310

3 different types of helices

- 310 helix: 3 residues per turn, 2.0 Å rise per turn, occurs only in short segments helix: 3.6 residues per turn, 1.5 Å rise per turn, abundant helix: 4.3 residues per turn, 1.1 Å rise per turn, hypothetical

310 helix: 10 atoms in the ring closed by a hydrogen bond. angles at the edge of an allowed region in the Ramachandran plot.Side chains are not as nicely staggered as in an helix.

Schultz & Schirmer, Principles of Protein Structure, Springer, 1985

Leucine zippers

Leucine zippers are heptad repeats containing a leucine at position 4 with almost complete conservation.

Example: transcription factor GCN4

Leucine zippers form -helices with a helicalrepeat of 3.5 residues per turn (instead of 3.6 residues per turn as in a conventional -helix).Plotted on a helical wheel, the leucine residuesall face the same side of the helix.Residues in positions a and d are hydrophobic or, at least, uncharged.

Carl Branden & John Tooze, Introduction to Protein Structure, Garland, 1998

Leucine zippers form two parallel coiled-coil helices, where the hydrophobic side chains in positions a and d of the heptad repeats form a hydrophobic core between the helices with the leucine residues facing each other. The side chains immediately outside this core (positions e and g) are frequently charged and can either promote or preventdimer formation.

The GCN4 basic region-leucine zipperbinds DNA as a dimer of two uninterrupted helices.

Leucine zipper homodimers and heterodimers can recognize differentDNA sequences, as indicated by the red and blue regions of the DNA.

Domain swapping

> 40 cases of domain swapping identified.Some of them are crystallization artifacts,while some of them are physiologicallyrelevant.

Prot. Sci. 11, 1285 (2002)

conventional conventionalnew

antibody binding sites with carbohydrates from HIV gp120

Science 300, 2065 (2003)

Domain swapping generatesa dimer with increasedbinding surface to multiplecarbohydrate chains.Furthermore, additionalnew binding sites are created, enhancing thebinding affinity.

Fab fragment of conventional antibody

Additional

antigen binding site

What is a domain?

- a segment of similar amino acid residues identified in different proteins (by sequence alignment)- an independent folding unit

Domains are often arranged like beads on a string, connected by flexible linkers.

Science, 291, 1279 (2001)

Example: domain architectures of apoptotic proteins

Compare, however, with PEST domains!(PEST: domains rich in Pro, Glu, Ser and Thr, which enhance the degradation rate of proteins by the proteasome)PEST domains are unstructured.

Science 285, 751 (1999)

Some pairs of interacting proteins have homologs in another organism fused into a single protein chain.Searching sequences from many genomes revealed 6809 such putative protein-protein interactions in Escherichia coli and 45,502 in yeast.

Protein function derived from genome comparison

Two nonsequential enzymes from the histidine biosynthesis pathway are fused in yeast.

Two homologous enzymesare fused in human.

data base of interacting proteins: http://dip.doe-mbi.ucla.edu/

Amino acid frequencies in %.

Asterisks identify amino acids that are at least two times more or less frequent than in an average globular protein in the Protein Data Bank.

Trends in Biochem. Sci. 27, 527 (2002)

Amino acid composition of unstructured versus structured proteins

Not all proteins have a defined 3D structure

p27Kip1 complexed with cyclin-dependent kinase 2 (Cdk2) and cyclin A [CycA; ProteinData Bank (PDB) number 1JSU]

These proteins are likely to assume a defined structure in complex with other proteins.

Up to 50% of eukaryotic proteins have at least one long (>50 residues) disordered region. 11% of proteins in Swiss-Prot are probably fully disordered

Two proteins can be unstructured by themselves,but can form a specific complex of defined globular structure, when together.

Nature 415, 549 (2002)

Trends in Biochem. Sci. 27, 527 (2002)

Protein folding-unfolding equilibria and denaturation

Folded protein structures are in equilibrium with partially or fully unfolded forms.The conformational stability equals the free energy change G of the unfolding reaction:

urea guanidinium ion

Urea and guanidinium chloride denature proteins because they can form more stable H-bonds to the backbone amides than water.

Unfolded polypeptide chains assume a ‘random coil conformation’.

Unfolding can be achieved by- increased temperature (= heat denaturation)- decreased temperature (= cold denaturation)- chemicals (e.g. 6 M urea or guanidinium chloride)

UF

G is small: 5-15 kcal/mol for proteins with 70-200 residues.For comparison: G = 4-5 kcal/mol for the formation of a single H-bond between two water molecules.

Circular Dichroism (CD) Spectroscopy

Circular Dichroism is observed when optically active matter absorbs left and right hand circular polarized light slightly differently.Linear polarized light can be viewed as a superposition of opposite circular polarized light of equal amplitude and phase.When this light passes through a sample with a different absorbance A for the two components, the amplitude of the stronger absorbed component will smaller than that of the less absorbed component. The consequence is that aprojection of the resulting amplitude now yields an ellipse instead of the usual line.

http://www-structure.llnl.gov/cd/cdtutorial.htm

The CD signal at 222 nm is often used to monitorfolding-unfolding equilibriaas a function of temperature.

Folding-Unfolding Equilibria by CD and Fluorescence

folded

unfolded

The sigmoidal shape of the curve is a consequence of the cooperativity of unfolding.

Fluorescence mostly relies on the fluorescence of tryptophan residues which is different for solvated versus buried side chains.

http://people.cryst.bbk.ac.uk/~ubcg16z/amyloid/amyloid.html

Amyloid Fibres

Many proteins can become insoluble by the formation of amyloid fibres. These fibres consist of 4x2 sets of infinitely longparallel sheets stacked in two layers.

Cryoelectron microscopy image

Model of a fibril

Chaperones help proteins fold

mcdb.colorado.edu/courses/ 3280/class03-3.htmScience 284, 823 (1999)l

GroEL and GroES are E. coli proteins. GroEL forms a cavity, where unfolded protein (I) can bind. ATP is required to form the complex with GroES. The release of the folded (N) or still unfolded protein requires again ATP.

GroEL acts by partial unfolding of misfolded proteins, presumably by stretching the polypeptide chainin the cycle between open and closed conformation of the GroEL ring.

cis-trans isomerization of peptide bonds preceding Pro

trans cis

In the peptide Ac-Ala-X-Pro-Ala-Lys-NH2, the amount of cis-peptide bond varies between6% for X=Pro and 38% for X=Trp. (Nat. Struct. Biol. 6, 910 (1999))

Despite the relative rigidity of the side chain of proline, frequent occurance of prolines reduces the chances for globular structures due to the cis-trans heterogeneity.

Many heat shock proteins are chaperones.

Proline-cis-trans isomerases can be considered as chaperones, as the cis-trans isomerization step is slow compared with bond rotation around single bonds.

Proteins with more than one disulfide bond often form non-native disulfide bonds during folding which must be corrected. DsbA and DsbC in E. coli containthioredoxin-like domains which assist in reduction and oxidation of disulfide bonds.

DsbA

DsbA contains two cysteinesseparated by two residues. The disulfide bond betweenthese two cysteines is very reactive and act as an oxidizingagent for other cysteines.

Enzymes can provide a non-aqueous environment

PGG2

arachidonic acid

cyclooxygenase

Enzymes function by stabilizing the transition state

Example: catalytic antibodies.The antigen-binding sites are hypervariable.Antibodies can be selected which bind to any molecular target of interest.

Hilvert lab, ETH-Zűrich

An antibody raised against a transition state analog catalyzes the reaction from chorismate to prephenate:

Note: catalytic antibodies occur naturally, e.g. with proteolyticfunction in autoimmune diseases.

The Role of Water in Intermolecular Recognition

NMR experiments and molecular simulations have shown that the residence times of buried hydration water molecules is shorter than 1 ms.

On protein and DNA surfaces, the residence times are shorter than 1 ns.

Solvation-desolvation processes present no kinetic impediment to protein function.

Science 254, 974 (1991)J. Mol. Biol. 282, 859 (1998)

Example: in the E. coli Trp-repressor/DNA complex, thespecificity determining contacts between the protein and the DNA bases are mediated by water molecules.

Nature 335, 321 (1988)

The repressor binds with a helix-turn-helix motif to the major groove of the DNA.

W denotes water molecules.Base-pairs on the left,protein on the right.

But: water molecules can play a crucial structural role!

The Trp-repressor does not recognize ‘hydrated DNA’.

It is the energetically favourable arrangement of the complex which determines the

importance of the interfacial water molecules in the protein-DNA complex.