unit 2. structure and function of proteins

UNIT 2.

Structure and function of proteins.

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.

OUTLINE

2.1. Amino acids.

STRUCTURE:

• 20 -amino acids = 20 common amino acids

• Uncommon amino acids

Proline (-imino acid)

Amino group

Carboxyl group

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.

pI = ½ (pK1 + pK2)

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

2.1. Amino acids.

• Amino acid with dissociable hydrogens in the side chain

Acidic amino acids(net negative charge at neutral pH):

pI = ½ (pK1 + pKR)

IONIC PROPERTIES/ACID-BASE PROPERTIES:

2.1. Amino acids.

• Amino acid with dissociable hydrogens in the side chain

Basic amino acids(net positive charge at neutral pH):

pI = ½ (pKR + pK2)

IONIC PROPERTIES/ACID-BASE PROPERTIES:

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

GABA (-aminobutyric acid):Neurotransmitter

Tiroxine:Hormone

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.

C N

O

C

C

HC N

O

C C

H

Trans Cis

Geometry of the peptide backbones.

PEPTIDE BOND:

Due to the double bond character, the six atoms of the peptide bond group are always planar!

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)

SerylglicylthyrosylalanylleucineSer-Gly-Tyr-Ala-Leu

SGYAL

PEPTIDES PROPERTIES:• Peptides show polarity (direction).

2.2. Peptides. Primary structure determination.

2.2. Peptides. Primary structure determination.

H3N-CH-C-N-CH-COOH+

CH3

H

‖OHS-H2C

H3N-CH-C-N-CH-COO-+

CH3

H

‖OHS-H2C

H2N-CH-C-N-CH-COO-

CH3

H

‖OHS-H2C

Cationic form (pH ) Anionic fom (pH )Zwitterion

(Cys-Ala)

• Minimal peptide solubilisation at pH= pI

• No migration (no movement) in an electrical field.

PEPTIDES PROPERTIES:

• Peptides ionic forms:

2.2. Peptides. Primary structure determination.

Amphoteric behaviour Tetrapeptide (Glu-Gly-Ala-Lys)

WHAT DO YO HAVE TO KNOW:- How to calculate the isoelectric point related to a peptide

PEPTIDES PROPERTIES:

• Titration curve:

• 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:

2.2. Peptides. Primary structure determination.

Thin layer chromatography.

Ion exchange chromatography.

Reverse-phase high-performance liquid chromatography (HPLC).

10-100 horas a 105-110 ºC

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

Acid hydrolysis liberates the amino acids of a protein

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

Hidrantine

Amino acid

Purple max = 570 nm

Proline: yellow complex able to absorb at 440 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 N-terminal 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.

or2-mercaptoethanol

HC – O - OHO‖

( 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:

(FDNB)peptide

FDNB (Sanger reagent)

2-dinitrophenyl-peptide

2-dinitrophenyl-N-terminal residue

Acid hydrolysis

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.

PhenylisothiocyanatePeptide

Peptide-PTC (phenylthiocarbamil)

PTH-alanine(PTH derivative)

Smaller peptide (one amino acid residue is released)

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 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

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

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 post-translational 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:

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:

• Biological role:

2.3. Three-Dimensional structure and function of proteins.

PROTEINS CLASSIFICATION:

On the basis of the shape and solubility:Fibrous proteinsGlobular proteinsMembrane proteins

On the basis of the chemical composition:SimplesConjugates: (it contains non peptidic component: prosthetic group)

Apoprotein: protein without prosthetic group.Holoprotein: protein + prosthetic group.

- Glucoproteins- Lipoproteins- Methaloproteins- Phosphoproteins- Haemoproteins

Conformation: Overall three-dimensional architecture of a protein (the radicals can modified their spatial position by rotation. Bonds are not cleavage during this process.

2.3. Three-Dimensional structure and function of proteins.

PROTEINS CLASSIFICATION:

Configuration: Geometric possibilities fro a particular ser of atoms. In going from one configuration to another, covalent bonds must be broken ant rearranged.

SECONDARY STRUCTURE. RAMACHANDRAN DIAGRAM:

2.3. Three-Dimensional structure and function of proteins.

Ramachandran diagram corresponding to L-Ala residues.

SECONDARY STRUCTURE. RAMACHANDRAN DIAGRAM:

2.3. Three-Dimensional structure and function of proteins.

The reasonable conformations are those avoiding steric crowding

and angles = 0º, no favourable conformation in proteins.

n = 3.6 residues (single turn)nº atoms/single turn = 13d = 0.15 nm = 1.5 ÅTravel along the helix axis per turn (pitch of the helix)(v) = 0,54 nm = 5,4 Å (v = n·d)

SECONDARY STRUCTURE. -HELIX:

2.3. Three-Dimensional structure and function of proteins.

Left-hand twists Right- hand twists

Hydrogen bonds

H2N

CH2

CH2

H2C

CH

COO-

+

N

CH2

CH2

H2C

CH

C - C

C – N - C

‖

‖O

O H

proline

2.3. Three-Dimensional structure and function of proteins.

SECONDARY STRUCTURE. -HELIX:

7 Å

SECONDARY STRUCTURE. -PLEATED SHEET:

2.3. Three-Dimensional structure and function of proteins.

Strands run in opposite directions

2.3. Three-Dimensional structure and function of proteins.

6.5 Å

SECONDARY STRUCTURE. -PLEATED SHEET:

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

SECONDARY STRUCTURE. -TURNS:

2.3. Three-Dimensional structure and function of proteins.

SECONDARY STRUCTURE.

2.3. Three-Dimensional structure and function of proteins.

Bovine Carboxipeptidase A, it contains 307 residues and consists of a -pleated sheet (8 strands) and 6 -helix.

and values corresponding to all the piruvate quinase amino acids

residues (except Gly).

Right-handed -helix

Rigth-handed -sheet

Collagen triple helix

antiparallel -sheet

Parallel -sheet

SECONDARY STRUCTURE. RAMACHANDRAN DIAGRAM:

2.3. Three-Dimensional structure and function of proteins.

Left-handed-helix

- 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.

2.3. Three-Dimensional structure and function of proteins.

SUPERSECONDARY STRUCTURES:

-- loop

- vertex chains right handed

conected

Conections between chains

Barrel

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.

SUPERSECONDARY STRUCTURES:

- Some globular proteins contains a combination of different super secondary structures called DOMAINS OR MODULES.

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).

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:

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:

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.

2.3. Three-Dimensional structure and function of proteins.

TERTIARY STRUCTURE: DENATURATION AND RENATURATION:

2.3. Three-Dimensional structure and function of proteins.

TERTIARY STRUCTURE: DENATURATION AND RENATURATION:

Protein denaturation under two kind of external stresses.

• 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.

2.3. Three-Dimensional structure and function of proteins.

TERTIARY STRUCTURE: DENATURATION AND RENATURATION:

Chaperonin from E. coli. GroEL/GroES complex

Oligomer: protein containing

several identical subnits.

Protomer: structural unit of an

oligomeric protein.

Haemoglobin Tetramer

containing two protomers.

2.3. Three-Dimensional structure and function of proteins.

QUATERNARY STRUCTURE:

1

1

2

2

• 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.

-helix

Coiled-coil superhelical structure

Protofilament

Protofibril

Cells

Intermediate filaments

keratin -helix

Coiled-coil superhelical structure

Protofilament

ProtofibrilHair transversal section

2.3. Three-Dimensional structure and function of proteins.

FIBROUS PROTEINS. -KERATIN:

• 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. SILK FIBROIN:

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 (XPro; Y Hyp).

2.3. Three-Dimensional structure and function of proteins.

FIBROUS PROTEINS. COLLAGEN:

Structure of the collagen fibrils de colágeno

Collagen.(right handed).

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.