Protein Structure & Folding(9 / 25 / 2008)

•Secondary Structure

•Tertiary Structure

•Quaternary Structure and Symmetry

•Protein Folding

Example of each level of protein structure

Fibrous Proteins

Nails, hair, horns and feathers or -forms

30 variants, tissue specific type I and type II

acidic negative charge basic positive charge

keratin• hair- 20 M diameter• macrofibril 2000 Å parallel to hair • microfibril 80 Å and high sulfur content protein• can break -S-S- with mercaptans and reconnect (i.e. can give hair a “permanent” wave)

Keratin - A Coiled Coil

keratin proteins are helical but spacing differs from a regular -helix

a 5.1 Å vs. 5.4 Å pitch. This change in pitch forms closely associated pairs of helices. Each pair consists of a type I and type II proteinLeft-handed coil coiled-coil310 AA residues 7-residue pseudo repeat.Helical wheel - Look down an helix and residues stick out from center of helix 3.6 residues/turn 360 = 100 per residue3.6

a - b - c - d - e - f - g - a repeat on side of helix

Helical wheel diagram

a and d residues are nonpolar.

Protofilaments antiparallel strands

A coiled coil

View down the coil axis

Tropomyosin

a & d are non-polar and face the same side of helix. 3.6 residues/turn3.5 residues hydrophobic repeat

The hydrophobic strip aligns between two helices with 18 inclination from one to another.

Dimer protofilament microfibril macrofibril hair

keratin rich in cys and form disulfides hard keratin cys content is high soft keratin cys content is lowPerms reduce R-S--S-R bonds to 2R-SH Curly hair has more Cys residues.

Higher order Keratin structure

Collagen -A Triple Helix

Bones, teeth, cartilage, tendon, ligament, blood vessels and skin matrix Strong, flexible, stretchySeveral types

I [1(I)]22(I) skin, bone tendon, cornea vessels

II [1(II)]3 cartilage

III [1(III)]3 vessels, fetal skin

Type I 285 kDA 14Å wide

3000 Å long 30 distinct peptide types 16 variants

1/3 gly; 15-30% 4-hydroxyproline (Hyp); some 3-hydroxyproline (3-Hyp), and some 5-hydroxylysine (Hyl)

4-hydroxyprolyl 3-hydroxyproyl 5-hydroxylysyl(4-Hyp) (3-Hyp) (Hyl)

Gly-X-Y X often Pro Y often Hyp like a poly Gly or poly Pro helix

Left-handed 3.0 residues/turn pitch 9.4 extended conformation the prolines avoid each other.

3 left handed helices combine in a triple right handed coil.

Rope twist or metal cable longitudinal force (pulling) is supported by lateral compression opposite twisted strands prevents twists from pulling out.

Vitamin C is required for hydroxyproline formation

Hydroxyproline gives collagen stability and strength by H-bonding.

Without prolyl hydroxylase, collagen denatures at 24C instead of 39 to form gelatin.

Scurvy-skin lesions, broken blood vessels, wounds don’t heal, teeth fall out, cannot stand.

Crosslinking requires lysine oxidase

N C - CH2 - CH2 - NH2 -Aminopropionitrile, from sweet pea

Inhibits lysine oxidase i.e. no crosslinking several diseases:Lathyrism (abnormalities in bones, joints, etc.)

Osteogenesis imperfecta, brittle bone, A single amino acid change could be lethal

Ehlers-Danlos syndromes, hyperextensible joints and skin, Indian rubberman

Osteoarthritis - cartilage.

Tertiary protein structure

• Describes the folding of its secondary structural elements

• Determined via nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography

• 3D structures of many proteins are available at http://www.rcsb.org/pdb

The Protein Data Bank (PDB)

“Most atoms in the biological molecules have a little magnet inside them. If we put any of these molecules in a big magnet, all the little magnet in the molecule will orient themselves to line up with the big magnet”, allowing to scientist to probe various properties of the molecule…

Nuclear Magnetic Resonance (NMR)

The calcyclin dimer. Potts et al., NSB 2, 790-796 (1995)

NMR at the UH

• The UH Keck/IMD NMR Center has the first 800 MHz NMR spectrometer installed within Texas.

• The latest facility enhancement, in January 2006, is the installation of a Bruker 5mm TXI CryoProbe for the 800 MHz instrument. – This NMR probehead is

cooled by cyrogenic helium gas to reduce thermal noise and improve the signal to noise ratio by as much as three times a conventional probe.

UH 800 MHz NMR Spectrometer

X-ray crystallography

X-rays are bounced off of the protein

X-rays are diffracted by electronsin the various atoms/bonds

The diffraction pattern of theX-rays is measured and anelectron density map is created (cyan in the figure to the left)

Attempts are made to fit amino acids into the electron density

Most Protein Crystal Structures exhibit less than atomic resolution

How the quality of (degree of focus) of an electron density map varies with its resolution limits?

Visualizing Proteins

Ball-and-stick Space-filling Ribbons

The course of the polypeptide chain can be followed by tracing the positions of its C atoms or by representing helices as helical ribbons or cylinders and sheets as sets of flat arrows pointing from the N- to the C-termini.

Motifs and Domains

-helixfrom whale hemoglobinhas non-polar residues(yellow) and polar residues(purple) on opposite sidesof the helix.

-sheetwith protein bindingdomain on the sidewith non-polarresidues (orange)leaving the polar ones(purple) facing solvent water

-hairpin

Most common

Supersecondary structural motifs

CommonNucleotide bindingRossman Fold

Greek key

Orange spheres aremetal ions

Jack Bean Concanavalin A

Examples of Globular Proteins (1)

Triose phosphate isomerase

A glycolysis pathway enzyme

Examples of Globular Proteins (2)

cytochrome b562 (E. Coli) Fab (human) Lactate dehydrogenase (dogfish)Helix bundle Immunoglobulin fold 6-stranded parallel sheet

Retinol binding protein Peptide Asn amidase F TIM (human) (F. meningosepticum) (chicken muscle)

-barrels

1

2

1

2

3

34

4

5

5

67

Many single polypeptide proteinsfold into multiple structural domains,each with their own function

This is glyceraldehyde-3-phosphate(GAP) dehydrogenaseThe red domain binds NAD+The green domain binds GAPA glycolysis pathway enzyme

Large polypeptides form domains

Quaternary protein structure

4o structure is the relativeplacement of different poly-peptide segments

Hemoglobin is shown to theleft (1-yellow, 2-green,1-cyan, 2-blue), hemegroups are in red - bind O2

Subunits usually associate noncovalently

Subunits are symmetrically arranged

Sidechain locations in proteins

• Non-polar sidechains (Val, Leu, Ile, Met, and Phe) occur mostly in the interior of a protein keeping them out of the water (hydro-phobic effect)

• Charged polar residues (Arg, His, Lys, Asp, and Glu) are normally located on the surface of the protein in contact with water.

• Uncharged polar residues (Ser, Thr, Asn, Gln, and Tyr) are usually on the protein surface but also occur in the interior of the protein.

Protein Stability

Forces that stabilize protein structure: 1, 2, 31. The Hydrophobic Effect

Zinc finger:Nucleic acid-binding proteins

2. Electrostatic Interactions

3. Chemical Cross-links

Ion pair (salt bridge) of myoglobin

Protein folding problem

• Levinthal paradox• Prediction of three dimensional

structure from its amino acid sequence

• Translate “Linear” DNA Sequence data to spatial information

Protein Folding

Protein Folding Pathways

Proteins can be unfolded/denatured.

Denatured proteins can be refolded, sometimes requiring helper proteins, and this refolding takes place via preferred pathways.

Common thought is that secondary structures form first, eventually collapsing due to the formation of hydrophobic cores.

Folding funnelEnergy-entropy relationship for protein folding

Molecular chaperons

GroEL GroES

Molecular chaperones:

(1) Hsp70 proteins function as monomer

(2) Chaperonins, large multisubunit proteins

(3) Hsp90 proteins for the folding of proteins involved with signal transduction

Reaction cycle of the GroEL/ES cycle

1. GroEL ring binding 7 ATP and a substrate (improperly folded protein). Then it binds a GroES cap to become the cis ring.

2. The cis ring catalyzes the hydrolysis of its 7 ATP.

3. A 2nd substrate binds to the trans ring followed by 7 ATP.

4. The binding of substrate and ATP to the trabs ring conformationally induces the cis ring to release its bound GroES, 7 ADP, and the better folded substrate.The trans ring becomes the cis ring.

Protein disulfide Isomerase

Diseases Caused by Protein Misfolding

Alzheimer’s disease Transmissible spongiform encephalopathies (TSE) Amyloidoses

Prion protein conformation

A model of an amyloid fibril

Once it has formed, an amyloid fibril is virtually indestructible (interchain H- bonds).

It seems likely that protein folding pathways have evolved not only to allow polypeptides to assume stable native structures but also to avoid forming interchain H-bonds that would lead to fibril formation .

The factors that trigger amyloid formation remain obscure, even when mutation (hereditary amyloidoses) or infection (TSEs) appear to be the cause.

Lecture 11Tuesday 9/30/08

Protein Structure and Purification