STRUCTURAL BIOLOGY

Martina MijuškovićETH Zürich, Switzerland

Cell- the basic unit of life

Protein synthesis in the cell

The central “dogma” of molecular biology

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DNA structure

DNA: deoxyribonucleic acid- genetic material of the cell

Building blocks: nucleotides (A, T, C, G)

GENES ARE DNA!

1 GENE= 1 PROTEIN

3 nucleotides of DNA= 1 amino acid in a protein!

DNA structure

A view along the helix axis

A view down the helix axis

Nucleotide: BASE SUGAR PHOSPHATE

DNA structure

Bases in a double stranded DNA are connected by hydrogen bonds!

(A-T or C-G)

Protein structure

Proteins:

• building blocks of cells and organisms

• constructed from amino acids (22 different types)

Diversity of functions:

• enzymes (proteins that catalyze chemical reactions in the cell)

• structural proteins (collagen in extracellular matrix, proteins of cytoskeleton...)

• transport proteins (hemoglobin...)

• messengers (neurotransmitters, hormones...)

• antibodies

• etc...

Protein structure

4 “levels” of protein structure:

PRIMARY- amino acid sequence (Met-Thr-Ala-Ser...)

Amino acids are connected by PEPTIDE BONDS!

That makes protein a POLYPEPTIDE.

SECONDARY- short distance interactions within main chain atoms (α-helix, β-sheet)

TERTIARY- the whole 3D structure of a protein, interactions between amino acids distant in sequence but close in space

QUATENARY- interactions between subunits of a protein (if a protein is built from more than 1 polypeptide chain)

Protein structure

Ferritin: 4 α-helices make a binding site for a iron ion

Cro repressor: special α-helices recognize DNA

Important: structure-function relationship!

Protein structure

Porin: a β-barrel, very stable protein channel in bacterial outer membrane

Plasma retinol binding protein: a β-sandwich, carrier of vitamin A in the blood

Why do we need to know 3D structures of proteins?

• deeper understanding of basic biological concepts and processes

• understanding the cause of diseases

• drug design

• protein engineering (design of proteins with novel properties)

• etc...

How to determine macromolecular 3D structures?

2 main methods:

A) X-ray crystallography

• a macromolecular crystal is necessary

• can be applied on very big structures (ribosome, viruses)

B) NMR (nuclear magnetic resonance spectroscopy)

• structure is determined from the solution

• limited on smaller proteins (about 100 amino acids)

How x-ray crystallography works?

Analogy with light microscopy:

• an object is seen in the microscope because light is reflected from its surface: this light is focused by lenses to form an image

• macromolecules can be “seen” by X-ray diffraction BUT there are no lenses which can focus X-rays

Why X-rays?

• resolution limit of light is ½ of the wavelength

• cells and organells are down to 200 nm in size (visible light)

• typical covalent bond is 0.12 nm (X-rays)

How x-ray crystallography works?

• X-rays are scattered from a regular repeating arrays of macromolecules (CRYSTAL!)

• a pattern of constructive and destructive interference is used to determine the structure

• mathematics is used as a lens to transform the diffraction pattern into an original structure

a diffraction pattern of a macromolecule

Protein x-ray crystallography- practical point of view

A) cloning B) expression

6.5

14.4

21.5

31.0

45.0

66.2 kDa

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pET3aTBPS1595153bp

TBP

NdeI (4088)

BamHI (4639)

Expression vector: a plasmid carrying the gene of interest

Protein SDS PAGE gel: each band corresponds to one protein

TBP

D) crystallization E) solving the structureC) purification

A protein crystalSDS PAGE showing a purified protein

Ribbon representation of a protein structure (violet) bound to DNA

Protein x-ray crystallography- practical point of view

Some important recent structures and what can we learn from them

K. Luger et al, Nature 1997.

The nucleosome core particle

Aquaporin

(water channel)

H. Sui et al, Nature 2001.

Some important recent structures and what can we learn from them

The ribosome

(large subunit)

N. Ban et al, Science 2000.

Some important recent structures and what can we learn from them

Recommended literature

1. Alberts et al. : Molecular biology of the cell

2. Voet and Voet: Biochemistry

3. Van Holde: Principles of physical biochemistry