ACDEFGHIKLMNPQRSTVWY

primary structure

Principles of Protein Structure

Different Levels of Protein Structure

NH2

Lysine

Histidine

Valine

Arginine

Alanine

COOH

Common Secondary Structure Elements

• The Alpha Helix

Properties of alpha helix

• 3.6 residues per turn, 13 atoms between H-bond donor and acceptor approx. -60º; approx. -40º• H- bond between C=O of ith residue & -NH of (i+4)th residue• First -NH and last C=O groups at the ends of helices do not participate in H-

bond• Ends of helices are polar, and almost always at surfaces of proteins• Always right- handed• Macro- dipole

Alpha Helix

Helical wheel

Residues i, i+4, i+7 occur on one face of helices, and hence show definite pattern of hydrophobicity/ hydrophilicity

Introduction to Molecular BiophysicsAssociation of helices: coiled coils

These coiled coils have a heptad repeat abcdefg with nonpolar residues at position a and d and an electrostatic interaction between residues e and g.

Isolated alpha helices are unstable in solution but arevery stable in coiled coil structures because of the interactions between them

The chains in a coiled-coil have the polypeptide chains aligned parallel and in exact axial register. This maximizes coil formation between chains.

The coiled coil is a protein motif that is often used to control oligomerization.

They involve a number of alpha-helices wound around each other in a highly organised manner, similar to the strands of a rope.

Introduction to Molecular BiophysicsThe Leucine Zipper Coiled Coil

Initially identified as a structural motif in proteins involved in eukaryotic transcription. (Landschultz et al., Science 240: 1759-1763 (1988).

Originally identified in the liver transcription factor C/EBP which has a Leu at every seventh position in a 28 residue segment.

Association of helices: coiled coils

The helices do not have to run in the same direction for this type of interaction to occur, although parallel conformation is more common.

Antiparallel conformation is very rare in trimers and unknown in pentamers, but more common in intramolecular dimers, where the two helices are often connected by a short loop.

Chan et al., Cell 89, Pages 263-273.

Since the dipole moment of a peptide bond is 3.5 Debye units, the alpha helix has a net macrodipole of:

n X 3.5 Debye units (where n= number of residues)

This is equivalent to 0.5 – 0.7 unit charge at the end of the helix.

Basis for the helical dipoleIn an alpha helix all of the peptidedipoles are oriented along the same direction.

Consequently, the alpha helix has a net dipole moment.

The amino terminus of an alpha helix is positive and the carboxy terminus is negative.

Structure of human TIMTwo helix dipoles are seen to play important roles:

1.Stabilization of inhibitor 2-PG2.Modulation of pKa of active site His-95.

Helical PropensitiesAla -0.77Arg -0.68Lys -0.65Leu -0.62Met -0.50Trp -0.45Phe -0.41Ser -0.35Gln -0.33Glu -0.27Cys -0.23Ile -0.23Tyr -0.17Asp -0.15Val -0.14Thr -0.11Asn -0.07His -0.06Gly 0Pro ~3

Common Secondary Structure Elements

• The Beta Sheet

Secondary structure: reverse turns

Secondary Structure:Phi & Psi Angles Defined

• Rotational constraints emerge from interactions with bulky groups (ie. side chains).

• Phi & Psi angles define the secondary structure adopted by a protein.

The dihedral angles at C atom of every residue provide polypeptides requisite conformational

diversity, whereby the polypeptide chain can fold into a globular shape

Ramachandran Plot

Structure Phi () Psi()

Antiparallel -sheet -139 +135Parallel -Sheet -119 +113Right-handed -helix +64 +40

310 helix -49 -26 helix -57 -70Polyproline I -83 +158Polyproline II -78 +149Polyglycine II -80 +150

Phi & Psi angles for Regular Secondary Structure Conformations

Table 10

Secondary Structure

Beyond Secondary StructureBeyond Secondary Structure

Supersecondary structure (motifs): small, discrete, commonly observed aggregates of secondary structures

sheet

helix-loop-helix

Domains: independent units of structure barrel four-helix bundle

*Domains and motifs sometimes interchanged*

Common motifs

Supersecondary structure:

Crossovers in ---motifs

Right handed

Left handed

• Consists of two perpendicular 10 to 12 residue alpha helices with a 12-residue loop region between

• Form a single calcium-binding site (helix-loop-helix). • Calcium ions interact with residues contained within the loop

region. • Each of the 12 residues in the loop region is important for

calcium coordination. • In most EF-hand proteins the residue at position 12 is a

glutamate. The glutamate contributes both its side-chain oxygens for calcium coordination.

EF Hand

Calmodulin, recoverin : Regulatory proteins Calbindin, parvalbumin: Structural proteins

EF Fold

Found in Calcium binding proteins such as Calmodulin

•Consists of two helices and a short extended amino acid chain between them.  •Carboxyl-terminal helix fits into the major groove of DNA.   •This motif is found in DNA-binding proteins, including repressor, tryptophan repressor, catabolite activator protein (CAP)

Helix Turn Helix Motif

Leucine Zipper

•The beta-alpha-beta-alpha-beta subunit•Often present in nucleotide-binding proteins

Rossman Fold

What is a Protein Fold?

Compact, globular folding arrangement of the polypeptide chain

Chain folds to optimise packing of the hydrophobic residues in the interior core of the protein

Common folds

Tertiary structure examples: All-

AlamethicinThe lone helix

Rophelix-turn-helix

Cytochrome Cfour-helix bundle

Tertiary structure examples: All-

sandwich barrel

Tertiary structure examples:

placental ribonucleaseinhibitor horseshoe

triose phosphateisomerase barrel

Four helix bundle

•24 amino acid peptide with a hydrophobic surface•Assembles into 4 helix bundle through hydrophobic regions•Maintains solubility of membrane proteins

Oligonucleotide Binding (OB) fold

TIM Barrel

•The eight-stranded / barrel (TIM barrel)

•The most common tertiary fold observed in high resolution protein crystal structures

•10% of all known enzymes have this domain

Zinc Finger Motif

Domains are independently folding structural units.

Often, but not necessarily, they are contiguous on the peptide chain. Often domain boundaries are also intron boundaries.

Domain swapping: Parts of a peptide chain can reach into neighboring

structural elements: helices/strands in other domains or whole domains in other subunits.

Domain swapped diphteria toxin:

• Helix bundlesLong stretches of apolar amino acidsFold into transmembrane alpha-helices“Positive-inside rule”

Cell surface receptorsIon channelsActive and passive transporters

• Beta-barrelAnti-parallel sheets rolled into cylinder Outer membrane of Gram-negative bacteria

Porins (passive, selective diffusion)

Transmembrane Motifs

Quaternary Structure

• Refers to the organization of subunits in a protein with multiple subunits

• Subunits may be identical or different

• Subunits have a defined stoichiometry and arrangement

• Subunits held together by weak, noncovalent interactions (hydrophobic, electrostatic)

• Associate to form dimers, trimers, tetramers etc. (oligomer)

• Typical Kd for two subunits: 10-8 to 10-16M (tight association)

–Entropy loss due to association - unfavorable

–Entropy gain due to burying of hydrophobic groups - very favourable

• Stability: reduction of surface to volume ratio

• Genetic economy and efficiency

• Bringing catalytic sites together

• Cooperativity (allostery)

Structural and functional advantages of quaternary structure

Quaternary structure ofmultidomain proteins