protein structure - ntnufolk.ntnu.no/audunfor/7. semester/biopolymerkjemi/komplementerende... ·...
TRANSCRIPT
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Protein Structure Marianne Øksnes Dalheim, PhD candidate
Biopolymers, TBT4135, Autumn 2013
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The presentation is based on the
presentation by Professor Alexander Dikiy,
which is given in the course compedium:
Part 4.4 on page 165
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Outline
• Part 1: Protein structure fundamentals
• Part 2: Determining the protein structure
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Part 1:
Protein structure fundamentals
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Polypeptides
• Biopolymer
• Monomers (building blocks): Amino Acids
• Monodisperse – DNA RNA Protein
– Defined sequence of amino acids
• A protein: one or more polypeptide chains folded into a structure, having a biological function
All proteins are polypeptides, but not all polypeptides are proteins
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Amino acids –the building blocks
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Amino acids – stereochemistry
The proteins are constituted by L-AA isomers
In Fischer projection: • Vertical bonds: stretch out in
the space behind the paper • Horizontal bonds: stretch up
and out of the plane of the paper
• L-configuration: Functional group (-NH3
+) to the left
• D-configuration: Functional group (-NH3
+) to the right
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Amino acids – chemistry of the side group
• Nonpolar, aliphatic • Polar, uncharged • Aromatic • Charged
• Positively • Negatively
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Chemistry of the side group → function
• 20 different amino acids → many different functional groups in one molecule
• The proteins are tailor made to specific biological functions and reactions
• Proteins from very different organisms, with the same biological function: almost identical or very similar primary structure (homology)
• Complex proteins • Glycoprotein
• Lipoprotein
• Phosphoproteins
Functions: • Catalysis • Regulation • Structure • Movement • Transport • Signaling
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The protein alphabet
• The protein alphabet is represented either in a one-letter or a three-letter code language
• Each AA has its own unique code definition
Alanine Ala A
Arginine Arg R
Asparagine Asn N
Aspartic acid Asp D
Cysteine Cys C
Glutamine Gln Q
Glutamic acid Glu E
Glycine Gly G
Histidine His H
Isoleucine Ile I
Leucine Leu L
Lysine Lys K
Methionine Met M
Phenylalanine Phe F
Proline Pro P
Serine Ser S
Threonine Thr T
Tryptophan Trp W
Tyrosine Tyr Y
Valine Val V
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Acid-base properties of amino acids
Monovalent acid: HA H+ + A-
Henderson – Hasselbach equation:
Ka
apH = pK - logacid
base
apH = pK + log1
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Ionization of Gly and His
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The isoelectric point (pI) of amino acids
Definition: pI = pH when net charge is zero
∑(+) = ∑(-)
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The properties of single amino acids are reflected on the protein functional peculiarities and structure
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Importance of the amino acid nature for protein structure
- The hemoglobin
Hemoglobin A: -Val-His-Leu-Thr-Pro-Glu-Glu-Lys-
Hemoglobin S: -Val-His-Leu-Thr-Pro-Glu-Val-Lys-
Mutation of Glu (hydrophilic) on Val (hydrophobic) results in complete alteration of the protein structure thus causing disease – Sickle cell anemia.
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The peptide bond
Formed by a condensation reaction: carboxyl + amine = amide + H2O
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Rotation flexibility of AA
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cis- and trans- AA
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Backbone dihedral (torsion) angles
Dihedral angle
- Angle between two planes - Determined from 4 atoms Phi angle (φ) The dihedral angle composed of the four atoms: C(i-1) - >N(i) - C(i) - C(i). - free rotation around N-C bond. Psi angle (ψ) The dihedral angle composed of the four atoms: N(i) - C (i) - C(i) >- N(i+1). - free rotation around C-C(O) bond Omega angle (ω) The dihedral angle decided by the four atoms: Cα(i)-C(i)-N(i+1)-Cα(i+1) - rotation around the C(O)-N bond (peptide bond - restricted rotation, 0°or 180°(cis or trans)
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Phi– and Psi- dihedral angles can not take any values combination, due to steric hindrance
Psi- angle
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Main area 1: - φ: -60 → -180 - Ψ: -75 → - 15 → α helix Main area 2: - φ: -60 → -180 - Ψ: 10→ 180 → β sheet
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Polypeptide chain
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Protein structure
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Primary structure
• The amino acid sequence.
• The nascent polypeptide chain should, in most cases, take the protein fold.
Let’s consider a protein with 100 AA. If each AA can assume 3 different conformations (in practice it is much more), it would exist for
this protein 3100 = 1047 possible conformations.
However, the proteins, during around
picoseconds, chooses its unique fold.
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Anfinsen’s experiment
→ Proteins adopt their native structure/information spontaneously
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• Proteins gets folded through the interaction of amino acids.
• Weak interactions: electrostatic, hydrophobic, hydrogen bonding, metal-AA coordination bonds
• Covalent bonds in a protein exist only within AA, peptide bond and disulfide bridges (S-S).
Protein folding
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Secondary structure
• Interaction between AA lead to different types of secondary structure.
• Local folding
-helix, -sheet and loops
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Different types of helixes
3.6 - 3 - 5 - residues per turn
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Hydrogen bonding network: i - i+3 residue (310 helix), i - i+4 residue (normal helix), i - i+5 residue (pi helix)
-helix
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-sheets
antiparallel
parallel
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Tertiary structure
Tertiary structure represents the protein folding and is a spatial arrangement of elements of secondary structure (-helixes, -sheets), as well as connecting loops, turns, unfolded (not structured) regions.
Total amount of different folds can be estimated as approximately 2000.
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Protein domains A protein domain is a part of protein sequence and structure that can evolve, function, and exist independently of the rest of the protein chain. Each domain forms a compact three-dimensional structure and often can be independently stable and folded. Many proteins consist of several structural domains. One domain may appear in a variety of different proteins.
Wikipedia
Pyruvate Kinase – 1pkn
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Quaternary structure Only multi chain proteins have quaternary structure. The inter-chain
interaction is based on weak and S-S interactions
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1) Membrane protein: Rhodopsin 2) Globular protein: SelW 3) Fibrous protein: Collagen
Some structural examples
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Membrane protein: Rhodopsin
Rhodopsin is the protein component of the light receptor in the retinal rods of the vertebrate eye. Similar molecules are found in the light-sensing structures of all animals
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Globular protein: The mammalian SelW protein
SelW is a selenoprotein involved in cellular redox reactions. • Small: 89 amino acids
• Motif: Cys-X-X-U, where U is
Selenocystein • Its structure reveals a -----
fold
• Globular
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Fibrous protein: Collagen
Collagen is the most abundant protein in mammals. About one quarter of all of the protein in your body is collagen. Collagen is the main protein of connective tissue. It has great tensile strength.
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Fibrous protein: Collagen
• Three polypeptide chains with the repeat sequence: Gly-X-Y
• X is often proline • Y is often hydroxyproline
(posttranslational modification) • Each chain is about 1000 amino acid
residues long • Synthesized as procollagens - globular
propeptides that are excised off by extracellular enzymes.
• Excision of propeptides allows the triple chain molecule to polymerize into fibrils
Branden C., Tooze, J. (1999) Introduction
to protein structure, 2nd ed., Garland
publishing, New York, p 284
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Fibrous protein: Collagen
Each of the three polypeptide chains are folded into an extended left-handed helix • 3.3 residues per turn (α-helix: 3.6) • Rise per residue: 2.9 Å (α-helix: 1.5) • Rise per turn: 9.6 Å (α-helix: 5.4)
→ More extended conformation than the α-helix. The three helices in collagen form a trimeric molecule by coiling about the central axis to form a right-handed superhelix The side chain of every third residue is close to the central axis, where there is no room for a side chain, consequently every third residue must be a glycine. Branden C., Tooze, J. (1999) Introduction
to protein structure, 2nd ed., Garland
publishing, New York, p 284
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Fibrous protein: Collagen
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Part 2:
Determining the protein structure
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What can we learn analyzing the protein structure?
• Protein function • Protein mechanism • Protein evolution • Protein system biology • Structure based drug design
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What does it mean to determine the 3D structure of a protein?
Determine either •ALL the distances between each atom and the remaining protein atoms or •ALL protein’s dihedral angles
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Experimental techniques for
macro-molecule structures determination
Low resolution techniques 1. Electron microscopy 2. SAXS (small angle X-ray scattering)
→ rough structure, topology, quarternary structure of large proteins. → Not position of each atom High resolution techniques 1. X-ray crystallography –first applied in 1961 (Kendrew and Perutz – Nobel
prize winners) 2. NMR spectroscopy –first applied in 1983 (Ernst and Wuthrich –Nobel
prize winners)
→ position of each atom
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X-ray Crystallography • Most widespread technique to determine high-resolution structure of molecules in
the solid state
• The method depends on directing a beam of x-rays onto a regular, repeating array of many identical molecules a crystal
• The x-rays diffract from the crystal in a diffraction pattern
• The diffraction data from the crystal is used to calculate an electron density map
• Interpret the map as a polypeptide chain with a particular amino acid sequence
Branden C., Tooze, J. (1999) Introduction
to protein structure, 2nd ed., Garland
publishing, New York, p 377
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X-ray Crystallography
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X-ray Crystallography Prerequisite • Have to obtain well ordered crystals that diffract x-rays
Proteins can be difficult: large spherical irregular surfaces that is impossible to pack into a crystal. • Large channels between the individual molecules, filled with disordered solvent
molecules • Only a few contact points between the protein molecules. This is also the reason why the structures determined by x-ray crystallography are the same as those for the proteins in solution
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NMR spectroscopy
A technique that relies on observation of energy absorption by nuclei in a external magnetic field under the influence of electromagnetic radio frequency irradiation
– Place the protein molecules in a strong magnetic field and the spin of their nuclei will align along the field. This process is an equilibrium process
– If you apply radio frequency pulses the equilibrium alignment will be changed to an excited state
– When the nuclei return to the equilibrium state, they emit radio frequency radiation that can be measured
– The frequency of the emitted radiation depends on the chemical environment of the nucleus and will therefor be different for each atom.
– The different frequencies are obtained relative to a reference signal and is what we call a chemical shift.
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NMR spectroscopy
• Distinguish various nuclei on the basis of their magnetic properties determined by their chemical environment
• The nuclei has to have an intrinsic magnetic moment (non zero spin): 1H, 13C, 15N
• The nature, duration, and combination of the applied RF pulses can be varied to probe different molecular properties of the sample
• Assign the spectrum of chemical shifts
• Measure distances and dihedral angles
• Solid state NMR or Solution NMR
→ Complementary techniques Crystallography: high resolution, fast technique, strong macromolecular complexes NMR: Structure in solution, dynamics(folding), weak macromolecular complexes
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How can I find whether the structure I am interested in is already determined?
Internet address: www.rcsb.org
→ all the determined structures are deposited in the protein data bank
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Statistics available at RCSB on October 9, 2012
85212 released atomic coordinate entries
Molecule Type:
78911 proteins, peptides, and viruses
2432 nucleic acids
3845 protein/nucleic acid complexes
24 other
Experimental Technique
78911 X-ray
9626 NMR
499 electron microscopy
165 other
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ATOM 1 N CYS A 1 -23.284 7.726 4.920 1.00 5.78 N ATOM 2 CA CYS A 1 -23.838 6.461 5.494 1.00 4.91 C ATOM 3 C CYS A 1 -22.786 5.345 5.449 1.00 3.96 C ATOM 4 O CYS A 1 -21.826 5.419 4.700 1.00 3.93 O ATOM 5 CB CYS A 1 -25.060 6.097 4.640 1.00 5.02 C ATOM 6 SG CYS A 1 -26.538 6.897 5.318 1.00 5.60 S ATOM 7 H2 CYS A 1 -24.029 8.449 4.870 1.00 6.28 H ATOM 8 HA CYS A 1 -24.152 6.627 6.514 1.00 5.16 H ATOM 9 HB2 CYS A 1 -24.908 6.431 3.625 1.00 5.62 H ATOM 10 HB3 CYS A 1 -25.201 5.025 4.645 1.00 4.44 H ATOM 11 HG CYS A 1 -26.624 7.759 4.904 1.00 5.70 H ATOM 12 H1 CYS A 1 -22.908 7.542 3.966 1.00 5.73 H ATOM 13 H3 CYS A 1 -22.516 8.073 5.530 1.00 6.16 H ATOM 14 N ALA A 2 -22.968 4.318 6.246 1.00 3.36 N ATOM 15 CA ALA A 2 -21.993 3.182 6.271 1.00 2.48 C ATOM 16 C ALA A 2 -22.085 2.364 4.975 1.00 1.96 C ATOM 17 O ALA A 2 -23.145 2.256 4.384 1.00 2.54 O ATOM 18 CB ALA A 2 -22.369 2.322 7.481 1.00 3.05 C ATOM 19 H ALA A 2 -23.753 4.294 6.832 1.00 3.63 H ATOM 20 HA ALA A 2 -20.991 3.564 6.403 1.00 2.30 H ATOM 21 HB1 ALA A 2 -22.564 2.957 8.333 1.00 2.93 H ATOM 22 HB2 ALA A 2 -23.252 1.744 7.252 1.00 3.30 H
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From the coordinates we can easily calculate the distance between two points:
D=
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What does it mean to determine the 3D structure of a protein?
Determine either •ALL the distances between each atom and the remaining protein atoms or •ALL protein’s dihedral angles
Therefore, the coordinates of each atom allows us to determine ALL the distances within the protein, and thus describe the structure of our protein
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References
Brandon, C., and Tooze, J., (1999) Introduction to protein structure, 2nd edition, Garland Publishing, New York Smidsrød, O., Moe, S.T., (2008) Biopolymer chemistry, Tapir academic press, Trondheim, chapter 3 & 8 Christensen, B.E., (2013) Compedium TBT4135 Biopolymers