4.

Structure and function

of proteins

Tertiary and Quaternary

Structure

1
 Globular proteins
Perform different functions:
Synthesis
Binding and Transport
Catalysis and Metabolism
They are compact structures folded on themselves
Different ways to show/study/display proteins

Bovin Pancreatic
Trypsin Inhibitor
BPTI
58 aa
Prevents trypsin
from catalysing
peptide hydrolysis

Skeletal model Ribbon model Space-filling model

Prof. G. Gilardi - Biological Chemistry 2
 Other examples
Many proteins have a prosthetic groups
These are small molecules non-covalently or covalently bound to the
protein to enable function - for example redox centres:

FMN FMN

Flavodoxin
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 Varieties of protein folds

sandwich

Parallel barrel

Antiparallel barrel Twisted sheet

Helix bundles Predominantly sheet Mixed /

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 Rules for 3ary folds

1. All globular proteins

have a define inside
and outside
Hydrophilic (green)
versus hydrophobic
(red)

Horse heart cytochrome c

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 -turn
Type I

2. -sheets are usually twisted or wrapped into H

barrel structures
3. There are different types of turns
-turns:
Type I: 4 residues where the C=O of residue 1 makes -turn
H-bond with the N-H of residue 4. Type II
Type II: same as type I but residue 2 has a bulky R, so
residue 3 is a Gly.
-turns:
Only 3 residues, of which only 1 is out of the H-
bonding sequence. Usually Pro is involved.
4. Some parts of proteins cannot be classified as
helix, -sheets or turns: these are random coil
regions. Usually are found at the N-ter and C-ter

-turns
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 Study of 3ary structure

Fluorescence
X-ray crystallography
NMR

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Absorbance spectroscopy:
Aromatic aminoacids
Absorbance

Detection of proteins
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 Fluorescence spectroscopy

Fluorimeter

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 Protein
Crystallography
A diffraction is observed when a radiation passes
through a regular, repeating structure;
The repeating unit in crystals is the unit cell that
may contain one or more molecules;
The scattered waves will be in phase only in
certain directions: these will give constructive
interference, the other will give destructive
interference;
The result is a diffraction pattern containing spots
of constructive interference;
The diffraction pattern will be sharp when the
repeating unit is very precise and when the
radiation used has a shorter than the regular
spacing in the elements of the structure: for this
reason X-rays are used (< nm).

Prof. G. Gilardi - Biological Chemistry 10

Prof. G. Gilardi - Biological Chemistry 11
 X-ray diffraction

The intensities of the diffraction spots

allow to calculate the 3D structure;
The steps to get a structure from X-
ray diffraction are:
Obtain good, regular crystals;
Record the diffraction pattern;
Apply the isomorphous replacement and
repeat the procedure
Calculate the structure factors, calculate
the electron density, energy minimise the
structure

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 From the electron density
to the 3D structure
The structure factors of all the spots allow to calculate
the electron density;
The structure is then fitted into the electron density: the
more spots we take into account, the more precise is
the structure (i.e. the higher is the resolution);

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Nuclear Magnetic Resonance

B0 nucleus

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Multi-dimensional NMR

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 How to study structures
Dowload the pdb (protein data bank) file from Brookhaven
National Laboratory (http://www.rcsb.org/pdb/)
For example search for cytochrome c: you will find a file named 1HRC

Load the molecule to Rasmol (see

http://www.umass.edu/microbio/rasmol/ ) or imol (see
http://www.pirx.com/iMol/) or PyMOL
(http://pymol.sourceforge.net/)

Learn how to display proteins and the real 3D structure of side

chains, helices, -sheets, turns, prosthetic groups.
imol
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Quaternary Structure

17
 Definitions
Proteins can associate to form complexes
made of multi-subunits
This level of structure is called 4ary
The interactions that provide the energy to
stabilize the multi-subunit structure are the
same kind that stabilize the 3ary structure:
Salt bridges, H-bonds, van der Waals interaction,
hydrophobic interactions, in some case di-sulphide
bridges
Depending on the type of subunits, we can
have:
Homotypic protein-protein interactions
Heterotypic protein-protein interactions
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 Homotypic protein-protein
interactions
When subunits associate, different levels of
symmetry can arise:

1. Helical symmetry
2. Point-group symmetry
These have different axis of symmetry and are classified
on that basis

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 Symmetry of
protein 4ary
structures

Herpesvirus with
icosahedral geometry

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 Pre-albumin dimer: Scheme of a dimer
2 monomers form a complete without symmetry:
-sandwich with a 2 fold symmetry A-C and B-D are blocked from
axis to the slide reaction with other monomers
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 Heterotypic protein-protein
interactions
Association of different proteins
Example: trypsin with the trypsin
inhibitor (BPTI)
This complex inhibits the activity of
trypsin in the pancreas
If structures are not available from X-
ray crystallography, docking
programmes (AUTODOCK) can be
BPTI-trypsin model
used found within 0.5 of the known

X-ray structure of the complex
The modelling of protein-protein
complexes is very successful, more
advanced than the 3D structure
prediction Prof. G. Gilardi - Biological Chemistry 22
 Outline of what
you must now know:

List variety of protein II-ary folds;

List and draw the type of turns that link II-ary structure motifs
to lead to III-ary structure;
Describe and discuss principles and potentials for methods
used to study protein structures.

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 Domande possibili
Proteine fibrose e proteine globulari: facendo uso di esempi
specifici di queste due classi di proteine discuti il ruolo della
struttura nel determinare la funzione di queste proteine.
Descrivi i due metodi di determinazione della struttura delle
proteine, cristallografia ai raggi X e risonanza magnetica
nucleare (NMR), discutendone i vantaggi e svantaggi.
Descrivi come funzionano le tecniche di assorbimento della
luce, di fluorescenza e di dicroismo circolare nello studio delle
proteine, e a quali amminoacidi e/o legami si riferiscono.

Prof. G. Gilardi - Biological Chemistry 24