protein structure (in a nutshell) guy ziv december 26 th, 2006 myoglobin (1958)
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Protein Structure (in a nutshell)
Guy Ziv
December 26th, 2006
Myoglobin (1958)
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Proteins
From the Greek “proteios” meaning “of first importance”
The basic building blocks of almost all life Constitutes the majority of the cell, and perform nearly all
enzymatic activities Composed of 20 naturally occurring amino-acids
varying moiety called “side chain”
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Protein Synthesis In-vivo
1. Transcription: DNA messenger RNA (mRNA)
2. Translation: mRNA Linear chain of a.a.(Ribosome)
3. Folding: Linear chain Structure
Peptide bond
Protein chains have direction N-terminal → C-terminal
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X-Rays crystallography – the tool of structural biology
Why X-ray?Wavelength of visible light: ~500 nmBond lengths in proteins: ~0.15 nmTypical X-ray wavelength: ~0.15 nm
X-ray are (weakly) scattered by electrons Diffraction from a single molecule is weak
so use a crystal:– Multiple copies of the molecule increases diffraction– Crystalline structure imposes constraints on diffraction
pattern
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Diffraction occurs at particular angles
Diffraction spots are the result of constructive interference from multiple scatterers satisfying Bragg’s Law: λ = 2 d sinθ
θ
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Bragg planes intersect the unit cell in particular “indices”
0,0
h=1, k=1
h
k
h=4, k=-2
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Each Spot Represents a Unique Set of Bragg Planes
h=2, k=1, l=3
h=10, k=3, l=8
1 2 3
detector
λ = 2 d sinθ
Points in k-space (Fourier Space)
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Modern X-Ray Crystallography
Need good crystals for better resolution, which is difficult in proteins (need right conditions)and sometimes nearly impossible (e.g. membranal proteins)
High resolution details are faint – requiresgood experimental apparatus
Recorded intensity give only the magnitudebut not the phase of the complex “form factor”
Error in density map lead to un-realisticatom assignment, requiring iterative refinementprocess
Early 1950’s
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Historical perspective to Pauling and Corey paper series
X-ray crystallography, invented in the beginning of the 20’th century, has been used to solve structures of some amino-acids, synthetic polymers (poly-glu) and small organic molecules
Some fibrous materials such as wool and α-keratin are sufficiently crystalline to give diffraction patterns
Evidence suggested that these proteins’ structure involve mainly translation and rotation
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Pauling and Corey
Robert Corey (1897-1971) Linus Pauling (1901-1994)
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Pauling and Corey papers series –PNAS April 1951
1. Pauling, L., Corey, R.B. and Branson H. R. The Structure of Proteins: Two Hydrogen-Bonded Helical Configurations of the Polypeptide Chain. PNAS, 37, 205-211, (1951).
2. Pauling, L. & Corey, R. B. Atomic Coordinates and Structure Factors for Two Helical Configurations of Polypeptide Chains. PNAS, 37, 235-240, (1951).
3. Pauling, L. & Corey, R. B. The Structure of Synthetic Polypeptides. PNAS, 37, 241-250, (1951).
4. Pauling, L. & Corey, R. B. The Pleated Sheet, A New Layer Configuration of Polypeptide Chains. PNAS, 37, 251-256, (1951).
5. Pauling, L. & Corey, R. B. The Structure of Feather Rachis Keratin. PNAS, 37, 256-261, (1951).
6. Pauling, L. & Corey, R. B. The Structure of Hair, Muscle, and Related Proteins. PNAS, 37, 261-271, (1951).
7. Pauling, L. & Corey, R. B. The Structure of Fibrous Proteins of the Collagen-Gelatin Group. PNAS, 37, 272-281, (1951).
8. Pauling, L. & Corey, R. B. The Polypeptide-Chain Configuration in Hemoglobin and Other Globular Proteins. PNAS, 37, 282-285, (1951).
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Pauling and Corey papers series –PNAS April 1951
1. Pauling, L., Corey, R.B. and Branson H. R. The Structure of Proteins: Two Hydrogen-Bonded Helical Configurations of the Polypeptide Chain. PNAS, 37, 205-211, (1951).
2. Pauling, L. & Corey, R. B. Atomic Coordinates and Structure Factors for Two Helical Configurations of Polypeptide Chains. PNAS, 37, 235-240, (1951).
3. Pauling, L. & Corey, R. B. The Structure of Synthetic Polypeptides. PNAS, 37, 241-250, (1951).
4. Pauling, L. & Corey, R. B. The Pleated Sheet, A New Layer Configuration of Polypeptide Chains. PNAS, 37, 251-256, (1951).
5. Pauling, L. & Corey, R. B. The Structure of Feather Rachis Keratin. PNAS, 37, 256-261, (1951).
6. Pauling, L. & Corey, R. B. The Structure of Hair, Muscle, and Related Proteins. PNAS, 37, 261-271, (1951).
7. Pauling, L. & Corey, R. B. The Structure of Fibrous Proteins of the Collagen-Gelatin Group. PNAS, 37, 272-281, (1951).
8. Pauling, L. & Corey, R. B. The Polypeptide-Chain Configuration in Hemoglobin and Other Globular Proteins. PNAS, 37, 282-285, (1951).
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Linus Carl PaulingThe Nobel Prize in Chemistry 1954
"for his research into the natureof the chemical bond and itsapplication to the elucidationof the structure of complexsubstances"
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Determinants of helical structure
Distances and anglesBetween atoms
Resonant partial double bond character of peptide bond induces planar arrangement of atoms
All hydrogen bonds should be satisfied,i.e. distance N-O of about 2.7Å and anglebetween C = O and H – N less then ~30°
superposition
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Building a model – similar to building with LEGO blocks
Start assembling monomers (amino-acids) with fixed translation and rotation
Look for configurations which have no steric hindrance (i.e. clashes)
Calculate N-H…O=C distances andangles (3-d trigonometry..)
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2 models satisfies all constraints
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The α-helix – one of the two common structural elements in proteins
Completes one turn every 3.7 residues
Rises ~5.4 Å with each turn
Has hydrogen bonds between the C=O of residue i and the N-H of residue i+4
Is right-handed
i
i+4
C
N
O
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Alpha-helices appear a lot in trans-membranal proteins
E.g. Lactose permease (LacY)
1pv6.pdbmembrane
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Why did Pauling and Corey succeed where others failed?
Understanding the importance of hydrogen bonds
Taking into account the planar peptide bond Better knowledge of covalent bond lengths
and angles MOST IMPORTANTLY – they were NOT
crystallographers, and did not consider only models with integer number of residues per turn!
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Proof came 7 years later…
Kendrew, J. C., Bodo, G., Dintzis, H. M. Parrish, R. G., Wyckoff, H.,
and Phillips, D. C. A Three-Dimensional Model of the Myoglobin Molecule
Obtained by X-ray Analysis. Nature, 181, 662 (1958).
John Cowdery Kendrew The Nobel Prize in Chemistry 1962
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Hierarchy of Protein Structure
Linear chain made of 20 possibleamino acids Alpha-helices, beta-sheets, turns
Motifs, domains
Oligomers, complexes
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The Protein Data Bank (www.pdb.org)
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The PDB contains over 40,000 structures (as of December 2006)
NMR - Nuclear magnetic resonanceAllows structure determination based ondistance and angular constraints in solution
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Proteins’ Structure is Dynamic
Fluctuations exists in all proteins Conformational changes ↔ Function
Adenylate kinase An enzyme that catalyzes the production of ATP from ADP
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Protein Folding – still an open question
1954 Christian B. Anfinsen proved that the protein structure is determined by it’s sequenceProtein Denatured (unfolded) Protein
1969 “Levinthal paradox” – For a 100 a.a. sequence there are 9100 possible configurations. If sampled randomly every nanosecond, it will take longer then the age of the universe to fold a single protein
+ Urea DilutionRNaseenzyme
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Protein folding – research continues
Late 1980’s - Wolynes et al. present the “Energy Landscape” or “Folding Funnel” model for protein folding
2006 – There is still no precise understanding how proteins fold fast (up to µsec!), reliably and accurately to their native structure
Energ
y
Entropy
Native(folded) state