Folding and flexibility

Outline What is protein folding ? How proteins fold in vivo ? What is protein flexibility ?

Structural features present in any folded globular protein: 1. Most mainchain hydrogen bonds are formed. 2. Core is formed of hydrophobic residues, but not all hydrophobic residues are buried. 3. Well-packed hydrophobic core. 4. Secondary structural elements (helices, sheet) show a hydrophobic and hydrophilic face - Amhipathic helices have hydrophobic residues every third/fourth residue - Amphipathic sheets alternate hydrophobic and hydrophilic residues. 5. Most polar residues are on the surface. 6. Buried polar residues have hydrogen bonded partners. 7. Almost all charged residues are on the surface. 8. Buried charged residues often have charges of opposite sign close by.

The molten globule state

Properties of molten globule Most secondary structure elements of native state have formed Less compact than native state and proper packing of hydrophobic core is not yet accomplished Interior is in more “liquid” than solid state Surface structures and loops largely unfolded

Single and multiple folding pathways

How do correct disulfides form ? Example: Bovine pancreatic trypsin inhibitor is unfolded before the disulfides have formed Protein disulfide isomerase catalyzes internal disulfide exchange During folding, first disulfides form in random More stable disulfides accumulate

Cis- and trans- prolines

Active site is on the side of barrel, which is quite unusual The cis-trans isomerization is increased 1,000,000-fold Prolyl peptide isomerase

Molecular chaperones Proteins, which help the other proteins to fold corrcetly How?

Chaperonin GroES-GroEL GroEL : 14 subunits GroES : 7 subunits

Structure of GroEL monomer Unfolded proteins bind to hydrophobic residues of apical domain ATP binds to equatorial domain

Binding of GroES and ATP to GroEL Upon binding of GroES and ATP, one side of GroEL gets extended and the central cavity increases in volume In central cavity now are less hydrophobic residues exposed The other side of GroEL looses affinity to another GroES molecule

Events upon protein folding inside GroES- GroEL complex unfolded protein folded protein

Chaperones, other than GroES-EL exist Principle is similar – chaperones bind to hydrophobic surfaces Other chaperones do not form enclosed structure

Can the 3D structure of a protein be predicted? Many proteins can be folded and unfolded reversibly in vitro This implies that practically all information necessary to determine the 3D structure is contained in the sequence

But... A general method for accurate fold prediction has not been discovered yet Available methods are at most 60% reliable and certainly not a replacement for x-ray or NMR studies

Flexibility Folded proteins are not static – structural rearrangements are common In most cases the structural rearrangements are minor and limited to loop regions Frequently, some particular secondary structure element (strand or helix) switches between ordered / disordered states (for example, upon ligand binding) Sometimes, major structural rearrangements are found

Causes of structural rearrangemenets Interaction with a ligand Interaction with other proteins Changes of pH and/or ionic strength Covalent modifications

Examples of minor structural rearrangements Different packing environments for icosahedral virus MS2 coat protein subunits Same loop adopts different conformations at vertices of icosahedron and in the middle of one triangular face

Ordered C-terminal helix upon substrate binding to glutathione transferase

Example of major structural rearrangements Conformational changes of calmodulin

Flexibility in multidomain proteins Example: fibonectin Large extracellular protein with several functions Composed from about 30 domains All linker regions are flexible