Diapositiva 1

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UNIT 2. Structure and function of proteins.
2
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.

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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.

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2.1. Amino acids.

• STRUCTURE
• 20 ?-amino acids 20 common amino acids
• Uncommon amino acids

Carboxyl group
Amino group
Side Chain
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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.

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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

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2.1. Amino acids.

• STRUCTURE
• pH of the cells ? 7,4 Zwitterion ionic forms
  of the amino acid (neutralnet charge 0). Soluble
  in water

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2.1. Amino acids.

• STRUCTURE
• Disulfide bridges between cysteine residues (S-S)

Thiol
Intrachain
Interchain
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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).

Dextrorotatory behaviour
Levorotatory behaviour
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2.1. Amino acids.

• STRUCTURE
• D,L-nomenclature is based on D- and
  L-glyceraldehyde

• L-amino acids predominate in nature

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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.
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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 dissociable hydrogens in
  the side chain)

Titration of Glycine
pI ½ (pK1 pK2)
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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)
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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
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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.
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2.1. Amino acids.

• UNCOMMON AMINO ACIDS
• They are produce by modifications of one of the
  20 amino acids already incorporated into a
  protein

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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
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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.

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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
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2.2. Peptides. Primary structure determination.

• PEPTIDE BOND

Due to the double bond character, the six atoms
of the peptide bond group are always planar!
Geometry of the peptide backbones.
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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-L
eu SGYAL
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2.2. Peptides. Primary structure determination.

• PEPTIDES PROPERTIES
• Peptides show polarity (direction).

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2.2. Peptides. Primary structure determination.

• PEPTIDES PROPERTIES
• Peptides ionic forms

• Minimal peptide solubilisation at pH pI
• No migration (no movement) in an electrical
  field.

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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
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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.

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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

• Thin layer chromatography.
• Ion exchange chromatography.
• Reverse-phase high-performance liquid
  chromatography (HPLC).

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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 411) 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.

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2.2. Peptides. Primary structure determination.

• ANALYSIS OF THE PRIMARY STRUCTURE OF A PROTEIN
  AMINO ACID SEQUENCE
• Ion exchange chromatography

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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

Proline yellow complex able to absorb at 440 nm
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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 N-terminal residue of a
protein. Successive reaction cycles can reveal
the amino acid sequence of a peptide
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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.

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2.2. Peptides. Primary structure determination.

• ANALYSIS OF THE PRIMARY STRUCTURE OF A PROTEIN
  AMINO ACID SEQUENCE
• Cleavage of disulfide bridges.

Met interferences
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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

FDNB (Sanger reagent)
peptide
(FDNB)
Acid hydrolysis
2-dinitrophenyl-peptide
2-dinitrophenyl-N-terminal residue
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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

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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)
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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).

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2.2. Peptides. Primary structure determination.

• ANALYSIS OF THE PRIMARY STRUCTURE OF A PROTEIN
  AMINO ACID SEQUENCE
• Fragmentation of the polypeptide chain

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2.2. Peptides. Primary structure determination.

• ANALYSIS OF THE PRIMARY STRUCTURE OF A PROTEIN
  AMINO ACID SEQUENCE

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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 A.
Terminal fragmentation 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 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 Rn1 ? Pro 3. Chymotrypsin
C-side of the Rn Rn Phe, Tyr, Trp,
Leu Rn1 ? 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
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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 post-translational
modifications
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2.2. Peptides. Primary structure determination.

• PEPTIDES OF BIOLOGICAL INTEREST

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2.3. Three-Dimensional structure and function of
proteins.

• PROTEIN STRUCTURE LEVELS OF ORGANIZATION

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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.

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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
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2.3. Three-Dimensional structure and function of
proteins.

• PROTEINS CLASSIFICATION

Conformation Overall three-dimensional
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.
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2.3. Three-Dimensional structure and function of
proteins.

• SECONDARY STRUCTURE. RAMACHANDRAN DIAGRAM

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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.
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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 nd)
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2.3. Three-Dimensional structure and function of
proteins.

• SECONDARY STRUCTURE. ?-HELIX

Right- hand twists
Left-hand twists
proline
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2.3. Three-Dimensional structure and function of
proteins.

• SECONDARY STRUCTURE. b-PLEATED SHEET

Strands run in opposite directions
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2.3. Three-Dimensional structure and function of
proteins.

• SECONDARY STRUCTURE. b-PLEATED SHEET

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2.3. Three-Dimensional structure and function of
proteins.

• SECONDARY STRUCTURE. b-TURNS

• Usually located in the protein surface.
• Stabilised by hydrogen bonds
• They allow the protein strands to change
  direction.
• Glycine and proline as predominant amino acids.

Proline isomers
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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 a-helix.
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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).
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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.

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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.
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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).

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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
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2.3. Three-Dimensional structure and function of
proteins.

• TERTIARY STRUCTURE

F. electrostáticas
F. van der Waals
P. hidrógeno
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2.3. Three-Dimensional structure and function of
proteins.

• TERTIARY STRUCTURE

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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
  thermophilic proteins).
• extreme pHs.
• Organic solvents(alcohol, acetone).
• Some detergents.
• Several salts ? chaotropic agents.
• Renaturation restoration of the native structure
  and biological role.

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2.3. Three-Dimensional structure and function of
proteins.

• TERTIARY STRUCTURE DENATURATION AND
  RENATURATION

Protein denaturation under two kind of external
stresses.
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2.3. Three-Dimensional structure and function of
proteins.

• TERTIARY STRUCTURE DENATURATION AND
  RENATURATION

• Anfinsens experiment (1957) Ribonuclease A
  RNase A (124 residues)

• Chaotropic compounds

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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
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2.3. Three-Dimensional structure and function of
proteins.

• QUATERNARY STRUCTURE

• Oligomer protein containing several identical
  subnits.
• Protomer structural unit of an oligomeric
  protein.
• Haemoglobin ? Tetramer containing two protomers.

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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.

Hair transversal section
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2.3. Three-Dimensional structure and function of
proteins.

• FIBROUS PROTEINS. SILK FIBROIN

• Antiparallel ?-pleated sheet.
• Tandem repetition GlyAla.
• Voluminous amino acids Val y Tyr.

Gly-Ala-Gly-Ala-Gly-Ser-Gly-Ala-Ala-Gly-(Ser-Gly-
Ala-Gly-Ala-Gly)8
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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, 4-Hydroxyproline
  (Hyp), 3-Hydroxyproline y 5-Hydroxylysins (Hyl).
• Tandem repetition Gly-X-Y (X?Pro Y? Hyp).

Structure of the collagen fibrils de colágeno
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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)

Top vision of the triple helix. Gly in red.
Collagen. (right handed).