into its 3D structure.

Ionized groups play role in the biological activity of the protein, e.g. in
enzyme-substrate interaction; in hormone-binding to its receptor; etc.

Nonpolar (hydrophobic) side chains are critical in protein folding

Hydrophobic amino acids, those less soluble in water, such as Leu, Val
and Ile, are mostly in the interior of a protein and help stabilize the
Polar and charged amino acids are found mostly on the surface and there
interact with each other and water.

When nonpolar amino acids are on the surface they are usually mixed
with polar amino acids to enhance solvation.
When nonpolar amino acid side chains are clustered, they create a
surface that may be a binding site for other proteins or ligands.



Why should we study the structure of proteins?
Structure determines the function of the protein. If structure changes, the function
of the protein can be altered or diminished resulting in the malfunctioning of the
organism. The severity of this phenomenon depends on the degree of the
change in protein structure.
Levels of protein organization:

Primary structure is the order of amino acid residues.

Secondary structure is the local three-dimensional folding of the chain.

Tertiary structure refers to the 3-D structure of the protein.

Quaternary structure refers to the non-covalent association of discrete

polypeptide subunits into a multi-subunit protein.
(Note: Not all proteins have quaternary structure.)

The native conformation is the one that has the lowest Gibbs free energy
kinetically accessible to the polypeptide chain.

Folding is the process how proteins reach their 3-D shape. It is

spontaneous but chaperone proteins may facilitate protein folding.

Figure 2.1. Lippincott. Four hierarchies of the protein structure



The primary structure of a protein is its amino acid sequence.

The gene for the protein encodes the primary structure.
The primary structure determines the higher levels of protein organization
(secondary, tertiary and quaternary structures) and through this the function
of the protein.
If primary sequence is changed, the conformation of the protein and its
function may also change. This can be positive (activating the protein) or
negative (diminishing the activity of the protein).

Changes in amino acids can be conservative and non-conservative:

Conservative change: when an amino acid is changed to another amino acid of
similar size and chemical nature.
E.g.: Val to Leu; Arg to Lys; Asp to Glu
Non-conservative change: when an amino acid is changed to another amino acid
of different size and/or chemical nature.
E.g.: Gly to Phe; Arg to Val; Asp to Lys
This change usually results in the change of the structure of the protein.
If the change occurs at the functionally active site of the molecule, even
conservative changes can result in the loss of function of that protein. These
amino acids are invariant to preserve the function of the protein.
Additionally, if the changed amino acid is on the surface of the protein, or it
triggered the change of conformation of the protein, the protein can be
recognized by the immune system as a foreign protein.

Example 1: Changing the primary structure of a protein brings about the active
Insulin is produced as proinsulin by the islet cells of the pancreas. This molecule
has 3 intra-chain disulfide bridges and is physiologically inactive.
When needed, proinsulin is proteolytically modified by proteases in the islet cells
that cleave between residues 30-31 and 65-66 to release a 35 amino acid
residue segment (C-peptide). (C-peptide is used in diagnosing which disease?)
Active insulin contains now two chains, A and B, joined by the same disulfide
bonds as in proinsulin. This molecule is active; can fold into the right
conformation that can bind to cell surface insulin receptors.


Figure 3.23, Devlin: Primary structures of human proinsulin, insulin and C-peptide.


Both porcine and bovine insulins are used in the treatment of human diabetes.
The primary structures of both porcine and bovine insulins are different from that
of human; however, these changes are small in number and conservative in
nature. The structure and function of insulin in different species is conserved.
In some patients, however, these differences in amino acid composition can
result in the development of immune response towards the foreign insulin, thus
insulin deficiency may occur due to high antibody titers. Porcine insulin is more
similar to human insulin than bovine, thus better tolerated by diabetic individuals.
Human insulin is now available from recombinant sources.


Example 2: A single amino acid change in hemoglobin brings about the loss of its


In hemoglobin S (HbS) a nonconservative substitution occurs in the sixth position
of the -globin chain. A polar Glu residue is replaced by a non-polar Val, which
creates a hydrophobic patch on the surface of HbS. HbS in its deoxy form, can
bind to other HbS molecules, leading to precipitation within the red blood cells.
This changes the red blood cells shape to sickle that results in high rate of
hemolysis and lack of elasticity as cells move through small capillaries.
See more details later.



Secondary structures can form because of the conformation of the peptide bond.
There is no rotation around the peptide bond due to its double bond character.
However, there is rotation allowed between the -carbon and the nitrogen (phi
bond) and the -carbon and the carbonyl carbon (psi bond) of the peptide.
Secondary structures are: -helix
-pleated sheet
random coil

A. -helical structure
L-amino acids form right-handed alpha helices.

Figure 2. 6. Lippincott. An alpha-helix.

The -helix is kept together by hydrogen bonds formed between the

carbonyl group (C=O) of a peptide bond and the imino (N-H) group of another
peptide bond directly under or above each other (roughly every 4th peptide
bond). The side chains of the amino acid residues project towards the outside
of the helix.
Pro disrupts the helix due to changes in chain direction and steric exclusion.


B. -structures
B/1. -pleated sheet:
Two polypeptide chains or segments of one polypeptide chain are aligned in a
parallel (same direction) or antiparallel (opposite direction) fashion.
-strands appear like pleated sheets.
Hydrogen bonds form between the carbonyl group (C=O) of a peptide bond
of a polypeptide chain and the imino (N-H) group of another peptide bond
from the other polypeptide chain directly next to each other. The side chains
project above and below the pleated sheet like structure.

Figure 2.7. Lippincott. Two polypeptide chains in a -sheet structure conformation (A); Example
of an antiparallel (B) and a parallel (C) sheet structure.

B/2. -turn: When the secondary structural elements, -helix or the -sheet are
disrupted, -turns are frequently formed to let the helix or sheet continue in
another direction. -turns contain 3 amino acid residues (peptide bonds) only,
and stabilized by hydrogen bonds. Proline and glycine are most frequently
found in these structures.


C. Random coil
Secondary structural segments are connected by random coils (loops). This
gives the basis for the formation of the tertiary structure.
Coils contain non-repetitive but well-defined primary amino acid sequences
and are longer than the -turns.
Many of the coils are functional, found in binding sites, active centers of
proteins. E.g. antigen-binding sites of the immunoglobulins are localized in
random coil segments.



The tertiary structure is the 3-D structure or shape of the protein.

Certain rules are followed when tertiary structure is formed:
Hydrophobic side chains are generally inside, away from the water interface.
Ionized side chains are on the outside and interact with water. Water forms a
solvation shell around the protein.
Some long polypeptide chains often fold into multiple semi-independent
folding domains; each with a compact geometry, with hydrophobic core and
polar outside. These are called multi-domain proteins.
Domains are connected by flexible segments (random coils); thus the
domains can move with respect to each other. Enzyme active sites are often
associated with these segments and situated between two domains.

Example: Hexokinase has two domains. When the substrate (glucose) binds to
this enzyme, the domains move closer, clamp down on the substrate.

Figure 3.32, p. 47, Devlin. Unliganded hexokinase with free glucose (top) and hexokinase with
glucose bound.



A quaternary structure is formed when several polypeptide chains form a specific

noncovalent association
Quaternary structure has subunits that are non-covalently attached to
Two or more polypeptide chains are synthesized and folded separately
and then come together to form a quaternary structure.
Homomultimers when subunits are of the same polypeptide.
Heteromultimers when subunits are of different polypeptides.
Hemoglobin (Hb) with four non-covalently attached subunits does have a
quaternary structure. It is usually a heteromultimer: in adults (HbA1) two
alpha and two beta subunits are bound together (22).
Pyruvate kinase (an enzyme in glucose metabolism) is a homomultimer
with 4 identical subunits.
The poliovirus protein contains 60 subunits and tobacco mosaic virus
protein has 2120 subunits in quaternary structure.
Advantages of forming a quaternary structure:
Stability- binding subunits together shields more hydrophobic residues from
water in the inside of the protein and this result in stronger interaction within
the multimer.
Genetic economy in homomultimers only one gene is needed to code for a
complex protein.
Efficiency in heteromultimers, active sites are brought in close proximity; the
substrate is channeled from one subunit to the other without
diffusion and/or transport.
- in homomultimers, multiple substrates can be processed in the same
Cooperativity subunit interactions can facilitate the reaction, e.g. one subunit
changes to active conformation can help the other subunit(s) to change.



contains the properties sufficient to
promote spontaneous protein folding
to its unique conformation.

Folding is initiated by short-range

structures in small regions.

These structures then fold upon each

other to form larger folded segments

The final conformation is the most

stable (with the lowest Gibbs free
energy) accessible within a limited
time frame.

After formation of the native structure,

disulfide bonds form to stabilize it.

Quaternary structure may form from

individually folded protein monomers.

Figure 2.12. Lippincott. Steps in protein folding


Chaperone proteins may assist the protein folding process

Chaperone proteins belong to the family of heat shock proteins whose

synthesis is increased at higher temperatures. When temperature is
increased, cellular proteins start to unfold. Chaperons assist these proteins to
refold into their native structure.
Chaperones bind to hydrophobic surfaces as proteins are synthesized and
thus block aggregation of the proteins in the cell where protein concentration
is high.
They prevent protein aggregation prior to the final folding process and prevent
formation of dead end or nonproductive folding intermediates.
Chaperonins are of the hsp60 family and form long cylindrical multisubunit
quaternary structures that bind unfolded proteins.
Chaperonins have ATPase activity, which supplies energy required to fold
Chaperones are also required for refolding proteins as they cross membranes
and also facilitate protein transport into the mitochondria and into and through
the ER. Proteins are unfolded when they cross these membranes.
Chaperons also participate in the assembly of multimeric proteins.

Figure 3.46. Devlin Chaperonin-directed protein folding in

E. coli A model for ATP-dependent release of an unfolded
polypeptide from its multiple attachment sites in GroEL.
ATP binding and hydrolysis mask the hydrophobic sites of
GroEL subunits (darker areas) that bind to unfolded
polypeptide, thus permitting it to fold in an isolated


Molecular interactions and forces during protein folding:

Noncovalent forces lead to protein folding and contribute to the proteins
(These bonds keep together the tertiary and quaternary structures of
Non-covalent forces are weak bonding forces (1-7 kcal/mol) as compared to a
typical covalent bond (50 kcal/mol).

Hydrophobic Interaction Forces are the most important for protein folding.
Hydrophobic groups are folded inside the protein to prevent interaction with
water. The presence of large number of hydrophobic residues within a protein
results in the fact that hydrophobic forces are the major contributors to protein
Hydrogen Bonds
A donor shares a proton with a second atom, called an acceptor.
H bonds help stabilize secondary (as well as tertiary and quaternary)
structures but are less important than hydrophobic interactions for the stability
of the protein.
Electrostatic (ionic) Interactions
Also called ionic or salt bridges, can be repulsive or attractive. They are
formed between a negatively and a positively charged amino acid residue.
Van der Waals-London Dispersion Forces
The weakest interaction, generated by dipole formation by close proximity.
Van der Waals forces are critical for tertiary structure formation. As proteins
fold into tertiary structure, the number of interactions may be in the


Figures 2.10 and 2.11. Lippincott. Interaction of amino acid side chains. A. Hydrophobic
interaction; B. Hydrogen bond and Ionic interaction.

Denaturation (unfolding) of proteins leads to loss of native structure

Denaturation disrupts native secondary, tertiary and quaternary structure

with no effect on primary structure.
Denaturation always causes loss of function; however loss of function can
also occur with small changes in conformation, short of denaturation.
Denaturation could be caused by change in pH, ionic strength, or
Denaturation in the laboratory can be caused by the addition of strong
acid, base or organic solvents, by heating the protein above 60 oC or by
the addition of denaturants such as urea, detergents or guanidine.
Denaturing agents will weaken primarily hydrophobic bonding. Some
detergents will break both ionic and hydrophobic interactions.
In the cell: denaturation or improper folding of a protein leads to enzymatic
removal of the protein in the proteasome.
Denatured, oxidized or improperly folded proteins or proteins that are not
necessary for the cell any more will be ubiquitinated and delivered to the
proteasome for elimination by proteinases.
Proteins that are taken up by the cell (extracellular proteins by
endocytosis) are degraded in the lysosome by specific proteinases.


CLINICAL CASES folding problems:

1. Proteins as Infectious Agents: Human Transmissible Spongiform
Encephalopathies (TSEs)
Proteins that act as infectious agents in the absence of DNA or RNA are known
as prion proteins.
CreutzfeldtJakob disease (CJD) is the most common of the prion diseases.
The disease is characterized by ataxia, dementia, and paralysis and is almost
always fatal. Pathological examination of the brain shows fibrous amyloid-like
plaques and spongiform (vacuolous) degeneration of the brain. The disease can
be generated by an inherited mutation (familial disease), by sporadic mutation, or
by an infectious process.
More recently, bovine spongiform encephalopathy or mad cow disease
(called scrapie in sheep) has been transmitted to humans through the ingestion
of meat from cattle infected by bovine spongiform encephalopathy. While there is
strong evidence that the infective agent is a protein, the direct infection of an
animal with pure recombinant prion protein has not been demonstrated.
The disease is believed to be caused by a conformational change in the prion
protein, from its normal soluble cellular conformation to a toxic conformation. The
toxic conformation polymerizes into insoluble amyloid fibers, which cause the
neurotoxic pathologies. The normal soluble domain fold is composed of three helical and two small -strand segments. The conversion to the toxic form is
characterized by the conversion of two of the -helical segments into -strand
conformations, which then can polymerize into amyloid fibers through -strand
interactions between monomers. Formation of the amyloid polymer is
An unknown factor, designated protein X (perhaps a chaperone protein),
facilitates the conversion of the -helical to -structure conformation and/or
promotes the polymerization of the -structure conformation into the amyloid
(Horwich, A. L. and Weissman, J. S. Deadly conformationsprotein misfolding in prion disease.
Cell 89:499, 1997; and Prusiner, S. B. Prion diseases and the BSE crises. Science 278:245,




2. Amyloidoses and Alzheimer disease

In this disease, normal intra- or extracellular proteins fold in an abnormal way
and form by spontaneous aggregation, long, fibrillar protein assemblies, called
amyloids. In Alzheimer disease, a normal transmembrane protein (called amyloid
precursor protein) is cleaved abnormally by an enzyme. The resulting A-beta
protein aggregates, forming insoluble fibrils of beta-sheet conformation,
precipitating as an amyloid plaque, found in the brain parenchyma and around
blood vessels. This protein is neurotoxic, leading to the cognitive impairment
characteristic of the disease.
A second biological factor involved in the development of Alzheimer disease is
the development of neurofibrillary tangles in the brain. A key player in this
phenomenon is an abnormal tau protein. The normal tau protein is responsible
for the assembly of the microtubular structure inside the cells. The abnormal tau
protein is an inhibitor of this process.


3. Defective protein folding may lead to other diseases (FYI only)

a. Inability to fold:
Cystic fibrosis 70% of the disease alleles has a deletion of Phe508 in a
fibroblast chloride channel gene. The mutant protein cannot fold into the active
conformation due to altered interaction with Hsp70. (When this mutant is folded
in the right way in vitro, the protein is active.)
Marfan syndrome mutation causes faulty folding of fibrillin
b. Toxic fold:
Alzheimers disease see above
Cataract aggregation of crystalline, the major protein in the lens
c. Mislocalization owing to misfolding:
Familial hypercholesterolemia mutation in the LDL-receptor locus results in the
production of the receptor protein, which is not properly folded and cannot be
transported from the ER to the Golgi.



Differences in physico-chemical characteristics of proteins can be used to
separate them from each other. The following list of methods serves as a
reference to re-visit when these methods are mentioned later.
1. Isolation and Separation of Proteins
Proteins can be separated on basis of their charge, molecular mass,
hydrophobicity and affinity to a specific binding partner. These separation
techniques can also be used when proteins are being purified. The net charge
of the protein depends on its amino acid composition and the pH of the
Gel filtration proteins are separated by size in a sieve, formed by gel beads that
have different pore size. Small molecules penetrate into the beads while
large molecules are excluded, thus come off the column sooner than that
of the smaller molecules.
Ion-exchange chromatography proteins are separated by their charge, thus
different ability to bind and being eluted from a charged gel matrix.
Affinity chromatography specific proteins with high binding affinity to the column
material can be separated from others. Example: an antibody is bound to
the column material and it selects out proteins from a mixture of proteins
that contains and epitope recognized by the antibody.
Electrophoresis proteins are separated in electric field according to their
charge. A gel or paper can be a support material.
Isoelectric focusing proteins are separated by their charge; they move in
electric field in a special buffer system until they reach their isoelectric
point (they become uncharged).
2-dimensional gel technique (proteomics) all proteins can be separated from
each other when electrophoresis and isoelectric focusing is combined in
two directions.
2. Determination of the Molecular Weight of a Protein
Ultracentrifugation proteins are sedimented by centrifugal force according to
their molecular mass.
Gel filtration (molecular sieve or exclusion chromatography) see above
Gel electrophoresis - When detergents are used (e.g. SDS), the gel as a sieving
support, will separate the proteins according to their mass.


3. Determination of the Amino Acid Composition of a Protein

Analysis of compositions may give information on abnormal proteins.
Proteins are hydrolyzed in strong acid to cleave peptide bonds and the resultant
amino acids are separated in ion-exchange columns and quantified by
reacting their amino groups with ninhydrin. This can be done in an amino
acid analyzer.
Amino acid sequencing combined with bioinformatics. Large proteins are
fragmented by enzymatic digestion into smaller fragments. These
segments are sequenced by the Edman degradation method. The Nteminal of the peptide is reacted with Edmans reagent
(phenylisothiocyanate), which results in instability of the peptide bond of
this first amino acid, thus can be selectively removed from the peptide and
analyzed. This process is automated and peptides as long as 100 amino
acid residues can be sequenced.
Determination of the sequence of the proteins by DNA sequencing- since the
primary structure of the protein is coded by DNA, sequencing the DNA will
give the sequence of the protein. However, posttranslational modification
of a protein cannot be determined by this method.
4. Determination of the 3D-Structure of a Protein
X-Ray Diffraction
Ultraviolet Light Spectroscopy
Fluorescence Spectroscopy
Optical Rotary Dispersion and Circular Dichroism Spectroscopy
Nuclear Magnetic Resonance Spectroscopy