Download Aminoacids. Protein structure and properties.

Amino Acids
Jana Novotna
Department of Medical Chemistry and Clinical
Biochemistry
2016
General characteristics of amino
acids (AA)
• Building blocks of proteins.
• 20 common AA - encoded by standard genetic code, construct
proteins in all species.
• Primary structure of AA determinates unique three-dimensional
structure, function, binding sites of proteins for different interactions.
• Important intermediates in metabolism (porphyrins, purines,
pyrimidines, creatin, urea etc).
• Some of AA have hormonal or catalytic function.
• Several genetic disorders are cause in amino acid metabolism errors
(aminoaciduria - presence of amino acids in urine)
The basic structure of amino acids
Simple monoamino monocarboxyl a-amino acid
- diprotic acid - can yield protons when fully
protonated
AA have characteristic titration curve
+1
+0.5
0
-0.5
-1
pKaCOOH + pKaNH3+
pI =
Proton
donor
Proton
acceptor
2.34 + 9.60
2
= 5.97
At the midpoint – pK=9.60 there
is equimolar concentration of
proton donor and proton
acceptor.
+
Isoelectric pH = pI
Dipolar ion
At the midpoint – pK1=2.34 there
is equimolar concentration of
proton donor and proton acceptor.
+
Fully protonated
form at wery low pH
pI =
2
Proton
donor
Proton
acceptor
Adopted from: D.L. Nelson, M.M. Cox Lehninger Principle of Biochemistry
Dissociation of the side chains of AA
Titration curve for (a) glutamate and (b) histidine
(the pKa of the R group is designed here as pKR)
pI =
pK1 + pKR
=
2.19 + 4.25
= 3.22
pI =
pKR + pK2
=
6.0 + 9.17
= 7.59
2
2
2
2
The isoelectric points reflect the nature of ionizing groups present. Glutamate has two –
COO- groups, histidine has two groups (a-NH3+ and imidazole group).
Adopted from: D.L. Nelson, M.M. Cox Lehninger Principle of Biochemistry
Binding interactions of AA
Electrostatic interactions
A disulfide bond
The stereochemistry of AA
Chiral molecules existing in two forms
http://www.imb-jena.de/~rake/Bioinformatics_WEB/gifs/amino_acids_chiral.gif
The two stereoisomers of alanine
a-carbon is a chiral center
Two stereoisomers are
called enantiomers.
The solid wedge-shaped bonds
project out of the plane of
paper, the dashed bonds
behind it.
The horizontal bonds project out
of the plane of paper, the vertical
bonds behind.
Classification based on chemical
constitution
Small amino acids – Glycine, Alanine
Branched amino acids – Valine, Leucine, Isoleucine
Hydroxy amino acids (-OH group) – Serine, Threonine
Sulfur amino acids – Cysteine, Methionine
Aromatic amino acids – Phenylalanine, Tyrosine, Tryptophan
Acidic amino acids and their derivatives – Aspartate, Asparagine,
Glutamate, Glutamine
Basic amino acids – Lysine, Arginine, Histidine
Imino acid - Proline
Essential and nonessential AA
Arginine*
Histidine*
Isoleucine
Leucine
Valine
Lysine
Methionine
Threonine
Phenylalanine
Tryptophan
* Essentials only for children
Alanine
Asparagine
Aspartate
Glutamate
Glutamine
Glycine
Proline
Serine
Cysteine** (from Met)
Tyrosine**(from Phe)
** Conditionally essentials
Uncommon amino acids
found in proteins
Intermediates of biosynthesis
of arginin and in urea cycle
Peptide bond formation
Proteins
How a sequence of AA in a polypeptide chain is
translated into a discrete, three dimensional
protein structure?



The three-dimensional structure is determined by
amino acid sequence.
The function depends on the structure.
The most important forces stabilizing the specific
structure are noncovalent interactions.
The peptide bond is rigid and planar
The peptide bond is a hybride between the resonance forms – the
carbonyl oxygen has a partial negative charge and the amide nitrogen
a partial positive charge, partial double form of peptide bond itself.
The N-Ca and Ca-C can rotate on angles f and j,
resp., the peptide C-N bond is not free to rotate.
Take over from: D. L. Nelson, M. M. Cox :LEHNINGER. PRINCIPLES OF BIOCHEMISTRY Fifth edition
Primary structure of proteins
Knowledge of primary structure of protein is require for
understanding of :



the protein´s structure
the mechanism of protein action on molecular level
the interrelationship with other proteins in evolution
Sequencing of protein is an aids for :


the study of protein modification
the prediction of the similarity between two proteins
The determination of the primary structure of a protein requires a
purified protein.
The cloning of the genes for many proteins and the sequencing of
gene is a much faster method to obtain the amino acid sequence.
The primary structure of peptides and proteins refers to
the linear number and order of the amino acids
present.


the N-terminal end - on the left (the end bearing the residue
with the free a-amino group)
the C-terminal end - on the right (the end with the residue
containing a free a-carboxyl group) .
Knowledge of primary structure of
insulin aids in understanding its
synthesis and action.
1. Pancreas produces single chain
precursor – proinsulin
2. Proteolytic hydrolysis of 35
amino acid segment – C
peptide
3. The remainder is active insulin
(two polypeptide chains A and B)
covalently joined by disulfide bonds
Amino acid identity in different animals:
Human, hors, rat, pig, sheep, chicken
insulin have differences only in residues 8,
9, and 10 of the A chain and residue 30 of
the chain B
Higher levels of protein organization
Secondary structure
The second level of protein structure determined by attractive and
repulsive forces among the amino acids in the chain. It is the
specific geometric shape caused by intra-molecular and
intermolecular hydrogen bonding of amide groups.
Tertiary structure
Three dimensional structure of polypeptide units (includes
conformational relationships in space of side chains R of
polypeptide chain).
Quaternary structure
Polypeptide subunits non-covalently interact and organize into
multi-subunit protein (not all proteins have quaternary structure).
The folding of the primary structure into secondary, tertiary and
quaternary structure appears to occur in most cases
spontaneously.
Cystein disulfide bonds are made after folding
Protein secondary structure
The a-helix
a-helix - right-handed coiled conformation.

Every backbone N-H group of peptide bond
donates a hydrogen bond to the backbone C=O
group of the amino acid four residues earlier.

3.6 amino acid residues are per 360o turn.
Region richer in Ala, Glu, Leu, Met, and poorer
in Pro, Tyr, Ser tend to form a-helix.
The formation of the a-helix is spontaneous.
b–sheets
2 strands (segments) of polypeptide chains are stabilized by H-bonding
between amide nitrogens and carbonyl carbons.
 Polypeptide segments are aligned in parallel or anti-parallel direction to
its neighboring chains.
b-structure gives plated sheet appearance – side chain groups are
projected above and below the plane generated by the hydrogen-bonded
polypeptide chains.
b–sheets
In parallel sheets adjacent peptide
chains proceed in the same direction
(i.e. the direction of N-terminal to Cterminal ends is the same).
In anti-parallel sheets adjacent
chains are aligned in opposite
directions.
The large number of hydrogen bonds
maintain the structure in a stretched
shape.
Protein tertiary structure
The folding pattern of the secondary structural element into
3D conformation
The tertiary structure of a protein
Forces that give rise to tertiary structure
Hydrophobic
interaction
b plated sheets
a helical regions
Examples of the tertiary structure
Examples of a,b-folded domains
in which b-structural strands form
a b barrel in the centre of the
domain
Examples of bfolded domains
Protein quaternary structure
The arrangement of the protein subunit in the threedimensional complex constitutes quaternary structure.
Hemoglobin
Four subunits (two a and two b subunits) are associated in the quaternary structure
Forces controlling protein structure
Hydrophobic interaction forces:


Interaction inside polypeptide chains (amino acids contain either
hydrophilic or hydrophobic R-groups).
Interaction between the different R-groups of amino acids in polypeptide
chains with the aqueous environment.
A non-polar residues dissolved in water induces in the water solvent a solvation shell in
which water molecules are highly ordered.
Two non-polar groups in the solvation shell reduce surface area exposed to solvent and
come very close come together.
Hydrogen bonds:


Proton donors and acceptors within and between polypeptide chains
(backbone and the R-groups of the amino acids).
H-bonding between polypeptide chains and surrounding aqueous
medium.
Electrostatic forces:

Charge-charge interactions between oppositely charged R-groups such
as Lys or Arg (positively charged) and Asp or Glu (negatively charged).

Ionized R-groups of amino acids with the dipole of the water molecule.
van der Waals forces:


Weak non-colvalent forces of great importance in protein structure, the
sum of the attractive or repulsive forces between molecules
Force is caused by the attraction between electron-rich regions of one
molecule and electron-poor regions of another
Protein denaturation and folding
Denaturation is a loss of the threedimensional. The protein loss of it
function.



Denaturation by heat has complex effect
on the weak interactions (primarily by
disrupting hydrogen bonds).
Extremes of pH alter the net charges on
the protein, causing electrostatic
repulsion and the disruption of some
hydrogen bonding.
Organic solvents and detergents act
primarily by disrupting hydrophobic
interactions
Renaturation is process in which protein
regains its native structure
Take over from: D. L. Nelson, M. M. Cox :LEHNINGER. PRINCIPLES OF BIOCHEMISTRY Fifth edition
Some proteins undergo assisted folding
Not all proteins fold spontaneously and require molecular chaperons. Chaperons
interact with partially or improperly folded polypeptides
Take over from: D. L. Nelson, M. M. Cox :LEHNINGER. PRINCIPLES OF BIOCHEMISTRY Fifth edition
Protein misfolding
Amyloid fibre is an insoluble extracellular formation (amyloidoses).
They arise from at least 18 inappropriately folded versions of proteins
and polypeptides present naturally in the body.
Alzheimer´s disease
b-sheet undergoes partial folding, associates
partially with the same region in another
polypeptide chain (the nucleus of amyloid).
Prion protein
Take over from: D. L. Nelson, M. M. Cox :LEHNINGER. PRINCIPLES OF BIOCHEMISTRY Fifth edition
Protein structure
1. Globular proteins are compactly folded and coiled.
2. Fibrous proteins are more filamentous or elongated.
3. Peptides
• Small peptides (containing less than a couple of
dozen amino acids) are called oligopeptides.
• Long peptides are called polypeptides.
• Peptides have a "polarity"; each peptide has only one
free a-amino group (on the amino-terminal residue)
and one free (non-side chain) carboxyl group (on the
carboxy-terminal residue)
Functional roles of proteins
1. Dynamic function
transport
metabolic control
contraction
catalysis of chemical transformation
2. Structural function
bone, connective tissue
Classification of proteins by bioloical
function
1. Enzymes (lactate dehydrogenase, DNA polymerase)
2. Storage proteins (ferritin, cassein, ovalbumin)
3. Transport proteins (hemoglobin, myoglobin, serum
albumin)
4. Contractile proteins (myosin, actin)
5. Hormones (insulin, growth hormone)
6. Protective proteins in blood (antibodies, complement,
fibrinogen)
7. Structural proteins (collagen, elastin, glycoproteins)
Types of proteins
Globular proteins
Spheroid shape
Variable molecular weight
Relatively high water solubility
Variety function roles – catalysts, transporters, control proteins (for the
regulation of metabolic pathways and gene expression)
Fibrous proteins
Rodlike shape
Low solubility in the water
Structural role in the organism
Lipoproteins
Complex of lipids with protein – the addition of lipids, fatty acylation
(palmitoylation)
Glycoproteins
Contain covalently bound carbohydrate – O- or N-glycosylation
Globular proteins
• Globular proteins, such as
most enzymes, usually
consist of a combination of
the two secondary structures.
• For example, hemoglobin is
almost entirely alpha-helical,
and antibodies are composed
almost entirely of beta
structures.
Fibrilar proteins
Collagen
Keratin
Lipoproteins
Multicomponent complexes of protein and lipids.
The lipids or their derivatives may be covalently or non-covalently
bound to the proteins.
Examples of lipoproteins: many enzymes, transporters, structural
proteins, antigens, adhesins and toxins.
The function of lipoprotein particles - transport of lipids (fats) and
cholesterol around the body in the aqueous blood, in which they
would normally dissolve
Glycoproteins
Glycoproteins have covalently attached sugar molecules at one or
multiple points along the polypeptide chain
Glycoproteins are:
• hormones
• extracellular matrix proteins
• proteins involved in blood coagulation
• antibodies
• mucus secretion from epithelial cells
• protein localized on surface of cells
• receptors (transmit signals of hormones or growth factors from
outside environment into the cell)
Sugar molecules are:
glucose, galactose, mannose, fucose, xylose, N-acetylglucosamine, N-acetylgalactosamine
Structure-Function Relationship of Protein
Families
Hemoglobin and myoglobin
Human hemoglobin occurs in
several forms.
Consist of four polypeptide chains
of two different primary
structure.
Bind oxygen in the lung and
transport the oxygen in blood to
the tissues and cells.

Myoglobin is a single polypeptide
chain with one oxygen binding site.
Binds and release oxygen in
cytoplasm of muscle cells.


Hemoglobin and myoglobin
molecules each contain a heme
prosthetic group.
 Protein without prosthetic group is
designated as apoprotein.
 Complete protein is a holoprotein
Oxygen binding to Fe2+ of heme in hemoglobin
Proximal histidine
binding pulls Fe2+ above
the plane of porphyrine
ring
O2 binding cause
conformational
changes which pulls
Fe2+ back
Contractile elements of muscles
Myosin – thick filament of the muscle
Actin – thin filament of the muscle G-actin (globular actin) F-actin (fibrilar
actin)
Tropomyosin
Troponin
One of the biologically important properties of myosin is its ability to combine with
actin to generate muscle contraction.
Biological membrane proteins
Integral membrane proteins
Peripheral membrane proteins
Channels and pores
Erythrocyte membrane
Diagram of a voltage-sensitive sodium channel α-subunit. G - glycosylation, P- phosphorylation, S - ion
selectivity, I - inactivation, positive (+) charges in S4 are important for transmembrane voltage sensing.
Membrane receptors
b-polypeptide stretch
extendings from a-helix.
2. Seven membrane-spanning
domains.
3. Recognize catecholamines,
principally norepinephrine.
1.
Hormone activates receptor.
Hormone-receptor mediated
stimulation of intracellular
signalling cascade.
Proposed model for insertion of the b2 adrenergic
receptor in the cell membrane
DNA binding proteins
Regulatory proteins binding to DNA sequence can promote
either an activation or repression of the rate of gene
transcription into mRNA



Helix-turn-helix binding proteins
The zinc finger motif
The leucine zipper motif
The zinc finger motif
Helix-turn-helix motif