Chapter 3

• Protein Structure

The SH2 domain

SEQUENCE DETERMINES STRUCTURE

Levels of protein structure

PRIMARY STRUCTURE

A peptide bond

The requirement that no two atoms overlap limits greatly the possible bond angles in a polypeptide chain

Each amino acid contributes three bonds (red) to thebackbone of the chain. The peptide bond is planar(gray shading) and does not permit rotation. Rotationcan occur about the C-C bond, whose angle of rotationis called psi (), and about the N-C bond, whose angleof rotation is called phi ()

The conformation of the main-chain atoms in a proteinis determined by one pair of and angles for eachacid. Because of steric collisions between atoms withineach amino acid, most pairs of and angles do notoccur. Each dot, in the Ramachandran plot shown here, represents an observed pair of angles in a protein. In-helices, the backbone dihedral angles, and have repeating values of -60°and -40° respectively.

some proteins containing proline.

SECONDARY STRUCTURE

The helix is one of the major elements of secondary structure in proteins.

Main-chain N and O atoms are hydrogen-bonded to each other within helices. (a) Idealized diagram of the path of the main chain in an helix. Alpha helices are frequently illustrated in this way. There are 3.6 residues per turn in an helix, which corresponds to 5.4 angstrom (1.5 angstrom per residue). (b) The same as (a) but with approximate positions for

main-chain atoms and hydrogen bonds included. The arrow denotes the direction from the N-terminus to the C-terminus. (c) Schematic diagram of an helix. Oxygen atoms are red, and N atoms are blue. Hydrogen bonds between O and N are

red and striated. The side chains are represented as purple circles.

The α-helix. The NH of every peptide bond is hydrogen-bonded to the CO ofa neighboring peptide bond located four peptide bonds away in the same

chain

The b-sheet. The individual peptide chains (strands) in a b-sheet are heldtogether by hydrogen-bonding between peptide bonds in different strands

The structure of a coiled-coil

TERTIARY STRUCTURE

Proteins fold into a conformation of lowest energy

Three types of noncovalent bonds that help proteins fold

A protein folds into a compact conformation

Hydrogen bonds in a protein molecule

A protein domain is a fundamental unit of organization

domains - structural units that fold more or less independently of each other

The Src protein

The Src protein

Ribbon models of three different protein domains

PROTEIN FOLDING

How does a protein chain achieves its native conformation?

As an example, E. coli cells can make a complete, biologically activeprotein containing 100 amino acids in about 5 sec at 37°C.

If we assume that each of the amino acid residues could take up 10 different conformations on average, there will be 10100 differentconformations for this polypeptide.

If the protein folds spontaneously by a random process in which ittries all possible conformations before reaching its native state, andeach conformation is sampled in the shortest possible time (~10-13 sec),it would take about 1077 years to sample all possible conformations.

There are two possible models to explain protein folding:

In the first model, the folding process is viewed as hierarchical, in whichsecondary structures form first, followed by longer-range interactions toform stable supersecondary structures. The process continues untilcomplete folding is achieved.

In the second model, folding is initiated by a spontaneous collapse of thepolypeptide into a compact state, mediated by hydrophobic interactionsamong non-polar residues. The collapsed state is often referred to as the‘molten globule’ and it may have a high content of secondary structures.

Most proteins fold by a process that incorporates features of both models.

Takada, Shoji (1999) Proc. Natl. Acad. Sci. USA 96, 11698-11700

Thermodynamically, protein folding can be viewed as a free-energy funnel

- the unfolded states are characterized by a high degree of conformational entropy and relatively high free energy- as folding proceeds, narrowing of the funnel represents a decrease in the number of conformational species present (decrease in entropy) and decreased free energy

Schematic pictures of a statistical ensemble of proteins embedded in a folding funnel for a monomeric  -repressor domain. At the top, the protein is in its denatured state and thus fluctuating wildly, where both energy and entropy are the largest. In the middle, the transition state forms a minicore made of helices 4 and 5 and a central region of helix 1 drawn in the right half, whereas the rest of protein is still denatured. At the bottom is the native structure with the lowest energy and entropy.

A schematic energy landscape for protein foldingNature 426:885

Steps in the creation of a functional protein

The co-translational folding of a protein

The structure of a molten globule – a molten globule form of cytochrome b562

Molecular chaperones help guide the folding of many proteins

A current view of protein folding

A unified view of some of the structures that can be formed by polypeptide chains

Nature 426: 888

The hsp70 family of molecular chaperones. These proteins act early,recognizing a small stretch of hydrophobic amino acids on a protein’s

surface.

The hsp60-like proteins form a large barrel-shaped structure that actslater in a protein’s life, after it has been fully synthesized

Nature 379:420-426 (1996)

Cellular mechanisms monitor protein quality after protein synthesis

Exposed hydrophobic regions provide critical signals for protein quality control

The ubiquitin-proteasome pathwayNature 426: 897

The proteasome

Processive cleavage of proteins

Figure 6-90 Molecular Biology of the Cell (© Garland Science 2008)

Processive protein digestion by the proteasome

Figure 6-91a Molecular Biology of the Cell (© Garland Science 2008)

The 19S cap – a hexameric protein unfoldase

Figure 6-91b Molecular Biology of the Cell (© Garland Science 2008)

Model for the ATP-dependent unfoldase activity of AAA proteins

Ubiquitin – a relatively small protein (76 amino acids)

Abnormally folded proteins can aggregate to cause destructive human diseases

(C) Cross-beta filament, a common type of protease-resistant protein aggregate

(D) A model for the conversion of PrP to PrP*, showing the likely change of two α-helices into four β-strands

Regulation of protein folding in the ER

Nature 426: 886

A schematic representation of the general mechanism of aggregation to form amyloid fibrils.

Nature 426: 887

Proteins can be classified into many families – each family memberhaving an amino acid sequence and a three-dimensional structure

that resemble those of the other family members

The conformation of two serine proteases

A comparison of DNA-binding domains (homeodomains) of the yeast α2 protein (green) and the Drosophila engrailed protein (red)

Sequence homology searches can identify close relatives

The first 50 amino acids of the SH2 domain of 100 amino acids compared for the human and Drosophila Src protein

Multiple domains and domain shuffling in proteins

Domain shuffling

An extensive shuffling of blocks of protein sequence (protein domains)has occurred during protein evolution

A subset of protein domains, so-called protein modules, are generallysomewhat smaller (40-200 amino acids) than an average domain

(40-350 amino acids)

Each module has a stable core structure formed from strands of β sheet and they can be integrated with ease into other proteins

Fibronectin with four fibronectin type 3 modules

Figure 3-18 Molecular Biology of the Cell (© Garland Science 2008)

Relative frequencies of three protein domains

Figure 3-19 Molecular Biology of the Cell (© Garland Science 2008)

Domain structure of a group of evolutionarily related proteins that have a similar function – additional domains in

more complex organisms

Quaternary structure

Lambda cro repressor showing “head-to-head” arrangement of identical subunits

Larger protein molecules often contain more than one polypeptide chain

DNA-binding site for the Cro dimer

ATCGCGATTAGCGCTA

The “head-to-tail” arrangement of four identical subunits that form a closed ring in neuraminidase

HEMOGLOBIN

A symmetric assembly of two different subunits

A collection of protein molecules, shown at the same scale

Protein Assemblies

Some proteins form long helical filaments

Helical arrangement of actin molecules in an actin filament

Helices occur commonly in biological structures

A protein molecule can have an elongated, fibrous shape

Collagen

Elastin polypeptide chains are cross-linked together to form rubberlike, elastic fibers

Disulfide bonds

Extracellular proteins are often stabilized by covalent cross-linkages

Proteolytic cleavage in insulin assembly