Upload
others
View
1
Download
0
Embed Size (px)
Citation preview
1
Proteins
Protein folding
10112010
Polypeptides and proteins 1
bull peptide short polymers of amino acidslinked by peptide bonds (lt ~50 amino acids)
bull protein long polymers of amino acidslinked by peptide bonds (gt ~50 amino acids)
Amino acid
bull A group of organic molecules that contains a basic amino group (-NH2) an acidic carboxyl group (-COOH) and an organic R group (or side chain) which is unique to each amino acid
Amino acidsbull The unit within a protein molecule
bull 20 amino acids building up the proteins within the human body
bull Essential amino acids (9 pcs) the human body can not poduce them in a proper amount (methionine)
bull To build a 100 amino acid long polymer from the 20 amino acids ndash the number of the variation is huge (20100
= ~ 1310130)
bull The order of the amio acids = amino acid sequence rarrprimary structure
Polypeptides and proteins 2
H2N ndash C ndash C
H
R1
O
OHH2N ndash C ndash C
H
R2
O
OH
amino-group carboxylic-group amino-group carboxylic-group
N ndash C ndash C
H
R2
O
OHH
H2N ndash C ndash C
H
R1
O
+ H2O
Dehydration or condensation
Amide
Peptide bondN-terminus C-terminus
Protein
1926 - James B Sumner (biochemist - USA) chrystallized an
enzyme called urease and showed that it is a protein rarr Nobel
prize in Chemistry (1946 - John Howard Northrop amp Wendell
Meredith Stanley)
1958 - Max Perutz amp Sir John Cowdery Kendrew solved the strucure of hemoglobin and myoglobin rarr Nobel prize in
Chemistry in 1962
(X-ray chrystallography rarr 3D structure)
2
bull structural (collagen)
bull transport (myosin)
bull biochemical reactions (enzyme)
bull immunology (antibodies)
bull signal transduction (hormones)
Protein functions Primary structure
The sequence of the monomeric units
within a chain of amino-acids connected by peptide bounds
Primary structure
C
N
O
C
Primary structure
Forming a spatial-structure (structure in space)Motifs bull alpha helix
bull beta sheet
Secondary structure Alpha helix
H
O
H O
Hydrogen-bond
Rolling up on the surface of an imaginary cylinder (+ or -)
3
Hydrogen bond
An electrostatic dipole-dipole interactionthat involves a hydrogen atom
δ+ δ+
δ-
δ+ δ+
δ-
Electronegativity a chemical property that describes the ability of an atom to attract electrons towards itself
H2O
H2O
Beta sheet
Parallel and antiparallel beta sheets
Tertiary structure
bull Folding Forming the final functional 3D forms of the proteins
bull Domain formation
bull Under physiological conditions a spontaneos disorder hArrorder transition = bdquofoldingrdquo
bull Chaperon A guide or companion to the protein that help to form its tertiary structure
bull Disulfide bridge hidrogen bond and hydrophobicinteractions are sabilizing the folded protein
Disulfide bond
A covalent bound (primary) forming between thiol groups (eg cysteine)
A link between two sulfur atoms
Hydrophobic interactions
bull No affinity for water (tending not to be dissolved in or mixed with water)
bull Usually non polar molecules are involved
bull one of the principal driving forces behind the protein folding
bull minimizing the number of hydrophobic side-chains exposed to water
bull the hydrophobic amino acids are shielded from the aqueous solvent
bull Very hydrophobic amino acids
Valine isoleucine isoleucine leucine methionine phenylalanine cysteine tryptophan
4
Tertiary structure Quaternary structure
The entire protein assembly
Two or more peptide chains forming the functinal form of a protein (eg hemoglobin)
wwwpdborg
Folding
A process where the tertiary structure (3D
shape) of a protein is formedThe functional form of the protein comes to
life
Misfolding
The folding is not succesfull (eg beta sheets instead of alpha helices) rarr misfolded proteins
The cell remove the wrong protein rarr the amount of the functional proteins decrease
The cell will not remove it rarr deposits (plaque) within the cells (Alzheimer disease)
Protein folding diseases
P53cancer
crystallinsCataract
rhodopsinRetinitis pigmentosa
Transthyretin lysosymeFamiliai amyloidoses
Prion proteinCreutzfeldt-Jakob disease
α-synucleinParkinsonrsquos disease
Amyloid β-peptidetauAlzheimerrsquos disease
CollagenScurvy
β-hexosaminidaseTay-Sachs disease
α1-Antitrypsin α1-Antitrypsin deficiency
HaemoglobinSickle cell anaemia
ProcollagenOsteogenesis imperfecta
FibrillinMarfan syndrome
HuntingtinHuntingtonrsquos disease
Phenylalanine hydroxilasephenylketonuria
Cystic fibrosis trans-membran regulatorCystic fibrosis
Low-density lipoprtotein receptorHypercholesterolaemia
PROTEINDISEASE
Christian Boehmer Anfinsen (biochemist-USA) March 26 1916 Monessen Pa USA - May 14 1995
Randallstown Md
Theories behind the Folding
5
Anfinsen experiment I
- SS -
- SS -
- SS -
- SS -- SS -
- SS -
RNase A
SH -SH -
- SH
- SH
- SH
- SH
Unfolded
(unstructured)
protein without
functional activity
Removing the denaturing agents rarr folded structured functional protein
Denaturation with
8M urea
β-mercaptoethanol
Interpretation thermodynamic hypothesis ndash under physiological conditions the native form of the protein tends to achieve a minimum in Gibbs free energy
Conlusion The proteins can fold spontaneously
The 3D structure of the proteins is encoded within their primary structure
rarr 1972 Nobel price in chemistry (Stanford Moore amp William H Stein)
Anfinsen experiment II
The energy landscape theories
1 Levinthalrsquos pradox
2 The folding funnel
The energy landscape theories
Conformationaldistribution
en
erg
y
A model describes the relationship between the different conformations and energy levels
Central hole (cavity) for the final
(functional) state of the protein
Levinthalrsquos paradox
1968 - Cyrus Levinthal very large number
of degrees of freedom in an unfolded
polypeptide chain rarr the number of the
possible conformations is huge
Is the protein sampling all the possible
conformations
conformation
en
erg
y
Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9
Levinthalrsquos paradox
conformation
en
erg
y
Calculation Forming a protein that contains 25 bonds and all bound can be in 5
different conformations
n=5
i=25
N=ni rarr 525
The length of forming one conformation 1 ns
(10-9s)
The length of trying all the possible
conformations rarr 52510-9 s = 298109s =
~95 year harr micromicromicromicros - ms
Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9
6
Levinthalrsquos paradox
conformation
en
erg
y
Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9
Conclusions1 an intensive purely random search
cannot succeed
2 the native state is achieved through
a directed search
The folding funnel
conformation
Energ
y
Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9
bull Large number of folding path with equal
probability ( harr one folding path in Levinthalrsquos idea)
bull All paths lead directly to the native state
(energetic minimum)
bull The depth of the well symbolize the energetic
stabilization of the native state versus the
denatured state
bull The width of the well symbolize the entropy of the
system
bull The surface outside the well can symbolize the heterogeneity of the random coil state
bull directed search
Thermal fluctuation of the chemical bonds (20degC)
bull Covalent-bond 2-10 eV
rarr n1n
0~ 138times10-85
bull H-bond 005-03 eV
rarr n1n
0~ 0005-16
bull Van der Waals bond lt 0025eV
rarr n1n
0~ 368
bull Dipole-dipole interaction~ 00125-005 eV
rarr n1n
0~ 135-60
Binding energy the net energy required to decompose a molecule to break up chemical bonds
Main stabilizing
forces within the
macromolecules
Conformational dynamics
bull Continuous conformational transitions within the macromolecular structures
bull Martin Karplus ndash 1986
Haemoglobin ndash structure from x-ray diffraction
+ O2
Flexibility and protein function
bull Proteins can adapt to their ligand (larrkey-lock)
ndash induced fit during the ligand binding both the
ligand and the protein can adjust its structure to the
presence of the other
bull Protein flexibility is necessary for their
biochemical function
The end
2
bull structural (collagen)
bull transport (myosin)
bull biochemical reactions (enzyme)
bull immunology (antibodies)
bull signal transduction (hormones)
Protein functions Primary structure
The sequence of the monomeric units
within a chain of amino-acids connected by peptide bounds
Primary structure
C
N
O
C
Primary structure
Forming a spatial-structure (structure in space)Motifs bull alpha helix
bull beta sheet
Secondary structure Alpha helix
H
O
H O
Hydrogen-bond
Rolling up on the surface of an imaginary cylinder (+ or -)
3
Hydrogen bond
An electrostatic dipole-dipole interactionthat involves a hydrogen atom
δ+ δ+
δ-
δ+ δ+
δ-
Electronegativity a chemical property that describes the ability of an atom to attract electrons towards itself
H2O
H2O
Beta sheet
Parallel and antiparallel beta sheets
Tertiary structure
bull Folding Forming the final functional 3D forms of the proteins
bull Domain formation
bull Under physiological conditions a spontaneos disorder hArrorder transition = bdquofoldingrdquo
bull Chaperon A guide or companion to the protein that help to form its tertiary structure
bull Disulfide bridge hidrogen bond and hydrophobicinteractions are sabilizing the folded protein
Disulfide bond
A covalent bound (primary) forming between thiol groups (eg cysteine)
A link between two sulfur atoms
Hydrophobic interactions
bull No affinity for water (tending not to be dissolved in or mixed with water)
bull Usually non polar molecules are involved
bull one of the principal driving forces behind the protein folding
bull minimizing the number of hydrophobic side-chains exposed to water
bull the hydrophobic amino acids are shielded from the aqueous solvent
bull Very hydrophobic amino acids
Valine isoleucine isoleucine leucine methionine phenylalanine cysteine tryptophan
4
Tertiary structure Quaternary structure
The entire protein assembly
Two or more peptide chains forming the functinal form of a protein (eg hemoglobin)
wwwpdborg
Folding
A process where the tertiary structure (3D
shape) of a protein is formedThe functional form of the protein comes to
life
Misfolding
The folding is not succesfull (eg beta sheets instead of alpha helices) rarr misfolded proteins
The cell remove the wrong protein rarr the amount of the functional proteins decrease
The cell will not remove it rarr deposits (plaque) within the cells (Alzheimer disease)
Protein folding diseases
P53cancer
crystallinsCataract
rhodopsinRetinitis pigmentosa
Transthyretin lysosymeFamiliai amyloidoses
Prion proteinCreutzfeldt-Jakob disease
α-synucleinParkinsonrsquos disease
Amyloid β-peptidetauAlzheimerrsquos disease
CollagenScurvy
β-hexosaminidaseTay-Sachs disease
α1-Antitrypsin α1-Antitrypsin deficiency
HaemoglobinSickle cell anaemia
ProcollagenOsteogenesis imperfecta
FibrillinMarfan syndrome
HuntingtinHuntingtonrsquos disease
Phenylalanine hydroxilasephenylketonuria
Cystic fibrosis trans-membran regulatorCystic fibrosis
Low-density lipoprtotein receptorHypercholesterolaemia
PROTEINDISEASE
Christian Boehmer Anfinsen (biochemist-USA) March 26 1916 Monessen Pa USA - May 14 1995
Randallstown Md
Theories behind the Folding
5
Anfinsen experiment I
- SS -
- SS -
- SS -
- SS -- SS -
- SS -
RNase A
SH -SH -
- SH
- SH
- SH
- SH
Unfolded
(unstructured)
protein without
functional activity
Removing the denaturing agents rarr folded structured functional protein
Denaturation with
8M urea
β-mercaptoethanol
Interpretation thermodynamic hypothesis ndash under physiological conditions the native form of the protein tends to achieve a minimum in Gibbs free energy
Conlusion The proteins can fold spontaneously
The 3D structure of the proteins is encoded within their primary structure
rarr 1972 Nobel price in chemistry (Stanford Moore amp William H Stein)
Anfinsen experiment II
The energy landscape theories
1 Levinthalrsquos pradox
2 The folding funnel
The energy landscape theories
Conformationaldistribution
en
erg
y
A model describes the relationship between the different conformations and energy levels
Central hole (cavity) for the final
(functional) state of the protein
Levinthalrsquos paradox
1968 - Cyrus Levinthal very large number
of degrees of freedom in an unfolded
polypeptide chain rarr the number of the
possible conformations is huge
Is the protein sampling all the possible
conformations
conformation
en
erg
y
Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9
Levinthalrsquos paradox
conformation
en
erg
y
Calculation Forming a protein that contains 25 bonds and all bound can be in 5
different conformations
n=5
i=25
N=ni rarr 525
The length of forming one conformation 1 ns
(10-9s)
The length of trying all the possible
conformations rarr 52510-9 s = 298109s =
~95 year harr micromicromicromicros - ms
Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9
6
Levinthalrsquos paradox
conformation
en
erg
y
Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9
Conclusions1 an intensive purely random search
cannot succeed
2 the native state is achieved through
a directed search
The folding funnel
conformation
Energ
y
Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9
bull Large number of folding path with equal
probability ( harr one folding path in Levinthalrsquos idea)
bull All paths lead directly to the native state
(energetic minimum)
bull The depth of the well symbolize the energetic
stabilization of the native state versus the
denatured state
bull The width of the well symbolize the entropy of the
system
bull The surface outside the well can symbolize the heterogeneity of the random coil state
bull directed search
Thermal fluctuation of the chemical bonds (20degC)
bull Covalent-bond 2-10 eV
rarr n1n
0~ 138times10-85
bull H-bond 005-03 eV
rarr n1n
0~ 0005-16
bull Van der Waals bond lt 0025eV
rarr n1n
0~ 368
bull Dipole-dipole interaction~ 00125-005 eV
rarr n1n
0~ 135-60
Binding energy the net energy required to decompose a molecule to break up chemical bonds
Main stabilizing
forces within the
macromolecules
Conformational dynamics
bull Continuous conformational transitions within the macromolecular structures
bull Martin Karplus ndash 1986
Haemoglobin ndash structure from x-ray diffraction
+ O2
Flexibility and protein function
bull Proteins can adapt to their ligand (larrkey-lock)
ndash induced fit during the ligand binding both the
ligand and the protein can adjust its structure to the
presence of the other
bull Protein flexibility is necessary for their
biochemical function
The end
3
Hydrogen bond
An electrostatic dipole-dipole interactionthat involves a hydrogen atom
δ+ δ+
δ-
δ+ δ+
δ-
Electronegativity a chemical property that describes the ability of an atom to attract electrons towards itself
H2O
H2O
Beta sheet
Parallel and antiparallel beta sheets
Tertiary structure
bull Folding Forming the final functional 3D forms of the proteins
bull Domain formation
bull Under physiological conditions a spontaneos disorder hArrorder transition = bdquofoldingrdquo
bull Chaperon A guide or companion to the protein that help to form its tertiary structure
bull Disulfide bridge hidrogen bond and hydrophobicinteractions are sabilizing the folded protein
Disulfide bond
A covalent bound (primary) forming between thiol groups (eg cysteine)
A link between two sulfur atoms
Hydrophobic interactions
bull No affinity for water (tending not to be dissolved in or mixed with water)
bull Usually non polar molecules are involved
bull one of the principal driving forces behind the protein folding
bull minimizing the number of hydrophobic side-chains exposed to water
bull the hydrophobic amino acids are shielded from the aqueous solvent
bull Very hydrophobic amino acids
Valine isoleucine isoleucine leucine methionine phenylalanine cysteine tryptophan
4
Tertiary structure Quaternary structure
The entire protein assembly
Two or more peptide chains forming the functinal form of a protein (eg hemoglobin)
wwwpdborg
Folding
A process where the tertiary structure (3D
shape) of a protein is formedThe functional form of the protein comes to
life
Misfolding
The folding is not succesfull (eg beta sheets instead of alpha helices) rarr misfolded proteins
The cell remove the wrong protein rarr the amount of the functional proteins decrease
The cell will not remove it rarr deposits (plaque) within the cells (Alzheimer disease)
Protein folding diseases
P53cancer
crystallinsCataract
rhodopsinRetinitis pigmentosa
Transthyretin lysosymeFamiliai amyloidoses
Prion proteinCreutzfeldt-Jakob disease
α-synucleinParkinsonrsquos disease
Amyloid β-peptidetauAlzheimerrsquos disease
CollagenScurvy
β-hexosaminidaseTay-Sachs disease
α1-Antitrypsin α1-Antitrypsin deficiency
HaemoglobinSickle cell anaemia
ProcollagenOsteogenesis imperfecta
FibrillinMarfan syndrome
HuntingtinHuntingtonrsquos disease
Phenylalanine hydroxilasephenylketonuria
Cystic fibrosis trans-membran regulatorCystic fibrosis
Low-density lipoprtotein receptorHypercholesterolaemia
PROTEINDISEASE
Christian Boehmer Anfinsen (biochemist-USA) March 26 1916 Monessen Pa USA - May 14 1995
Randallstown Md
Theories behind the Folding
5
Anfinsen experiment I
- SS -
- SS -
- SS -
- SS -- SS -
- SS -
RNase A
SH -SH -
- SH
- SH
- SH
- SH
Unfolded
(unstructured)
protein without
functional activity
Removing the denaturing agents rarr folded structured functional protein
Denaturation with
8M urea
β-mercaptoethanol
Interpretation thermodynamic hypothesis ndash under physiological conditions the native form of the protein tends to achieve a minimum in Gibbs free energy
Conlusion The proteins can fold spontaneously
The 3D structure of the proteins is encoded within their primary structure
rarr 1972 Nobel price in chemistry (Stanford Moore amp William H Stein)
Anfinsen experiment II
The energy landscape theories
1 Levinthalrsquos pradox
2 The folding funnel
The energy landscape theories
Conformationaldistribution
en
erg
y
A model describes the relationship between the different conformations and energy levels
Central hole (cavity) for the final
(functional) state of the protein
Levinthalrsquos paradox
1968 - Cyrus Levinthal very large number
of degrees of freedom in an unfolded
polypeptide chain rarr the number of the
possible conformations is huge
Is the protein sampling all the possible
conformations
conformation
en
erg
y
Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9
Levinthalrsquos paradox
conformation
en
erg
y
Calculation Forming a protein that contains 25 bonds and all bound can be in 5
different conformations
n=5
i=25
N=ni rarr 525
The length of forming one conformation 1 ns
(10-9s)
The length of trying all the possible
conformations rarr 52510-9 s = 298109s =
~95 year harr micromicromicromicros - ms
Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9
6
Levinthalrsquos paradox
conformation
en
erg
y
Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9
Conclusions1 an intensive purely random search
cannot succeed
2 the native state is achieved through
a directed search
The folding funnel
conformation
Energ
y
Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9
bull Large number of folding path with equal
probability ( harr one folding path in Levinthalrsquos idea)
bull All paths lead directly to the native state
(energetic minimum)
bull The depth of the well symbolize the energetic
stabilization of the native state versus the
denatured state
bull The width of the well symbolize the entropy of the
system
bull The surface outside the well can symbolize the heterogeneity of the random coil state
bull directed search
Thermal fluctuation of the chemical bonds (20degC)
bull Covalent-bond 2-10 eV
rarr n1n
0~ 138times10-85
bull H-bond 005-03 eV
rarr n1n
0~ 0005-16
bull Van der Waals bond lt 0025eV
rarr n1n
0~ 368
bull Dipole-dipole interaction~ 00125-005 eV
rarr n1n
0~ 135-60
Binding energy the net energy required to decompose a molecule to break up chemical bonds
Main stabilizing
forces within the
macromolecules
Conformational dynamics
bull Continuous conformational transitions within the macromolecular structures
bull Martin Karplus ndash 1986
Haemoglobin ndash structure from x-ray diffraction
+ O2
Flexibility and protein function
bull Proteins can adapt to their ligand (larrkey-lock)
ndash induced fit during the ligand binding both the
ligand and the protein can adjust its structure to the
presence of the other
bull Protein flexibility is necessary for their
biochemical function
The end
4
Tertiary structure Quaternary structure
The entire protein assembly
Two or more peptide chains forming the functinal form of a protein (eg hemoglobin)
wwwpdborg
Folding
A process where the tertiary structure (3D
shape) of a protein is formedThe functional form of the protein comes to
life
Misfolding
The folding is not succesfull (eg beta sheets instead of alpha helices) rarr misfolded proteins
The cell remove the wrong protein rarr the amount of the functional proteins decrease
The cell will not remove it rarr deposits (plaque) within the cells (Alzheimer disease)
Protein folding diseases
P53cancer
crystallinsCataract
rhodopsinRetinitis pigmentosa
Transthyretin lysosymeFamiliai amyloidoses
Prion proteinCreutzfeldt-Jakob disease
α-synucleinParkinsonrsquos disease
Amyloid β-peptidetauAlzheimerrsquos disease
CollagenScurvy
β-hexosaminidaseTay-Sachs disease
α1-Antitrypsin α1-Antitrypsin deficiency
HaemoglobinSickle cell anaemia
ProcollagenOsteogenesis imperfecta
FibrillinMarfan syndrome
HuntingtinHuntingtonrsquos disease
Phenylalanine hydroxilasephenylketonuria
Cystic fibrosis trans-membran regulatorCystic fibrosis
Low-density lipoprtotein receptorHypercholesterolaemia
PROTEINDISEASE
Christian Boehmer Anfinsen (biochemist-USA) March 26 1916 Monessen Pa USA - May 14 1995
Randallstown Md
Theories behind the Folding
5
Anfinsen experiment I
- SS -
- SS -
- SS -
- SS -- SS -
- SS -
RNase A
SH -SH -
- SH
- SH
- SH
- SH
Unfolded
(unstructured)
protein without
functional activity
Removing the denaturing agents rarr folded structured functional protein
Denaturation with
8M urea
β-mercaptoethanol
Interpretation thermodynamic hypothesis ndash under physiological conditions the native form of the protein tends to achieve a minimum in Gibbs free energy
Conlusion The proteins can fold spontaneously
The 3D structure of the proteins is encoded within their primary structure
rarr 1972 Nobel price in chemistry (Stanford Moore amp William H Stein)
Anfinsen experiment II
The energy landscape theories
1 Levinthalrsquos pradox
2 The folding funnel
The energy landscape theories
Conformationaldistribution
en
erg
y
A model describes the relationship between the different conformations and energy levels
Central hole (cavity) for the final
(functional) state of the protein
Levinthalrsquos paradox
1968 - Cyrus Levinthal very large number
of degrees of freedom in an unfolded
polypeptide chain rarr the number of the
possible conformations is huge
Is the protein sampling all the possible
conformations
conformation
en
erg
y
Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9
Levinthalrsquos paradox
conformation
en
erg
y
Calculation Forming a protein that contains 25 bonds and all bound can be in 5
different conformations
n=5
i=25
N=ni rarr 525
The length of forming one conformation 1 ns
(10-9s)
The length of trying all the possible
conformations rarr 52510-9 s = 298109s =
~95 year harr micromicromicromicros - ms
Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9
6
Levinthalrsquos paradox
conformation
en
erg
y
Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9
Conclusions1 an intensive purely random search
cannot succeed
2 the native state is achieved through
a directed search
The folding funnel
conformation
Energ
y
Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9
bull Large number of folding path with equal
probability ( harr one folding path in Levinthalrsquos idea)
bull All paths lead directly to the native state
(energetic minimum)
bull The depth of the well symbolize the energetic
stabilization of the native state versus the
denatured state
bull The width of the well symbolize the entropy of the
system
bull The surface outside the well can symbolize the heterogeneity of the random coil state
bull directed search
Thermal fluctuation of the chemical bonds (20degC)
bull Covalent-bond 2-10 eV
rarr n1n
0~ 138times10-85
bull H-bond 005-03 eV
rarr n1n
0~ 0005-16
bull Van der Waals bond lt 0025eV
rarr n1n
0~ 368
bull Dipole-dipole interaction~ 00125-005 eV
rarr n1n
0~ 135-60
Binding energy the net energy required to decompose a molecule to break up chemical bonds
Main stabilizing
forces within the
macromolecules
Conformational dynamics
bull Continuous conformational transitions within the macromolecular structures
bull Martin Karplus ndash 1986
Haemoglobin ndash structure from x-ray diffraction
+ O2
Flexibility and protein function
bull Proteins can adapt to their ligand (larrkey-lock)
ndash induced fit during the ligand binding both the
ligand and the protein can adjust its structure to the
presence of the other
bull Protein flexibility is necessary for their
biochemical function
The end
5
Anfinsen experiment I
- SS -
- SS -
- SS -
- SS -- SS -
- SS -
RNase A
SH -SH -
- SH
- SH
- SH
- SH
Unfolded
(unstructured)
protein without
functional activity
Removing the denaturing agents rarr folded structured functional protein
Denaturation with
8M urea
β-mercaptoethanol
Interpretation thermodynamic hypothesis ndash under physiological conditions the native form of the protein tends to achieve a minimum in Gibbs free energy
Conlusion The proteins can fold spontaneously
The 3D structure of the proteins is encoded within their primary structure
rarr 1972 Nobel price in chemistry (Stanford Moore amp William H Stein)
Anfinsen experiment II
The energy landscape theories
1 Levinthalrsquos pradox
2 The folding funnel
The energy landscape theories
Conformationaldistribution
en
erg
y
A model describes the relationship between the different conformations and energy levels
Central hole (cavity) for the final
(functional) state of the protein
Levinthalrsquos paradox
1968 - Cyrus Levinthal very large number
of degrees of freedom in an unfolded
polypeptide chain rarr the number of the
possible conformations is huge
Is the protein sampling all the possible
conformations
conformation
en
erg
y
Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9
Levinthalrsquos paradox
conformation
en
erg
y
Calculation Forming a protein that contains 25 bonds and all bound can be in 5
different conformations
n=5
i=25
N=ni rarr 525
The length of forming one conformation 1 ns
(10-9s)
The length of trying all the possible
conformations rarr 52510-9 s = 298109s =
~95 year harr micromicromicromicros - ms
Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9
6
Levinthalrsquos paradox
conformation
en
erg
y
Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9
Conclusions1 an intensive purely random search
cannot succeed
2 the native state is achieved through
a directed search
The folding funnel
conformation
Energ
y
Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9
bull Large number of folding path with equal
probability ( harr one folding path in Levinthalrsquos idea)
bull All paths lead directly to the native state
(energetic minimum)
bull The depth of the well symbolize the energetic
stabilization of the native state versus the
denatured state
bull The width of the well symbolize the entropy of the
system
bull The surface outside the well can symbolize the heterogeneity of the random coil state
bull directed search
Thermal fluctuation of the chemical bonds (20degC)
bull Covalent-bond 2-10 eV
rarr n1n
0~ 138times10-85
bull H-bond 005-03 eV
rarr n1n
0~ 0005-16
bull Van der Waals bond lt 0025eV
rarr n1n
0~ 368
bull Dipole-dipole interaction~ 00125-005 eV
rarr n1n
0~ 135-60
Binding energy the net energy required to decompose a molecule to break up chemical bonds
Main stabilizing
forces within the
macromolecules
Conformational dynamics
bull Continuous conformational transitions within the macromolecular structures
bull Martin Karplus ndash 1986
Haemoglobin ndash structure from x-ray diffraction
+ O2
Flexibility and protein function
bull Proteins can adapt to their ligand (larrkey-lock)
ndash induced fit during the ligand binding both the
ligand and the protein can adjust its structure to the
presence of the other
bull Protein flexibility is necessary for their
biochemical function
The end
6
Levinthalrsquos paradox
conformation
en
erg
y
Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9
Conclusions1 an intensive purely random search
cannot succeed
2 the native state is achieved through
a directed search
The folding funnel
conformation
Energ
y
Dill KA Chan HS From Levinthal to pathways to funnels Nat Struct Biol 1997 Jan4(1)10-9
bull Large number of folding path with equal
probability ( harr one folding path in Levinthalrsquos idea)
bull All paths lead directly to the native state
(energetic minimum)
bull The depth of the well symbolize the energetic
stabilization of the native state versus the
denatured state
bull The width of the well symbolize the entropy of the
system
bull The surface outside the well can symbolize the heterogeneity of the random coil state
bull directed search
Thermal fluctuation of the chemical bonds (20degC)
bull Covalent-bond 2-10 eV
rarr n1n
0~ 138times10-85
bull H-bond 005-03 eV
rarr n1n
0~ 0005-16
bull Van der Waals bond lt 0025eV
rarr n1n
0~ 368
bull Dipole-dipole interaction~ 00125-005 eV
rarr n1n
0~ 135-60
Binding energy the net energy required to decompose a molecule to break up chemical bonds
Main stabilizing
forces within the
macromolecules
Conformational dynamics
bull Continuous conformational transitions within the macromolecular structures
bull Martin Karplus ndash 1986
Haemoglobin ndash structure from x-ray diffraction
+ O2
Flexibility and protein function
bull Proteins can adapt to their ligand (larrkey-lock)
ndash induced fit during the ligand binding both the
ligand and the protein can adjust its structure to the
presence of the other
bull Protein flexibility is necessary for their
biochemical function
The end