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Erez Podoly
Introduction to Molecular Neurobiology
From Primary to Quanternary Structure
1º
2º
3º
There are three major 2º components: helices, β-sheets
and what’s in-between them (turns and loops).
 helices and  sheets are stabilized by hydrogen
bonds between backbone oxygen and hydrogen atoms.
4º
The Protein Folding Problem
In the 1960’s, C.B. Anfinsen performed a series of in vitro
experiments that lead him to the “Thermodynamic Hypothesis”: As he
stated ten years later, in his 1972 Nobel acceptance speech, “The
native conformation is determined by the totality of interatomic
interactions and hence by the amino acid sequence, in a given
environment”.
Anfinsen’s hypothesis has
some exceptions: Some
proteins
have
multiple
conformations and some
proteins get folding help
from chaperones.
Chou and Fasman
Amino Acid
Ala
Cys
Leu
Met
Glu
Gln
His
Lys
Val
Ile
Phe
Tyr
Trp
Thr
Gly
Ser
Asp
Asn
Pro
Arg
-Helix
β-Sheet
Turn
1.29
1.11
1.30
1.47
1.44
1.27
1.22
1.23
0.91
0.97
1.07
0.72
0.99
0.82
0.56
0.82
1.04
0.90
0.52
0.96
0.90
0.74
1.02
0.97
0.75
0.80
1.08
0.77
1.49
1.45
1.32
1.25
1.14
1.21
0.92
0.95
0.72
0.76
0.64
0.99
0.78
0.80
0.59
0.39
1.00
0.97
0.69
0.96
0.47
0.51
0.58
1.05
0.75
1.03
1.64
1.33
1.41
1.23
1.91
0.88
Favors
-Helix
Favors
β-strand
Favors
turn
Leventhal Paradox
How much time does it take to a given protein (100aa) to fold into
a single stable native conformation (assuming three
conformations/peptide bond)?
Leventhal Paradox
• 3100 = 5.15 × 1047 conformations.
• Fastest motions 10-15 sec.
• Sampling all conformations would take 5.15 × 1032 sec.
• 60 × 60 × 24 × 365 = 3.15 × 107 seconds in a year.
• Sampling all conformations will take 1.6 × 1025 years.
• The age of the universe is ~ 11-20 × 109 years.
The Leventhal Paradox: proteins are able to quickly fold into
their conformations despite such an overwhelming number of
possibilities.
Quick Overview of Energy
Bond
H-bonds
Ionic bonds
Hydrophobic
interactions
Van der vaals
interactions
Disulfide
bridge
Strength
(kcal/mole)
3-7
10
1-2
1
51
The Protein Folding Problem
Proteins could fold
more quickly if they
retain
native-like
intermediates along
the way.
The Protein Folding Problem
Much of conformation space is already
restricted by allowed phi/psi angles
(Ramachandran plot).
Gopalasamudram
Narayana Iyer
Ramachandran
Y
(1922 – 2001)
F
The Dihedral Angles – Φ, Ψ
Each unit can rotate
around two such bonds:
Cα-N (phi) & Cα-C (psi).
Most combinations of Φ & Ψ angles
are not allowed due to steric collisions.
Cα -N bond (phi)
Cα -C bond (psi)
Ramachandran Plot
The angle pairs Φ & Ψ are usually plotted against each other in a
diagram called a Ramachandran plot.
Most of Ramachandran plot area
includes values that are not allowed.
Colored areas show sterically allowed
regions.
Y
F
Secondary Structures
The
major
allowed
regions
in
Ramachandran
conformational components of proteins.
plot
define
Structural Classification of Proteins
CLASS: , β, /β, +β (but also: multi-domain, membrane
and cell surface, small proteins, coiled coil proteins).
FOLD: secondary structures in same arrangement.
SUPERFAMILY: function/structure similarity.
FAMILY: >30% sequence similarity, and similar known
structure/function.
Protein Class
Protein Fold
Protein Superfamily
Protein Family
Structural Classification of Proteins
• SCOP Manual classification http://scop.berkeley.edu/
• CATH Semi-manual classification http://www.cathdb.info/latest/index.html
• FSSP Automatic classification http://ekhidna.biocenter.helsinki.fi/dali/start
Class
Folds
Superfamilies
Families
All 
218
376
608
All β
144
290
560
/β
136
222
629
+β
279
409
717
Multi-domain
46
46
61
Membrane & cell surface
47
88
99
Small proteins
75
108
171
Total
945
1539
2845
Proteins’ Classes
All 
All β
/β
Proteins’ Folds
Proteins are defined as having a common fold if they have the same
major secondary structures in the same arrangement and with the
same topological connections.
A structural domain is an element of overall structure that is selfstabilizing and often folds independently of the rest of the protein
chain; Most domains can be classified into "folds".
Because they are self-stabilizing,
domains can be "swapped" by genetic
engineering between one protein and
another to make chimera proteins.
http://pawsonlab.mshri.on.ca/index.php?option=com_content&task=view&id=30&Itemid=63
The Structure/function paradigm
In parallel with the growth in structural knowledge, there has been
an increasing conviction that the biological function of proteins is
encoded in their 3D structure. Most molecular biologists believe
that determining protein functions depends on the protein structure.
The Structure/function paradigm: the
amino acid sequence determines
protein 3D structure and the structure
determines the function.
Intrinsically Disordered Proteins
A significant proportion of proteins contain regions, sometimes
quite large, that apparently don't fold into specific structure, but
rather remain as flexible ensembles.
These regions are termed "intrinsically disordered”, but also
“intrinsically unstructured” or “Naturally unfolded”.
Disordered regions are sequences within
proteins that fail to fold into one fixed
structure. It doesn’t mean they don’t
have a structure, on the contrary: we
may consider them as “multiple folded”.
Expansion of the Structure/Function Paradigm
“Creating a new theory is not like destroying an old barn and
erecting a skyscraper in its place. It is rather like climbing a
mountain, gaining new and wider views, discovering unexpected
connections between our starting points and its rich environment.
But the point from which we started out still exists and can be seen,
although it appears smaller and forms a tiny part of our broad view
gained by the mastery of the obstacles on our adventurous way up”.
-- Albert Einstein, The Evolution of Physics.
Function can arise from the two protein forms (the
ordered state, but also the random coil state) and
transitions between them. Thus, proteins that lack a
3D structure may carry out function.
The History of Structural Biology
1895: W. C. Roentgen discovers X rays.
1912: Max von Laue discovers X-ray diffraction by crystals.
1913: W. L. Bragg reports the crystal structure of NaCl .
1935: J. M. Robertson solves the structure of pthalocyanin.
1948: Bijvoet solves strychnine (cryst. decides bet. alternatives).
1958: Kendrew reports the crystal structure of Myoglobin.
1962: M. F. Perutz and Sir J. C. Kendrew win the Nobel Prize
for their studies on the structures of globlular proteins.
1965: Lysozyme. 1968: Haemoglobin . 1971: Insulin.
1971: PDB is established at Brookhaven National Lab., NY.
Methods for Structure Determination
Methods for Structure Determination
X-Ray
NMR
Rate limiting Step
Crystallization
Resonance assignment
Physical principle
Electron diffraction
Spin of nuclei in magnetic field
Hydrogens
Invisible
The only detectable atom
# structures in solution
1
15-20
Size limitation
None (MDa)
64KDa
Wavelength Range
0.1 – 100 Å
0.6 – 10 m
Quality measure
Resolution and R factor
RMSD
The result
Snapshot
Protein movements
Thermodynamics
Not possible
Kinetic measurements
Methods for Structure Determination
NMR
X-Ray