Download Macromolecules. Folding of proteins.

Biopolymers
Macromolecules.
Macromolecules.
Folding of proteins.
proteins.
Mitotic spindle of a dividing cell
November 14th, 2011.
Huber Tamá
Tamás
Actin filament network in
the epidermal cell of
Tobacco leaf.
Biopolymers
DNA
Polimer: Chains constructed of similar building blocks
(monomers, subunits)
Number of monomers: tipically 102-104
Titin: 3.435*104 aminoacid C132983H211861N36149O40883S693
Human chromosoma 1: 2.25*108 nucleotide
Biopolymer
DNA strand released from bacteriophage
Subunit
1953 - Rosalind Franklin – DNA double helix
X-ray diffraction
pattern of DNA
Bond
Protein
Amino acid
Covalent
(peptide bond)
Nucleic acid
(RNA, DNA)
Nucleotide
(CTUGA)
Covalent
(phosphodiesther)
1953 James D. Watson and
Francis Crick – DNA model
Polysaccharide
(e.g., glycogen)
Sugar
(e.g.,
glucose)
Covalent
(e.g., -glycosidic)
T-A (2 hydrogen bonds)
Protein polymer
(e.g., microtubule)
Protein
(e.g.,
tubulin)
Secondary
C-G (3 hydrogen bonds)
DNA primary structure
NATURE-1953
Phosphodiester bonds
between 2 deoxyriboses
1962 Nobel prize
Robert Cecil Olby - The path to
the double helix: The Discovery
of DNA
DNA double helix
James D. Watson - The Double
Helix
Hydrogen bonds between
the complementary bases.
1
DNA secondary structure
Structure of proteins
Primary structure: amino acid sequence
Major groove
Minor groove
Frederick Sanger
1958 Nobel prize
Determining the
sequence of insuline
1955
A-DNA: short (2.4 nm), wide
Hydrogen bonds
B-DNA: long (3.4 nm), narrow
Electrostatic forces
Z-DNA: elongated (alternation of purinepirimidine bases)
Van der Waals
Secondary structure of proteins
Secondary structural
elements are stabilized
by hydrogen bonds.
Peptide bond
Tertiary and quaternary structure of proteins
Spatial reletionship of secondary
structural elements relative to
each other.
Spatial assembley of subunits
β-sheet
Hemoglobin α-subunit
α-helix
Hemoglobin A
(2α- and 2β- subunit)
Protein folding
Driving force of the folding
Hydrophobic core
Unfolded protein
Native state of protein
Hydrophil amino
acids on the surface
2
Levinthal paradox
Anfinsen experiment, RNase A (1961)
Wrong conformation
Native structure
Cyrius Levinthal - 1976
Every peptid unit has ~ 10
conformational states
In the case of an 100 aa.-long polipeptide
chain
10100 variations
id
a
tio
n
1 conformational state: 10-13 s
-mercapto
ethanol
fa
st
ox
Conformation
In the reality the folding occurs within
slow
oxidation
Denaturated structure
Cyrius Levinthal
1089 s, ~ 1081 years are needed for
reaching the native state (longer than
the age of the universum)
Energy
-mercapto
ethanol
1 second!
Native structure
Conclusions:
The 3D structure of proteins is determinated by their amino acid sequence.
The native structure is thermodynamically the most stabilized state.
Energy landscape for protein folding
Two-state system
General case
Misfolded proteins
Prion: propageted, misfolded proteins,
infectious agents
- Transmissible spongiform encephalopaty
(Kuru or laughing sickness)
Daniel Carleton Gajdusek – 1976 Nobel prize
-Creutzfeldt-Jakob disease
-bovine spongiform encephalopaty (mad cow
disease)
Accumulation of β-amyloid – Alzheimer’s disease
The depth of the well symbolizes the energetic stabilization of the native state
versus the denaturated state.
All paths lead to the native state (energetic minimum).
Amiloyd plaques in mice
brain
Models to use to describe the felexibility of
polymers
Polymer shape resembles random walk
(Brownian motion)
rN
R 2  Nl 2  L  LP
ri = elementary vector
R = ”end-to-end” distance
r1
The polymer chain is not rigid, due to its flexibility!
“Square-root law”:
R
Reason of flexibility
•
•

ri  LP
Diffusion:
<x2>=2D
<x2> = mean squared displacement
D = diffusion constant
 = diffusion time (duration of
observation)
= persistence length
N = number of elementary vectors
Nl = L = contour length
• Persistence length informs
about the bending stiffnes of
the polymer!
Members of Fore tribe of New
Guinea suffering in Kuru
•
•
1. Rotation around C-C
bonds,
2. Rigid segments
connected with flexible
(frictionless) joints
3.Torsion of bonds
Titin, DNA
1
2
Describing model
• 1. Freely Rotating Chain
(FRC)
• 2. Freely Joint Chains
(FJC),
• 3. Worm Like Chain
(WLC).
3
• Contour length is the streched
out length of the polymer!
3
Flexibility of biopolymers
Biopolymer mechanics
LP=Persistence length
L= Contour length
Elasticity of the entropic chain
Rigid chain
LP>>L
LP=1-6 mm
Entropic elasticity
Microtubule
Correlation length
Semiflexible chain
LP~L
LP=0.1-20 μm
End-to-end distance (R)
Actin filament
configurational entropy increases
(orientation entropy of elementary vectors).
Flexible chain
LP<<L
LP=9-16 nm
Force (F)
The polymer chain exhibits thermally
driven bending motions
F = force
l = correlation length
(persistence length)
k B = Boltzmann’s constant
T = absolute temperature
L = contour length
R/L = relative extension
Titin
Mechanical manipulation of polymers
(Optical tweezer)
Tying a knot on an single actin filament by
optical tweezer!
Laser
focus
DNA molecule
Latex
bead
Highly focused laser beam
can trap particles.
Moveable
micropipette
Mechanical manipulation of polymers (Atomic
Force Microscopy)
Mechanical manipulation of Titin by AFM.
Stretching of titin by
AFM.
F ~ stability of domains
Distance between peaks ~
contour length
10μm
Deflection of the cantilever can be
calibrated as the force used to
manipulate the protein.
Cantilever is extremely flexible, it
deflects under some pN force (10-12)
Force extension curve
4
Biological relevance of Titin
(Molecular spring)
 -amiloid filaments on mica surface, scanning mode
It consists of
over 300 serially
linked Ig-domain.
Main source of elasticity
in the myofibril
The End!
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