a

1
3 different types of helices

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

310
a
p
310 helix 10 atoms in the ring closed by a
hydrogen bond. fy angles at the edge of an
allowed region in the Ramachandran plot. Side
chains are not as nicely staggered as in an a
helix.
Schultz Schirmer, Principles of Protein
Structure, Springer, 1985
2
Leucine zippers
Leucine zippers are heptad repeats containing a
leucine at position 4 with almost complete
conservation.
Example transcription factor GCN4
Leucine zippers form a-helices with a
helical repeat of 3.5 residues per turn (instead
of 3.6 residues per turn as in a conventional
a-helix). Plotted on a helical wheel, the leucine
residues all 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
3
Leucine zippers form two parallel coiled-coil a
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 prevent dimer formation.
The GCN4 basic region-leucine zipper binds DNA as
a dimer of two uninterrupted a helices.
Leucine zipper homodimers and heterodimers can
recognize different DNA sequences, as indicated
by the red and blue regions of the DNA.
4
Domain swapping
antigen binding site
Fab fragment of conventional antibody
antibody binding sites with carbohydrates from
HIV gp120
new
conventional
conventional
Domain swapping generates a dimer with
increased binding surface to multiple carbohydrate
chains. Furthermore, additional new binding
sites are created, enhancing the binding
affinity.
gt 40 cases of domain swapping identified. Some of
them are crystallization artifacts, while some of
them are physiologically relevant.
Science 300, 2065 (2003)
Prot. Sci. 11, 1285 (2002)
5
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.

Example domain architectures of apoptotic
proteins
Science, 291, 1279 (2001)
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.
6
Protein function derived from genome comparison
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.
Two homologous enzymes are fused in human.
Two nonsequential enzymes from the histidine
biosynthesis pathway are fused in yeast.
data base of interacting proteins
http//dip.doe-mbi.ucla.edu/
Science 285, 751 (1999)
7
Amino acid composition of unstructured versus
structured proteins
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)
8
Not all proteins have a defined 3D structure
Up to 50 of eukaryotic proteins have at least
one long (gt50 residues) disordered region. 11 of
proteins in Swiss-Prot are probably fully
disordered
These proteins are likely to assume a defined
structure in complex with other proteins.
Two proteins can be unstructured by
themselves, but can form a specific complex of
defined globular structure, when together.
p27Kip1 complexed with cyclin-dependent kinase 2
(Cdk2) and cyclin A CycA ProteinData Bank (PDB)
number 1JSU
Nature 415, 549 (2002)
Trends in Biochem. Sci. 27, 527 (2002)
9
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 DG of the unfolding reaction
U
F
DG is small 5-15 kcal/mol for proteins with
70-200 residues. For comparison DG 4-5
kcal/mol for the formation of a single H-bond
between two water molecules.

• 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)

urea
guanidinium ion
Urea and guanidinium chloride denature proteins
because they can form more stable H-bonds to the
backbone amides than water.
10
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 a projection
of the resulting amplitude now yields an ellipse
instead of the usual line.
The CD signal at 222 nm is often used to
monitor folding-unfolding equilibria as a
function of temperature.
http//www-structure.llnl.gov/cd/cdtutorial.htm
11
Folding-Unfolding Equilibria by CD and
Fluorescence
unfolded
folded
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.
12
Amyloid Fibres
Many proteins can become insoluble by the
formation of amyloid fibres. These fibres consist
of 4x2 sets of infinitely long parallel b-sheets
stacked in two layers.
Cryoelectron microscopy image
Model of a fibril
http//people.cryst.bbk.ac.uk/ubcg16z/amyloid/amy
loid.html
13
Chaperones help proteins fold
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 chain in the cycle between open and
closed conformation of the GroEL ring.
mcdb.colorado.edu/courses/ 3280/class03-3.htm Scie
nce 284, 823 (1999)l
14
cis-trans isomerization of peptide bonds
preceding Pro
cis
trans
In the peptide Ac-Ala-X-Pro-Ala-Lys-NH2, the
amount of cis-peptide bond varies between 6 for
XPro and 38 for XTrp. (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.
15
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 contain thioredoxin-like domains
which assist in reduction and oxidation of
disulfide bonds.
DsbA contains two cysteines separated by two
residues. The disulfide bond between these two
cysteines is very reactive and act as an
oxidizing agent for other cysteines.
DsbA
16
Enzymes can provide a non-aqueous environment
arachidonic acid
cyclooxygenase
PGG2
17
Enzymes function by stabilizing the transition
state
Example catalytic antibodies. The
antigen-binding sites are hypervariable. Antibodie
s can be selected which bind to any molecular
target of interest.
18
An antibody raised against a transition state
analog catalyzes the reaction from chorismate to
prephenate
Note catalytic antibodies occur naturally, e.g.
with proteolytic function in autoimmune diseases.
Hilvert lab, ETH-Zurich
19
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-desolvati
on processes present no kinetic impediment to
protein function.
Science 254, 974 (1991) J. Mol. Biol. 282, 859
(1998)
20
But water molecules can play a crucial
structural role!
Example in the E. coli Trp-repressor/DNA
complex, the specificity determining contacts
between the protein and the DNA bases are
mediated by water molecules.
W denotes water molecules. Base-pairs on the
left, protein on the right.
The repressor binds with a helix-turn-helix motif
to the major groove of the DNA.
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.
Nature 335, 321 (1988)