Proteins

Proteins are polypeptides

Biochemistry Lectures

Amino Acids, Proteins, Enzymes

DR. M. Sasvári

Polypeptides: The peptide bond

Peptide bond: Condensation reaction

between the -carboxyl group and the -amino group:

-H2O

How to write a dipeptide?

+H3N

C

C

N

C

COO-

R

R

O

H

N terminal C-terminal

H

H

-glutamyl-cystein

Glu -Cys

+ H2N-Gly

A biologically important tripeptide: Glutathion

+ Glu-COOH H2N-Cys

-glutamyl-cystein synthetase

-glutamyl-cysteinyl-glycine

glutathion

Glu -Cys-Gly

glutathion synthetase

Glu -Cys

ATP

ATP

N

C

H

O

C

CH2SH

HN

C

H

O

C

O

CC

CH2SH

HH

N

H

C

H

COO-

H

N

H

C

H

COO-

H

+ H 3 N

H2C

CH 2

C

O

C - COO

-

H

+ H 3 N CH 2

C

O

C

O O

C - COO

-

H H

Function of Glutathion

2 G-SH + H2O2 G-S-S-G + 2H2O

reduced glutathion (thiol group) oxidized glutathion (disulphide bridge)

Importance: Maintaining of cellular redox state

Reductive power against oxidative stress

Red blood cells: high O2 concentration

oxidative effect

free radical formation

hydrogen peroxide formation

lipid peroxidation

NutraSweet® (aspartame), an artificial sweetener

Dipeptide of Asp and Phe (carboxyl terminal is esterified)

+H3N

C

C

N

C

CO-O- CH3

CH2COO-

O

H

N terminal C-terminal

H

H

CH2

methylester

Calculation of Ip:

Polypeptides: Isoelectric point

Sequence of a tetrapeptide: Glu-His-Arg-Gly

Charges at pH 7:

Isoelectric form:

+ + + -

Ip:basic Ip: (6.0 + 9.7)/2

+

Arg (guanidino) (pKa = 12.5)

- -carboxyl group (pKa = 2.3)

-amino group (pKa = 9.7) + +

His (imidasol) (pKa = 6.0)

- Glu -carboxyl group) (pKa = 4.3)

Example:

+ + -

-

-

Lost Next

Other biologically important peptides

Peptide hormones:

thyrotropin releasing factor (3 amino acid residues)

oxytocin (9) – uterin contraction

bradykinin (9) – inhibits inflammation

enkephalins (CNS)

small proteins – large peptides

insuline (30+21)

glucagon (29)

Levels of protein

structure

Primary structure

Quaternary structure

Main molecular forces

- Amino acid sequence - covalent bonds

(peptide bond and disulphide bridge)

Secondary structure -H-bonds between the atoms of the peptide group

Tertiary structure - Secondary bonds between the side chains of the same

polypeptide

- Secondary bonds between the side chains of different

polypeptides (subunits)

CONFORMATION

Chemical:

e.g. cooking the proteins in strongly base

result: mixture of amino acids

Enzymatic:

proteases (endopeptidases)

result: smaller peptides

Primary structure: peptide bond

Cutting the peptide bond: Hydrolysis

The peptide group is rigid and planar

RESONANCE INTERACTION

Consequences:

1. Planarity

2. Trans conformation

C-N bond will have some double bond character

C

O

N

H

.. C

C

carbonyl group

p bond

lone electron pair

of N

Six atoms of peptide group: C1 – CO-NH-C2

Rotation is permitted at the N-C (f) and C- C () bonds

Limited rotation of atoms around the peptide bond

Ramachandran plot: Prediction of secondary structure

possible angles of free rotation

C f N

H

O

C N

H

O

C H

R

b strands

helix

Molecular forces:

H-bond between the atoms of the peptide groups

O

H

C N

O

H

C N

Backbone structure

Side chains has only small influence

Secondary structure

Conformation of the polypeptide backbone

Examples:

-helix

parallel and antiparallel b-sheets

collagen helix (see later)

The conformational relationship of neighboring amino acids

Right handed -helix

R groups protrude outward from the helical backbone

Space-filling

model

Ball-and-stick model

backbone

Left Right

handed helix

Stability of an -helix is decrease by

1. electrostatic repulsion (or attraction)

of adjacent positive (or negative) side chains

2. bulkiness of adjacent side chains

3. (ionic) interaction between amino acid side chains

spaced tree (four) residues apart

4. positively charged amino acid at the C-terminal, or

negatively charged amino acids at the N terminal

5. presence of Proline

Stability of the -helix and b-sheet is based on

- the minimized steric repulsion between the side chains

- the maximized H-bonds between the peptide groups

Stability of the -helix

Antiparallel chains of b-conformation

b-sheets: extended, zizag structure

Alternating pattern of R groups

H-bond

b-turn

Other secondary structures

A connection between

the ends of antiparallel b-chains

• a tight connection

• involves 4 amino acid residues

• H bond between the peptide groups

of the 1st and 4th amino acids

• Gly and Pro occur frequently

• often found near to the surface of the protein

Protein folding: Tertiary structure

Amino acid side chain interactions of the same polypeptide chain

Longer-range aspects of amino acid sequence

Types of interactions:

nonpolar (Van der Waals)

polar

H bonds

ionic interactions

hydrophobic

pocket (hydrophobic

side chain

interactions)

hydrophilic surface (polar interaction

of side chains with water)

Conformations: many helices

Conformations: b sheets

Quaternary structure – subunit structure

Amino acid side chain interactions of different polypeptide chains

Longer-range aspects of amino acid sequence

Types of interactions:

nonpolar

polar

H bonds

ionic interactions

A leucin zipper (DNA binding protein)

2 polypeptides (all-alpha subunits)

2 identical (all-beta) subunit

Enzymes are proteins

Cofactors

Prostetic groups

Metal ions

Coenzymes

Coordinative bond

Covalent bond

Secondary interactions

Optimal

Conditions:

Ionic strength

Temperature

Solvent

pH

Native conformation

Maximal catalytic activity

Temperature dependence of enzyme activity

T

Enzyme

activity

Heat denaturation

changes in conformation

Heat sensitivity is different

Heat-shock proteins

Q10 temperature coefficient

Optimal temperature

pH

Enzyme

activity

5 6 4

pH profiles

pH optimum Isoelectric pont =

pH dependence of enzyme activity

Active center:

Glu COOH pKa = 5.9

- Lysozyme

Asp bCOOH pKa = 4.5

Optimal pH?

Example:

pH

4 5 6 protonated deprotonated

4.5 5.9

Asp - Glu

optimal pH

Analysis and separation

of proteins

Appendix

Gel electrophoresis (practice)

SDS-PAGE

isoelectric focusing

two-dimensional electrophoresis

Gel filtration (practice)

Dialysis (desalting)

Affinity chromatography

Ion exchange chromatography

Analysis of protein mixtures – SDS-PAGE

1. SDS-PAGE (see: practice book) – separation of protein mixtures

Denaturation of proteins, negative charges

Crude extract (1st lane)

Proteins after the purification steps (others)

four subunits (after denaturation)

Purified protein

SDS-PAGE: Cotrolling the purification procedure

SDS-PAGE: Determination of molecular weight of proteins

Analysis of protein mixtures: Isoelectric focusing

Analysis of protein mixtures: Two dimensional electrophoresis

1st dimension:

Isoelectric focusing

2nd dimension:

SDS-PAGE

Identification of more than 1000 proteins from E. Coli

Dialysis: Separation of proteins from small molecules (desalting)

Gel filtration: Separation of proteins from small molecules (desalting)

Protein purification: Affinity chromatography

Separation is based on the

specific binding

between the LIGAND and the protein

The ligand is cross-linked to the beads

Protein purification: Ion exchange chromatography

Separation is based on

charges of the proteins

(could be used for amino acids, too)

Chromatography

paper ~ (see prctice)

thin-layer ~ (see prctice)

column ~