Download proteins - Technische Universität München - Physik

Structure formation and
association of biomolecules
Prof. Dr. Martin Zacharias
Lehrstuhl für Molekulardynamik (T38)
Technische Universität München
Motivation
• Many biomolecules are chemically synthesized in a cell as
linear chain molecules.
• Chain molecules (proteins, RNA) need to form a defined
three-dimensional structure:
– Structure formation or folding process
• The folded three-dimensional structure of a biomolecule is
directly related to its function.
Biomolecule function depends on threedimensional biomolecular structure.
Protein-peptide complex
Protein folding
• Proteins can fold from an extended chain into a compact globular structure
(some proteins need help to adopt the correct structure in vivo).
•
The fold is determined by the amino acid sequence.
•
The final protein three-dimensional (3D) structure has a well packed core.
•
The 3D-structure has limited stability
•
Folded structure can be disrupted by heat and changes of solution conditions
Interactions of biomolecules
•
Most biomolecules form
functional complexes
•
Many functions are mediated
by multiple-protein-protein
interactions
•
What are the driving forces
for such interactions?
•
Specific vs. non-specfic
interactions
•
What happens during a
binding process?
•
Can we predict which
proteins interact and how?
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Outline
•
Protein structure
–
Folded and unfolded state
•
Molecular interactions and forces in solution
•
Thermodynamics of protein folding
•
Experimental methods to study protein folding
•
Theories of protein folding
•
Structure formation of nucleic acids
•
Computer simulation of structure formation
•
Mechanism of biomolecular association
•
Prediction of binding (geometry and affinity)
•
Talks on interesting papers
Aim
• Qualitative and quantitative understanding of folding and
binding processes
– What can be observed?
– What are the current theories on folding and association
• What are the forces that drive structure formation and
folding?
• Protein folding problem
• Folding of nucleic acids
• Prediction of 3D structures
• Predicion of how biomolecules interact
• Computer simulation of folding and binding
Overview on protein structure (folded state)
•
Primary structure
– Sequence of the protein
•
Secondary structure
– Regular local structural
elements stabilized by
hydrogen bonds
•
Tertiary structure
– Three-dimensional fold of the
protein
•
Quaternary structure:
– Non-covalent assembly of
several proteins to form a
functional complex
-Gly-Ala-Arg-Phe-ValG A R F V
Primary structure of proteins
- Amino acid sequence of a protein: primary protein structure.
- 20 naturally amino acids occur in proteins.
- Amino acids have a common chemical structure:
Tetrahedral (sp3) carbon atom (Cα) bound to four asymmetric groups:
1. Amino group (NH2)
2. Carboxy group (COOH)
3. H atom
4. Functional chemical group: characteristic for the amino acid
- Naturally occuring amino acids
are typically L amino acids.
Cβ
Cα
NH2
Hα
COOH
The geometry of the peptide bond
•Amino acids connect via a peptide bond -> poly-peptide structure.
•Peptide bond formation by a condensation reaction
•Peptide unit NH-CO (omega: ω) is planar (small deviations are possible)
and is mostly in a trans configuration (except for Pro).
R1
O
phi
N
H
O
H
psi
O
N
omega
R2
Amino acids can be grouped based on side chain character
Non-polar side-chains (hydrophobic):
Alanine (Ala), Valine (Val), Isoleucine (Ile), Leucin (Leu), Methionine (Met),
Phenylalanine (Phe), Proline (Pro) and Tryptophan (Trp), also Glycine (Gly)
Polar side-chains (hydrophilic):
Serine (Ser), Threonine (Thr), Cysteine (Cys), Asparagine (Asn), Glutamine (Gln),
Tyrosine (Tyr) and Histidine (His)
Charged side-chains (hydrophilic):
Positive charge: Arginine (Arg) and Lysine (Lys)
Depending on the pH, Histidine (His) can also be positively charged.
Negative charge: Glutamic acid (Glu) and Aspartic acid (Asp)
Secondary structure of proteins
•
Regular structural
elements which are
formed by hydrogen
bonds between relatively
small segments of the
protein sequence.
•
α-helix :
– hydrogen bonds
between the CO of
residues i and the
NH of residues i+4.
– 3.6 residues per
helical turn.
– helical rise of 1.5 Å
per residue.
• Important secondary
structures:
– α-helix
– β-sheet
– turns
– coil structures
β-sheet + β-turn
Super-secondary structures
•
•
•
To some degree tertiary structure can be viewed as a 3D
'packing' of secondary structural elements.
Analysis of 3D protein structures indicates the existence of
recurring patterns of secondary structure arrangements->
called motifs or super-secondary structure.
common super-secondary structures (motifs) are:
– β-hairpin
– β-meander
– βαβ-motif
– Helix-loop-helix motif
Examples:
β-hairpin
βαβ-motif
helix-loop-helix motif
Tertiary structure of proteins
•
The tertiary structure of a protein are the 3D coordinates to which
it is folded. This tertiary structure is directly related to its function.
•
Some combinations of super-secondary elements are frequently
found in proteins:
– Four α-helix bundle (two α-helix-loop-α-helix motifs connected
by a loop)
– βαβαβ (Rossmann) fold (two βαβ motifs sharing one β-strands)
•
Not all helices and strands in a folded protein belong to supersecondary structural elements
•
Data base of protein structures:
Brookhaven Protein Structure data base
http://www.rcsb.org/pdb
Classification of protein tertiary structures
•
Tertiary structure describes the
packing of structural elements within
one domain.
•
classification:
– All (or mostly) α-helical: also
called all-α
– All (or mostly) β-sheet containing:
all-β
– α/β proteins: contain both helices
and β-sheets
– (sometimes additional class, α + β:
segregated α-helices + β-sheets)
– proteins with very little secondary
structure content may form
another class
Spatial distribution of amino acids in folded proteins
• The spatial distribution of amino acids with respect to
the center of a folded globular protein is not random.
• Hydrophobic amino acids are found preferentially
inside the folded protein.
• Hydrophilic and charged amino acids are more
frequently located at the surface of the protein.
• This observation indicates that the solvent plays a
dominant role for the structure formation of a protein.
• 3D-fold can be further stabilized by disulfide bonds.
Protein domains
•
Domains: contiguous portions of the protein chain that fold into
compact (semi-independent) tertiary structures (definition by
Richardson, 1981).
•
Some proteins consists of only one domain. However, especialy
proteins with several hundred residues often consists of several
domains.
•
In some cases different functions of one protein can be associated
with different domains.
SHP2: Phosphotyrosine phosphatase 2
N-SH2-domain
PTP-domain
C-SH2-domain
Is the number of possible 3D protein folds limited?
• Observation
– Many sequences form
similar structures
Atomic resolution structures of biomolecules are
stored at the Protein Data Bank
•
•
Contains ~35000 structures (mostly determined by X-ray
crystallography)
Several new structures per day
Methods to determine the threedimensional structure of proteins
•
X-ray crystallography is the most powerful and most successful
technique to obtain high resolution structures of a biomolecule
– ~80% of all structures in the PDB have been solved by X-ray
crystallography (X-ray diffraction of protein/nucleic acid
crystals)
– ~20% have been solved by NMR spectroscopy
– Very few by other experimental techniques
•
A wide range of biomolecules and complexes can be analyzed by
X-ray crystallography
•
X-ray crystallography requires high-quality (well ordered) single
crystals of the biomolecule
•
NMR spectroscopy allows to determine the structure of a (small)
protein in solution
Examples of structures solved by X-ray
crystallography
nucleosome
lysozyme
K+ channel
RNA-polymerase
•
Structures between a few
hundred to up to million daltons
can be solved if high-quality
crystals can be obtained.
ribosome
The unfolded state of proteins
• The term unfolded state can have several different
meanings
– Random coil structure of a peptide with negligible residual
interactions (except chain connectivity)
– The state formed immediately after synthesis (extended
chain)
– The state(s) formed after “denaturation” of a protein
• Denaturation can be achieved by changing the
temperature or the solution conditions.
• What is the unfolded state of a protein?
Random walk or stochastic chain model
• Random walk or stochastic chains
– Freely joined chain with constant bond
length and no restriction of orientation
of segments
• The average (root mean square)
end-to-end distance R for the
chain scales with N0.5.
• Very common (but also most
unrealistic) model of an unfolded
protein chain
•
•
Experiment: Rg = Ro Nν, ν= 0.598+/-0.028
theoretical prediction for random coil according to rotational isomeric state model
(Flory, 1969) for Θ-solvent: ν= 0.588
• Rigid segment simulations (connected by flexible linkers)
• Rg = Ro Nν, ν= 0.602+/-0.028 (RIS for Θ-solvent: ν= 0.588)
Energetic evaluation with a continuum solvent model
Contributions to the total energy of a molecule conformation:
Esolv-nonpolar
+
Esolv-polar
~surface area
Poisson equation
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Force field
+
+ +
+
Esolv-salt
Poisson-Boltzmann eq
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Evacuum
- - + +
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ΔGsolv= ΔGsolv(elec)+ΔGsolv(nonpolar)
ΔGsolv(elec)= EPB(εin=2; εex=80)-EPB(εin=2; εex=1)
ΔGsolv(nonpolar)= b + γ * SASA
b = 0.86 kcal mol-1
γ = 0.005 kcal mol-1Å-2
Good agreement
with experiment
for PARSE set of
optimized charges
and radii
„pure“ SASA-model
Authors use two different
solvation models to
calculate σ and s for a
zipper-model of (polyAla)
α-helix folding
All models give good agreement of
calculated solvation free energy
with experiment for a model
compound (N-Methylacetamid):
dGsolv(exp) = -10.0 kcal mol-1
Plotting ΔGi vs. i gives RTln(s)
and RTln(s) as intercepts and
slope, respectively.
Improved continuum modelling of non-polar
(hydrophobic) solvation
•
Continuum modelling of hydrophobic
contribution using a single surface tension
parameter:
good predictions for solvation of linear alkanes,
poor results for cyclic alkanes
•
Separation into two parts:
A.) water reordering at molecule-water
interface: (proportional to surface area)
B.) molecule-water van der Waals
interaction (surface integral)
•
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Significant improvement for cyclic alkanes
Significant improvement for conformational
changes
Zacharias, M., J.Phys.Chem. 2003
cyclo-alkane
branched+
n-alkanes