Download 2.3. Three-Dimensional structure and function of proteins.

UNIT 2.
Structure and function of proteins.
OUTLINE
2.1. Amino acids.
 Structure.
 Ionic properties/Acid-base properties
 Uncommon amino acids.
2.2. Peptides. Primary structure determination.
 Peptide bond.
 Nomenclatures of the peptides.
 Characteristics of the peptides.
 Analysis of the primary structure of a protein
 Protein sequencing.
 Peptides of biological interest.
OUTLINE
2.3. Three-Dimensional structure and function of proteins.
 Proteins classification.
 Secondary structure: Ramachandran Diagram. -Helix. -pleated
sheet. -loops.
 Motives or super secondary structures.
 Tertiary structure.
 Denaturation and renaturation.
 Quaternary structure.
 Fibrous proteins: -keratins. Fibroin. Collagen.
2.1. Amino acids.
STRUCTURE:
• 20 -amino acids = 20 common amino acids
• Uncommon amino acids
Carboxyl group
Amino group
Proline (-imino acid)
Side Chain
2.1. Amino acids.
STRUCTURE:
WHAT DO YOU HAVE TO KNOW?
- Name of the 20 common amino acids
- Chemical composition of the 20 common amino acids
-Three-letter code used to represent the amino acids
- Amino acids classification
- Main properties of the amino acids grouped into each category.
2.1. Amino acids.
STRUCTURE:
•
•
•
•
You should know names, structures, pKa
values, 3-letter and 1-letter codes
Non-polar amino acids
Polar, uncharged amino acids
Acidic amino acids
Basic amino acids
2.1. Amino acids.
STRUCTURE:
• pH of the cells  7,4: Zwitterion = ionic forms of the amino acid
(neutral=net charge 0). Soluble in water
Zwitterion (neutral)
2.1. Amino acids.
STRUCTURE:
• Disulfide bridges between cysteine residues (S-S)
Thiol
Intrachain
Interchain
2.1. Amino acids.
STRUCTURE:
• Asymmetric/chiral
carbon.
Amino
acids
show
optical
and
stereochemical properties. All but glycine are chiral
• Stereoisomers:
same
chemical
composition,
different
spatial
organization.
• Enantiomers: type of steroisomers. Nonsuperimposable mirror-image
(L and D).
Levorotatory
behaviour
Dextrorotatory
behaviour
2.1. Amino acids.
STRUCTURE:
•D,L-nomenclature is based on D- and L-glyceraldehyde
• L-amino acids predominate in nature
2.1. Amino acids.
IONIC PROPERTIES/ACID-BASE PROPERTIES:
• Amino Acids are Weak Polyprotic Acids.
All the amino acids contain at least two dissociable hydrogens.
2.1. Amino acids.
IONIC PROPERTIES/ACID-BASE PROPERTIES:
• Isoelectric point (pI) = pH where the amino
acids have a net charge of 0.
• Simple amino acid (no
hydrogens in the side chain):
pI = ½ (pK1 + pK2)
dissociable
Titration of Glycine
2.1. Amino acids.
IONIC PROPERTIES/ACID-BASE PROPERTIES:
• Amino acid with dissociable hydrogens
in the side chain
Acidic amino acids
(net negative charge at neutral pH):
pI = ½ (pK1 + pKR)
2.1. Amino acids.
IONIC PROPERTIES/ACID-BASE PROPERTIES:
• Amino acid with dissociable
hydrogens in the side chain
Basic amino acids
(net positive charge at neutral pH):
pI = ½ (pKR + pK2)
Titration of Histidine
2.1. Amino acids.
IONIC
PROPERTIES/ACID-BASE
PROPERTIES:
You should know these numbers and
know what they mean!
Alpha carboxyl group  pKa = 2
Alpha amino group  pKa = 9
These numbers are approximate, but
entirely suitable for our purposes.
2.1. Amino acids.
UNCOMMON AMINO ACIDS:
• They are produce by modifications of one of the 20 amino acids
already incorporated into a protein :
2.1. Amino acids.
UNCOMMON AMINO ACIDS:
• Amino acids with specific biological functions. They occur only rarely in
proteins:
Dopamine:
Neurotransmitter
Histamine:
Allergy reactions
Tiroxine:
Hormone
GABA (-aminobutyric acid):
Neurotransmitter
Citrulline:
Urea cycle intermediate
L-ornithine:
Urea cycle intermediate
2.2. Peptides. Primary structure determination.
PEPTIDE BOND:
• Peptide bond: covalent amide bond establish between the -COOH
and the -NH3+ groups of two amino acids.
• One water molecule is eliminated during this reaction.
• It allows the polymerisation of the amino acids to form peptides and
proteins.
2.2. Peptides. Primary structure determination.
PEPTIDE BOND:
• Properties of the peptide bond:
- It is usually found in the trans conformation
- It has partial (40%) double bond character
- It is about 0.133 nm long - shorter than a typical single bond
but longer than a double bond
- N partially positive; O partially negative
Peptide bond is best described as a resonance hybrid f these two structures
2.2. Peptides. Primary structure determination.
PEPTIDE BOND:
O
C
C
N
Trans
H
C
H
C
O
N
C
C
Cis
Due to the double bond character, the
six atoms of the peptide bond group are
always planar!
Geometry of the peptide
backbones.
2.2. Peptides. Primary structure determination.
PEPTIDES CLASSIFICATION ACCORDING TO THE NUMBER OF AMINO ACIDS :
Dipeptide (2)
Tripeptide (3)
Oligopeptide (more than 12 and less than 20)
Polipeptide (many)
Serylglicylthyrosylalanylleucine
Ser-Gly-Tyr-Ala-Leu
SGYAL
2.2. Peptides. Primary structure determination.
PEPTIDES PROPERTIES:
• Peptides show polarity (direction).
2.2. Peptides. Primary structure determination.
PEPTIDES PROPERTIES:
• Peptides ionic forms:
+
H
H
H3N-CH-C-N-CH-COOH
‖
HS-H2C O CH3
Cationic form (pH )

+
H3N-CH-C-N-CH-COO‖
HS-H2C O CH3
(Cys-Ala)

Zwitterion
• Minimal peptide solubilisation at pH= pI
• No migration (no movement) in an electrical field.
H
H2N-CH-C-N-CH-COO‖
HS-H2C O CH3
Anionic fom (pH )
2.2. Peptides. Primary structure determination.
PEPTIDES PROPERTIES:
• Titration curve:
Amphoteric behaviour
Tetrapeptide (Glu-Gly-Ala-Lys)
WHAT DO YO HAVE TO KNOW:
- How to calculate the isoelectric point related to a peptide
2.2. Peptides. Primary structure determination.
ANALYSIS OF THE PRIMARY STRUCTURE OF A PROTEIN: AMINO ACID SEQUENCE:
• Amino acids sequence comparison
(haemoglobin from human beings and
sperm whale) :
 84% identical amino acids
(They determine the biological role of
the protein).
 94% homologous.
2.2. Peptides. Primary structure determination.
ANALYSIS OF THE PRIMARY STRUCTURE OF A PROTEIN: AMINO ACID SEQUENCE:
Acid hydrolysis liberates the amino acids of a protein
10-100 horas a
105-110 ºC
Thin layer chromatography.
Ion exchange chromatography.
Reverse-phase high-performance liquid chromatography (HPLC).
2.2. Peptides. Primary structure determination.
ANALYSIS OF THE PRIMARY STRUCTURE OF A PROTEIN: AMINO ACID SEQUENCE:
• Chromatographic methods used to separate amino acids:
Ion exchange chromatography: the charged molecules of interest (amino
acids) are exchanged for another ion (salt ion) on a charged solid support (resins).
Resins containing negatively charged groups interact with positive charge
molecules, which elute from the resins by changing the pH buffer or the salt ion.
Thin layer chromatography: amino acids absorbed on a thin layer of silica
gel are separated thanks to the solvent migration (buthanol: water: acetic acid
4:1:1) by capillarity.
Reverse-phase high-performance liquid chromatography (HPLC): amino
acids are separated on the base of their polarity by the used of a column having a
nonpolar liquid immobilised on an inert matrix (stationary phase). A more polar
liquid serves as the mobile phase. Amino acids are eluted in proportion to their
solubility in this more polar liquid.
2.2. Peptides. Primary structure determination.
ANALYSIS OF THE PRIMARY STRUCTURE OF A PROTEIN: AMINO ACID
SEQUENCE:
Ion exchange chromatography:
2.2. Peptides. Primary structure determination.
ANALYSIS OF THE PRIMARY STRUCTURE OF A PROTEIN: AMINO ACID
SEQUENCE:
• Methods for amino acids identification:
1. UV absorbance
2. Ninhidrine reaction
Ninhidrine
Amino acid
Hidrantine
Proline: yellow complex able to
absorb at 440 nm
Purple max = 570 nm
2.2. Peptides. Primary structure determination.
ANALYSIS OF THE PRIMARY STRUCTURE OF A PROTEIN: AMINO ACID
SEQUENCE:
• Methods for amino acids identification:
3. Fluorescence (Edman degradation): Phenylisothiocyanate (=Edman reagent)
combines with the free amino terminus of a protein.
Not only identifies the Nterminal residue of a
protein. Successive reaction
cycles can reveal the amino
acid sequence of a peptide
2.2. Peptides. Primary structure determination.
ANALYSIS OF THE PRIMARY STRUCTURE OF A PROTEIN: AMINO ACID
SEQUENCE
• Amino acid sequence:
1. If the protein contains more than one polypeptide, the chains are
separated and purified.
2. Cleavage of disulfide bridges (intrachain).
3. Determination of the N-terminal and C-terminal.
4. The polypeptide chain is cleaved into smaller fragments
(proteolytic enzymes).
5. Analysis of the amino acid composition and sequence of each
fragment (Edman degradation).
6. The overall amino acid sequence of the protein is reconstructed
from the sequences in overlapping fragments.
2.2. Peptides. Primary structure determination.
ANALYSIS OF THE PRIMARY STRUCTURE OF A PROTEIN: AMINO ACID
SEQUENCE
Cleavage of disulfide bridges.
O
‖
HC – O - OH
or
2-mercaptoethanol
( ICH2COO- )
Met interferences
2.2. Peptides. Primary structure determination.
ANALYSIS OF THE PRIMARY STRUCTURE OF A PROTEIN: AMINO ACID
SEQUENCE
• Identification of the N-terminal residue:
1. Sanger reagent:
peptide
FDNB
(Sanger
(FDNB)
reagent)
Acid
hydrolysis
2-dinitrophenyl-peptide
2-dinitrophenyl-N-terminal residue
2.2. Peptides. Primary structure determination.
ANALYSIS OF THE PRIMARY STRUCTURE OF A PROTEIN: AMINO ACID
SEQUENCE
• Identification of the N-terminal residue:
2. Edman reagent:
2.2. Peptides. Primary structure determination.
ANALYSIS OF THE PRIMARY STRUCTURE OF A PROTEIN: AMINO ACID
SEQUENCE
• Identification of the N-terminal residue:
2. Edman reagent:
Phenylisothiocyanate
Peptide
Peptide-PTC (phenylthiocarbamil)
PTH-alanine
(PTH derivative)
Smaller peptide (one amino acid residue is released)
2.2. Peptides. Primary structure determination.
ANALYSIS OF THE PRIMARY STRUCTURE OF A PROTEIN: AMINO ACID
SEQUENCE
• Identification of the C-terminal residue:
1. Carboxipeptidases:
- Carboxipeptidase A: Hydrolyses the C-terminal peptide bond
of all amino acids except Pro, Arg and Lys.
- Carboxipeptidase B: Hydrolyses the C-terminal peptide bond
of the basic amino acids residues (Arg or Lys).
2.2. Peptides. Primary structure determination.
ANALYSIS OF THE PRIMARY STRUCTURE OF A PROTEIN: AMINO ACID
SEQUENCE
• Fragmentation of the polypeptide chain:
2.2. Peptides. Primary structure determination.
ANALYSIS OF THE PRIMARY STRUCTURE OF A PROTEIN: AMINO ACID
SEQUENCE
2.2. Peptides. Primary structure determination.
Methods to fragmentise the polypeptide chains in order to analyse de amino aid sequence of a protein
Method
Cleavage target
Specificity
1. Sanger reagent
C-side of the N-terminal
Rn = all aa
2. Edman Degradation
idem
idem
3. Carboxipeptidase A
N-side of the C-terminal
Rn  Arg, Lys, Pro
A. Terminal fragmentation:
Rn-1  Pro
4. Carboxipeptidase B
N-side of the C-terminal
Rn = Arg, Lys
Rn-1  Pro
B. Intrachain cleavage:
1. Cyanogen bromide
C-side of the Rn
Rn = Met
2. Trypsin
C-side of the Rn
Rn = Lys, Arg
Rn+1  Pro
3. Chymotrypsin
C-side of the Rn
Rn = Phe, Tyr, Trp, Leu
Rn+1  Pro
4. Thermolysin
N-side of the Rn
Rn = Phe, Tyr, Trp, Leu, Ile, Val
Rn-1  Pro
5. Pepsin
N-side of the Rn
Rn = Phe, Tyr, Trp, Leu, Asp, Glu
Rn-1  Pro
2.2. Peptides. Primary structure determination.
OTHER METHODS OF PROTEIN SEQUENCE ANALYSIS:
• Amino acid sequence determined by the analysis of the gene
sequence (nucleotides).
It is possible to obtain the
sequence of the protein directly
produced during the translation
process, but not the posttranslational modifications
2.2. Peptides. Primary structure determination.
PEPTIDES OF BIOLOGICAL INTEREST:
2.3. Three-Dimensional structure and function of proteins.
PROTEIN STRUCTURE: LEVELS OF ORGANIZATION:
2.3. Three-Dimensional structure and function of proteins.
PROTEINS CLASSIFICATION:
• Biological role:
1. Catalysis: enzymes.
2. Structural role (protection and support): collagen, fibroin, elastin.
3. Movement: actin, tubulin.
4. Defence: keratin (against mechanical or chemical damage), fibrinogen and
thrombin (avoid blood loosing), immunoglobulins (immunosytem
proteins).
5. Regulation: hormones, growth factors.
6. Transport: membrane transporters, haemoglobin, lipoproteins.
7. Storage: ovalbumin, casein (from milk), ferritin.
8. Adaptations to environmental changes: cytochrome P450, heat chock
proteins.
2.3. Three-Dimensional structure and function of proteins.
PROTEINS CLASSIFICATION:
 On the basis of the shape and solubility:
Fibrous proteins
Globular proteins
Membrane proteins
 On the basis of the chemical composition:
Simples
Conjugates: (it contains non peptidic component: prosthetic group)
Apoprotein: protein without prosthetic group.
Holoprotein: protein + prosthetic group.
- Glucoproteins
- Lipoproteins
- Methaloproteins
- Phosphoproteins
- Haemoproteins
2.3. Three-Dimensional structure and function of proteins.
PROTEINS CLASSIFICATION:
Conformation:
Overall
threedimensional architecture of a protein
(the radicals can modified their
spatial position by rotation. Bonds are
not cleavage during this process.
Configuration: Geometric possibilities
fro a particular ser of atoms. In going
from one configuration to another,
covalent bonds must be broken ant
rearranged.
2.3. Three-Dimensional structure and function of proteins.
SECONDARY STRUCTURE. RAMACHANDRAN DIAGRAM:
2.3. Three-Dimensional structure and function of proteins.
SECONDARY STRUCTURE. RAMACHANDRAN DIAGRAM:
The reasonable conformations are
those avoiding steric crowding
Ramachandran diagram
corresponding to L-Ala residues.
 and  angles = 0º, no favourable
conformation in proteins.
2.3. Three-Dimensional structure and function of proteins.
SECONDARY STRUCTURE. -HELIX:
n = 3.6 residues (single turn)
nº atoms/single turn = 13
d = 0.15 nm = 1.5 Å
Travel along the helix axis per turn (pitch of the helix)
(v) = 0,54 nm = 5,4 Å (v = n·d)
2.3. Three-Dimensional structure and function of proteins.
SECONDARY STRUCTURE. -HELIX:
Left-hand twists
COOH
C

Right- hand twists
+
H2 N
CH2
H2 C
CH2
proline
O
Hydrogen bonds
‖
C – N - C
H
C
O
‖
C - C
H
N
CH2
H2C
CH2
2.3. Three-Dimensional structure and function of proteins.
SECONDARY STRUCTURE. -PLEATED SHEET:
7Å
Strands run in opposite directions
2.3. Three-Dimensional structure and function of proteins.
SECONDARY STRUCTURE. -PLEATED SHEET:
6.5 Å
2.3. Three-Dimensional structure and function of proteins.
SECONDARY STRUCTURE. -TURNS:
 Usually located in the protein
surface.
 Stabilised by hydrogen bonds
 They allow the protein strands
to change direction.
 Glycine
and
proline
predominant amino acids.
Proline isomers
as
2.3. Three-Dimensional structure and function of proteins.
SECONDARY STRUCTURE.
Bovine Carboxipeptidase A, it contains 307
residues and consists of a -pleated sheet (8
strands) and 6 -helix.
2.3. Three-Dimensional structure and function of proteins.
SECONDARY STRUCTURE. RAMACHANDRAN DIAGRAM:
Collagen
triple helix
antiparallel
-sheet
Parallel sheet
Rigth-handed sheet
Left-handedhelix
Right-handed helix
 and  values corresponding to all
the piruvate quinase amino acids
residues (except Gly).
2.3. Three-Dimensional structure and function of proteins.
SUPERSECONDARY STRUCTURES:
- Combinations of few secondary structures giving a characteristic
geometric shape
- They are the base of the structural classification of the proteins
- Some of them show specific biological roles, but in other cases they
are just part of the main structural and functional peptide.
-- loop
 chains right handed
conected
Conections between
 chains
- vertex
Barrel 
2.3. Three-Dimensional structure and function of proteins.
SUPERSECONDARY STRUCTURES:
- Some globular proteins contains a
combination of different super secondary
structures called DOMAINS OR MODULES.
Gliceraldehyde-3-phoste dehydrogenase from
Bacillus stearothermophilus. It is possible to
distinguished two domains in the folded peptide.
2.3. Three-Dimensional structure and function of proteins.
TERTIARY STRUCTURE:
The location of the amino acids’ side chain in a globular proteins
depends on their polarities:
1. Val, Leu, Ile or Phe (nonpolar) are inside the protein.
2. Lys, Arg, His, Asp and Glu (charged), are usually located in
the surface of the protein.
3. Ser, Thr, Tyr, Trp, Asn or Gln (polar and uncharged) ca be
located inside the protein structure or in the surface (usually).
2.3. Three-Dimensional structure and function of proteins.
TERTIARY STRUCTURE:
Interactions allowing tertiary structure stabilization
• Charge-charge.
• Van der Waals repulsion.
• Hydrogen bonds.
• Hydrophobic interactions.
• Disulfide bridges.
Thermodynamic driving force for
folding of globular proteins
2.3. Three-Dimensional structure and function of proteins.
TERTIARY STRUCTURE:
F. electrostáticas
F. van der Waals
P. hidrógeno
2.3. Three-Dimensional structure and function of proteins.
TERTIARY STRUCTURE:
2.3. Three-Dimensional structure and function of proteins.
TERTIARY STRUCTURE: DENATURATION AND RENATURATION:
Denaturation: loss of protein structure and function.
Factors:
 Increase of the temperature (exception:
proteins).
 extreme pHs.
 Organic solvents(alcohol, acetone).
 Some detergents.
 Several salts  chaotropic agents.
thermophilic
Renaturation: restoration of the native structure and biological role.
2.3. Three-Dimensional structure and function of proteins.
TERTIARY STRUCTURE: DENATURATION AND RENATURATION:
Protein denaturation under two kind of external stresses.
2.3. Three-Dimensional structure and function of proteins.
TERTIARY STRUCTURE: DENATURATION AND RENATURATION:
• Anfinsen’s
experiment
(1957):
Ribonuclease A = RNase A (124 residues)
• Chaotropic compounds:
2.3. Three-Dimensional structure and function of proteins.
TERTIARY STRUCTURE: DENATURATION AND RENATURATION:
• The conformation of a protein is the one of lowest Gibbs
free energy accessible to its sequence within a
physiological time frame. Folding is under thermodynamic
and kinetic control.
• Molten-globule: condensed intermediate on the folding
pathway that contains much of the secondary structure
elements of the native conformation but many incorrect
tertiary structure interactions.
CHAPERONES (also
called chaperonins)
proteins may assist the
protein folding process.
Chaperonin from E. coli.
GroEL/GroES complex
2.3. Three-Dimensional structure and function of proteins.
QUATERNARY STRUCTURE:
Oligomer: protein containing
2
several identical subnits.
1
Protomer: structural unit of an
oligomeric protein.
Haemoglobin  Tetramer
containing two protomers.
2
1
2.3. Three-Dimensional structure and function of proteins.
FIBROUS PROTEINS. -KERATIN:
• Epidermal layer, nails, hair,
feathers.
• Phe, Ile, Leu, Val, Met and Ala as
the main amino acids.
• -helix right handed.
• Different grade of hardness on the
basis of the % Cys. Disulfide bridges.
Cells
Intermediate filaments
Protofibril
Protofilament
keratin -helix
Coiled-coil superhelical
structure
Protofilament
Protofibril
Coiled-coil superhelical structure
-helix
Hair transversal section
2.3. Three-Dimensional structure and function of proteins.
FIBROUS PROTEINS. SILK FIBROIN:
• Antiparallel -pleated sheet.
• Tandem repetition: Gly–Ala.
• Voluminous amino acids: Val y
Tyr.
[Gly-Ala-Gly-Ala-Gly-Ser-Gly-Ala-Ala-Gly-(Ser-Gly-Ala-Gly-Ala-Gly)8]
2.3. Three-Dimensional structure and function of proteins.
FIBROUS PROTEINS. COLLAGEN:
 Most abundant protein in vertebrates.
 Provides the framework that gives the
tissues their form and strength (bone,
tooth, cartilage, tendon…).
 Simple helical structure (left handed).
 3,3 residues/turn.
 35% Gly, 11% Ala; other: Pro, 4Hydroxyproline (Hyp), 3-Hydroxyproline
y 5-Hydroxylysins (Hyl).
 Tandem repetition: Gly-X-Y (XPro;
Y Hyp).
Structure of the collagen fibrils de colágeno
2.3. Three-Dimensional structure and function of proteins.
FIBROUS PROTEINS. COLLAGEN:
 Hyp and Hyl give stability.
 Carbohydrates: Glucose, galactose
and disaccharides.
 In bones:
- Organic form Collagen.
- Inorganic form  Hydroxyapatite
[Ca5(PO4)3OH)]
Collagen.
(right handed).
Top vision of the triple helix.
Gly in red.