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Amino Acid Structure
Dr. Azin Nowrouzi
Peptides and proteins
• Polymers of amino acids.
• Two amino acids join covalently through a peptide bond.
• Another name for peptide bond is amide bond or linkage.
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Peptide bond formation
water is removed
This is a condensation reaction.
•
1.
2.
3.
•
There are only 3 known ways to make a peptide bond
Chemical abiotic synthesis in the laboratory.
Genetic engineering cloning mechanisms.
Biologically in cells.
Peptide bonds in proteins are quite stable, with an average half-life (t1/2) of
about 7 years under most intracellular conditions.
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Peptides are chains of amino acids
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• Dipeptide: 2 amino acids (aas) joined by 1 peptide
bond.
• Tripeptide: 3 aas joined by 2 peptide bonds.
• Tetrapeptide: 4 aas joined together by 3 peptide
bonds.
• Peptapeptide and so forth….
• Oligopeptide: Few aas joined together by peptide
bonds.
• Polypeptide: Many aas joined. Molecular weights
generally below 10000.
• Proteins: Many aas joined. Generally have high
molecular weights.
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Nomenclature
• The pentapeptide serylglycyltyrosylalanylleucine,
or Ser–Gly–Tyr–Ala–Leu.
• Peptides are named beginning with the amino terminal residue, which by
convention is placed at the left.
• The peptide bonds are shaded in yellow; the R groups are in red.
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Ionization behavior of peptides
amino-terminal (C-terminal) residue
carboxyl-terminal (C-terminal) residue
Alanylglutamylglycyllysine
Like free amino acids, peptides have characteristic titration curves and a
characteristic isoelectric pH (pI) at which they do not move in an electric field.
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Length of polypeptide chains
•
Lengths vary considerably.
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How to calculate the number
of amino acids in a protein
• We can calculate the approximate number of amino
• acid residues in a simple protein containing no other chemical
constituents by dividing its molecular weight by 110.
• Although the average molecular weight of the 20 common
amino acids is about 138, the smaller amino acids predominate
in most proteins.
• If we take into account the proportions in which the various
amino acids occur in proteins, the average molecular weight of
protein amino acids is nearer to 128.
• Because a molecule of water (Mr 18) is removed to create each
peptide bond, the average molecular weight of an amino acid
residue in a protein is about 128 -18 = 110.
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Polypeptides Have Characteristic
Amino Acid Compositions
• The 20 common amino
acids almost never occur
in equal amounts in a
protein.
• Some amino acids may
occur only once or not at
all in a given type of
protein; others may occur
in large numbers.
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Levels of Protein Structure
Rigidity of the
peptide bond
•
-carbons of adjacent amino acids are
separated by three covalent bonds
C-C-N-C
•
Tetrahedral angles:
N-C bond is labeled  (phi).
C-C bond is labeled 
Primary
structure
Secondary
structures
•
Amino acid
sequence
•
(psi).
Tertiary
structure
Quaternary
structure
Repetitive structures
•-Helix
•-Sheet
•parallel
•antiparallel
Non-repetitive structures
•-turn
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Primary structure
• A description of all covalent
bonds (mainly peptide and
disulfide bonds) linking amino
acid residues in a polypeptide
chain.
• The linear sequence of amino
acids within a peptide
• Written from NC,
– either in three-letter code,
– or, more often, in one-letter
code.
• Example: Glu-Gly-Ala-Lys or EGAK
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Insulin’s primary structure
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Determination of amino acid
composition of a polypeptide
•
•
•
•
Primary structure determination
Pure sample must be used.
Acid hydrolysis: strong acid, 110 C, 24hr.
Chromatography: Cation exchange, each amino
acid exits the column at a specific pH and ionic
strength.
• Quantitative analysis by heating with ninhydrin
• This analysis is done by amino acid analyzer.
Sequencing the peptide from
N-terminal
• Phenylisothiocyanate (Edman’s reagent) is used to
label the amino-terminal residue under mildly alkaline
conditions.
• The resulting phenylthiohydantoin (PTH) derivative
causes an instability in the N-terminal peptide bond.
• This bond can be hydrolyzed without cleaving the other
peptide bonds.
• This procedure can be applied repeatedly to the
shortened peptide.
• Good for about 100 aa.
• Has been automated (sequenator)
Cleavage of polypeptide chain into
smaller fragments
• When more than 100 aa are present in a
polypeptide chain.
• By using more than one cleaving agent on
separate samples of the purified polypeptide,
overlapping fragments are generated.
• Enzyme cleavage, such as trypsin and other
digestive enzymes
• Chemical cleavage (cyanogen bromide)
• Overlapping peptides
Sequencing a protein
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Specific cleavage of polypeptides
1.
2.
3.
4.
5.
Proteins larger than 50 aa are
first hydrolyzed into shorter
peptides.
Chemical or enzymatic
methods hydrolyze proteins at
specific sites.
Peptides are separated by
chromatography
Peptides generated by 2 or
more cleavage methods are
each sequenced separately.
Sequences of individual
peptides are overlapped
together to deduce the entire
protein sequence
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Protein Sequencing Example
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Secondary structure:
Five models of the helix
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-Helix
• H-bonds are inside the chain
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Description of -helix
• The polypeptide backbone is tightly wound around
an imaginary axis drawn longitudinally through the
middle of the helix.
• The R groups of the amino acid residues protrude
outward from the helical backbone.
• Each helical turn includes 3.6 amino acid residues.
• About one-fourth of all amino acid residues in
polypeptides are found in -helices.
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Stabilization of -helix
• The structure is stabilized by a hydrogen bond between the
hydrogen atom attached to the electronegative nitrogen
atom of a peptide linkage (amino acid n) and the
electronegative carbonyl oxygen atom of the fourth amino
acid (amino acid n+4) on the amino-terminal side of that
peptide bond.
• Each successive turn of the -helix is held to adjacent turns
by three to four hydrogen bonds.
• All the hydrogen bonds combined give the entire helical
structure considerable stability.
• Naturally occurring L-amino acids can form either right- or
left-handed helices, but extended left-handed helices
have not been observed in proteins.
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Factors affecting stability
1. Electrostatic repulsion (or attraction) between successive amino acid residues
with charged R groups.
2. Bulkiness of adjacent R groups.
3. The interactions between R groups spaced three (or four) residues apart.
4. Presence of Pro or Gly residues.
– Proline is only rarely found within an helix
•
In proline, the nitrogen atom is part of a rigid ring and rotation about the N-C bond is not
possible. Thus, a Pro residue introduces a destabilizing kink in an helix.
•
In addition, the nitrogen atom of a Pro residue in peptide linkage has no substituent
hydrogen to participate in hydrogen bonds with other residues.
– Glycine occurs infrequently in helices for a different reason
•
•
It has more conformational flexibility than the other amino acid residues.
Polymers of glycine tend to take up coiled structures quite different from an -helix.
5. Interaction between amino acid residues at the ends of the helical segment and
the electric dipole inherent to the helix.
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Helix dipole
• A net dipole extends along the
helix that increases with helix
length.
• Negatively charged amino acids
are often found near the amino
terminus of the helical segment,
where they have a stabilizing
interaction with the positive
charge of the helix dipole.
• A positively charged amino acid at
the aminoterminal end is
destabilizing.
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Secondary structure: Fully extended chains
can form -sheets
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Types of -sheets
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Two Types of -Pleated Sheets
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Non-repetitive secondary structures
1.
2.
3.
4.
Turns
Connections
Loops
Coils or random coils
•
These are well ordered but non repeating
configurations.
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 turns
• type I turns occur more than twice as frequently as type II.
• Type II turns always have Gly as the third residue.
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Tertiary structure
•
Amino acids that are far apart in the
polypeptide sequence and that reside in
different types of secondary structure may
interact within the completely folded
structure of a protein.
•
The location of bends (including turns) in
the polypeptide chain and the direction
and angle of these bends are determined
by the number and location of specific
bend-producing residues, such as Pro, Thr,
Ser, and Gly.
•
Interacting segments of polypeptide chains
are held in their characteristic tertiary
positions by different kinds of weak
bonding interactions (and sometimes by
Leptin
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Stabilization of tertiary Structure
• overall threedimensional
arrangement of all
atoms in a protein is
referred to as the
protein’s tertiary
structure.
• Stabilized primarily
through weak bonds.
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Folding of a polypeptide chain
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Three-dimensional structures
of some small proteins
Myoglobin
PDB ID 1MBO
Cytochrome c PDB
ID 1CCR
Lysozyme
PDB ID 3LYM
RibonucleasePDB
ID 3RN3
• PDB; www.rcsb.org/pdb
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Interactions stabilizing tertiary structure
• Specific overall shape of a protein
• Cross links between R groups of amino acids in
chain
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Domains
• When molecular weight is larger than 20000.
• The ratio of surface area to volume is small.
• A protein with multiple domains may appear to have
a distinct globular lobe for each domain.
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Example
• Crystal structure of the
heterodimeric enzyme
Rab Geranylgeranly
Transferase.
• It is a dimer of a alpha
(blue, red, yellow) and a
beta subunit (orange).
• The alpha subunit is a
multi domain protein.
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Quaternary structure
• Protein Quaternary Structures Range from Simple Dimers to Large
Complexes:
• A multisubunit protein is also referred to as a multimer.
• Multimeric proteins can have from two to hundreds of subunits.
• A multimer with just a few subunits is often called an oligomer.
• The repeating structural unit in such a multimeric protein, whether it is a
single subunit or a group of subunits, is called a protomer.
• The first oligomeric protein for which the three dimensional structure was
determined was hemoglobin (Mr 64,500), which contains four polypeptide
chains.
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Hemogloin
A tetrameric protein
two -chains (141 AA)
two -chains (146 AA)
four heme cofactors, one in each chain
The  and  chains are homologous to myoglobin.
Oxygen binds to heme in hemoglobin with same
structure as in Mb but cooperatively: as one O2 is
bound, it becomes easier for the next to bind.
Types of hemoglobin
Form
Chain
composition
Fraction of the total
hemoglobin
Hb A
HB F
HB A2
Hb A1c
α2β2
α2γ2
α2δ2
α2β2-glucose
90%
<2%
2-5%
3-9%
Simple and conjugated proteins
• Some proteins contain chemical groups other than amino acids.
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Spectroscopy of amino acids
• Aromatic amino-acids are strong chomophores in the far-uv.
• Only the aromatic amino acids absorb light in the UV region
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Absorbance can be measured
by UV-spectrophotometer
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Ninhydrin-detection of amino acids
• Complete hydrolysis for 24 hr at 110 oC in 6 M HCl.
• Amino acids can be detected on the chromatogram by using ninhydrin.
A solution of ninhydrin is sprayed onto the paper and heated. The
amino acids show up as purple spots (proline appears yellow).
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Paper chromatograms
2D chromatogram
eluting with a
different solvent
mixture in each
direction.
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Electrophoresis
•
Electrophoresis is a technique that uses the net charge of peptides
(amino acids) as a basis for separation.
 A potential difference is applied across a solid material (e.g. paper for
amino acid analysis) permeated by an electrolyte.
 Anions migrate to the anode and cations to the cathode. The rate of
diffusion is related to the size and net charge. Small highly charged
proteins migrate more quickly.
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