Lecture 13 Protein Structure II Chapter 3

PROTEIN FOLDING

How does a protein chain achieves its native conformation? As an example, E. coli cells can make a complete, biologically active protein 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 different conformations for this polypeptide. If the protein folds spontaneously by a random process in which it tries all possible conformations before reaching its native state, and each conformation is sampled in the shortest possible time (~ sec), it would take about 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 which secondary structures form first, followed by longer-range interactions to form stable supersecondary structures. The process continues until complete folding is achieved. In the second model, folding is initiated by a spontaneous collapse of the polypeptide into a compact state, mediated by hydrophobic interactions among 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, 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 folding Nature 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 b 562

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 acts later in a protein’s life, after it has been fully synthesized

Nature 379: (1996)

Cellular mechanisms monitor protein quality after protein synthesis Exposed hydrophobic regions provide critical signals for protein quality control

The ubiquitin-proteasome pathway Nature 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 member having 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 α 2 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 generally somewhat smaller ( amino acids) than an average domain ( 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 ATCGCGAT TAGCGCTA

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