Protein Structure (in a nutshell)

Guy Ziv

December 26th, 2006

Myoglobin (1958)

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”

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

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

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

Bragg planes intersect the unit cell in particular “indices”

0,0

h=1, k=1

h

k

h=4, k=-2

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)

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

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

Pauling and Corey

Robert Corey (1897-1971) Linus Pauling (1901-1994)

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).

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).

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"

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

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..)

2 models satisfies all constraints

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

Alpha-helices appear a lot in trans-membranal proteins

E.g. Lactose permease (LacY)

1pv6.pdbmembrane

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!

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

Hierarchy of Protein Structure

Linear chain made of 20 possibleamino acids Alpha-helices, beta-sheets, turns

Motifs, domains

Oligomers, complexes

The Protein Data Bank (www.pdb.org)

The PDB contains over 40,000 structures (as of December 2006)

NMR - Nuclear magnetic resonanceAllows structure determination based ondistance and angular constraints in solution

Proteins’ Structure is Dynamic

Fluctuations exists in all proteins Conformational changes ↔ Function

Adenylate kinase An enzyme that catalyzes the production of ATP from ADP

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

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