Transcript Introduction to Structure Biology

Introduction to Protein Structure
Q:
Whats that?
A:
Something, you
get Noble prize
for...
John Kendrew & Max Perutz 1962
Structures of myoglobin & hemoglobin
Subjects, covered in this lecture
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Amino acids and their properties
Peptide geometry
Secondary structure
Motifs
Domains
Quaternary structure
Why bother about protein
structure?
• Gives you an visual image
of how proteins look like.
• Study of protein structures
allows to gain an insight
into how protein really
accomplish their function.
• Nobel prizes...
Amino Acids
• 20 different ones, sharing a common backbone
but varying side chain.
• Classed according to their chemical properties
• L-form
nonpolar amino acids
-R group consists of carbon chains
leucine and isoleucine
are structural isomers
nonpolar amino acids
- R group consists of carbon chains
Methionine has a sulphur
atom in its sidechain
sulphur has the same
valence as oxygen
Phenylalanine and tryptophan
have aromatic rings which are
flat due to the double bond
network
Tryptophan is often classified
as being polar because of the
NH group. In practice, however
it has more of hydrophobic
properties
Proline has its R group
bound to the amino
nitrogen to form a ring
network
polar amino acids
- R group consists of carbon, oxygen and nitrogen atoms
together they make the sidechain more hydrophilic
Ser and thr are a mix of carbon
chains and hydroxyl functional
groups (-OH). Cysteine has a
thiol group (-SH) which is
otherwise structurally similar to
serine
but not chemically similar
Asn and gln have an
amide functional group
charged amino acids
- R group has a charge at physiological pH (7.4). pK of the charged groups vary
carboxyl
group
carboxyl
group
amino
group
guanidinio
imidazole
group group, sometimes
charged
Most often classified as a
polar amino acid
Cysteine and disulphides
• The almost exclusively only way to covalently link two nonsequential residues is by forming a disulphide bridge
• Formation of disulphide requires an oxidative environment,
threfore disulphides are very rare in intracellular proteins but
quite abundant in secretory proteins
Peptide units
• A peptide is a set of
covalently bonded
amino acids.
• The covalent bond
is usually referred
as peptide bond
Biochemist’s peptide unit –from N to
C – all main chain atoms within the
unit lie in the same residue
Structural biologist’s
peptide unit – from Ca to next Ca
- all main chain atoms within the
unit lie in a plane
– Angles phi (f) N-Ca
– Angle psi (y) Ca-CO
– Angle omega (w) C-N
The w angle, cis- and trans- peptides
• Because of the partly double nature of peptide bond, w is
always close to 180o for trans- peptides or 0o for cispeptides (±30o in exterme cases)
• Cis- peptides are energetically extremely unfavourable
(~1000 fold) because of steric clashes between the
neighbouring Ca atoms
• The only exception is peptide bond before proline,
where cis- peptide is just 4 times less favourable
than trans- peptide, because there are some steric
clashes in both cis- and trans- forms
• Proline cis-trans isomerization is an important
factor in protein folding, which is why there are
special enzymes – prolylpeptydyl isomerases to
catalyze the transition from one form to another
• According to statistics, 0.03% of nonproline peptides and 5.2% of X-Pro peptides
are in cis- conformation, resulting in a total
of 0.3 % cis-peptides
• In most cases cis- peptides, especially nonproline, occur for a good reason, for
example to maintain some particular
conformation in the active site of enzyme
Main chain conformations (I)
• Only certain combinations of y and f are allowed, due to
steric clashes of backbone atoms and Cb atom. Plot of these
combinations yields the Ramachandran plot.
• All amino acids clusters in specific regions (called allowed
regions) except Gly (explains why Glycine is an important
amino acid).
• In good quality structures only about 2% of
amino acid residues are found in the
disallowed regions of Ramachandran plot
• Of course, residues with disallowed
conformations often have some important
function in proteins
Side chain conformations
• Side chains can have
in principle different
conformations
(rotation of Ca-Cb...)
• The observed
conformations in
protein structures are
the ones which are
more energetically
favourable (rotamers).
Name three amino acids which
are very different from others!
Proline
• No free amino group
• Very rigid
• Introduces breaks in a helices and b strands
Glycine
• Lacks a side chain
• Can be found anywhere in Ramachandran plot
• In proteins often found in flexible regions with unusual
backbone conformations
Cysteine
• Disulphides
Primary, secondary, tertiary and
quaternary structures
The hydrophobic core
• The hydrophobic sidechains of protein has a tendency to
cluster together in order to avoid unfavourable contacts
with polar water molecules
• As a result, in general, hydrophobic sidechains are located
in the interior of protein, forming the hydrophobic core
• Polar and charged amino acids usually are located on the
surface of the protein
• Polar and charged residues also can make hydrophobic
contacts with their aliphatic carbon atoms
• Polar and charged residues are seldom completely buried
within the core and even when they are, the polar groups
are almost invariably involved in hydrogen bond formation
The reasons of secondary
structure formation
• Since sidechains of hydrophobic residues are
located in the hydrophobic core, the mainchain
atoms of the same residues in most cases are also
within the hydrophobic core
• Since the presence of polar groups in hydrophobic
environment is very unfavourable, the main chain
N- and O- atoms have to be neutralised by
formation of hydrogen bonds
• The two most efficient ways of hydrogen bond
formation is to build an alpha helix or a beta sheet
The alpha helix
• 3.6 residues per turn
• the hydrogen bonds are made between
residues n and n+4
Variants of alpha helix
• In regular a helix, residue n makes a H-bond with
residue n+4
• In 310 helix, residue n makes a H-bond with
residue n+3. There are 3 residues per turn,
connected by 10 atoms, hence the name 310
• In p helix, residue n makes a H-bond with residue
n+5
• In p helix there is a hole left in the middle of
helix and in 310 helix the main chain atoms are
packed very tightly. None of above is energetically
favourable
• 310 and especially p helices occur rarely and
usually only at the ends of regular a helix or as a
separate single-turn helix
Handedness of alpha helix
• The a helices as well as 310 and p helices ale
almost exclusively right-handed
• In very rare occasions, left handed a and 310
helixes can occur. They are always very short (4- 6
residues) and normally involved in some
important function of protein like in active site or
ligand binding
• There are about 30 reported cases of left-handed
helices. In contrast, the number of known right
handed helices is of order of hundreds of
thousands
The dipole moment of a helix
Good and bad helix formers
• Different side chains have been found to have
weak but definite preferences for helix forming
ability
• Ala, Glu, Leu and Met are good helix formers
• Pro, Gly, Tyr and Ser are very poor helix formers
• The above preferences are not strong enough to be
used in accurate secondary structure predictions
Periodic patterns in a helices
• The most common location of an a
helice is along the outside of protein,
with one side of the helix facing the
hydrophobic core and other side facing
the solvent
• Such a location results in a periodic
pattern of alterating hydrophobic and
polar residues
• On itself, however, the pattern is not
reliable enough for structure prediction,
since small hydrophobic residues can
face the solvent and some helices are
completely buried or completely
exposed
Beta sheets
Antiparallel
Parallel
A mixed b sheet
A mixed b sheet is far less common than antiparallel or parallel
Twist in b sheets
• Almost all b sheets in the known protein
structures are twisted
• The twist is always right-handed
Loops
• Loops connect secondary structure elements
• Loops are located on the surface of protein
• In general, main chain nitrogen and carbonyl oxygen atoms do not
make H-bonds each to other in loops
• Loops are rich in polar and charged residues
• The lenght of loops can vary from 2 to more than 20 residues
• Loops are very flexible, which makes them difficult to see in either xray or NMR studies of proteins
• Loops frequently participate in forming of ligand binding sites and
enzyme active sites
• In homologous protein families loop regions are far less conserved
than secondary stucture elements
• Insertions and deletions in homologous protein families occur almost
exclusively in loop regions
Hairpin loops and reverse turns
• Loops, which connect two adjacent antiparallel
beta strands are called hairpin loops
• 2 residues long hairpin loops are often called
reverse turns, beta turns or simply turns
Type I turn
Hairpin loop
Strand1
Strand2
Type II turn
Motifs
• Simple combinations of a few secondary structure
elements occur frequently in protein structures
• These units are called supersecondary structure or
motifs
• Some motifs can be associated with a specific
biological function (e.g. DNA binding)
• Other motifs have no specific biological function
alone, but are part of larger structural and
functional assemblies
Helix-loop-helix motifs
DNA binding motif
Calcium binding motif
The hairpin b motif
• Two adjacent anti-parallel b strands, joined
by a loop
• The hairpin motif can occur both as an
isolated unit or as a part of bigger b sheet
Bovine trypsin inhibitor
Snake venomerabutoxin
24 different ways to connect two b hairpins
• Only the first 8
arrangements exist in
known proteins
The Greek key motif
• The most common way to connect 4
adjacent antiparallel b strands
The Greek key motif in
Staphilococcus nuclease
The b-a-b motif
• A convinient way to connect two paralel
beta strands
• b-a-b motif is a part of almost all
proteins, containing a paralel beta sheet
The handedness of b-a-b motif
• Theoretically, two distinct “hands” can exist
in b-a-b motif, with a helice above or
below the plane of beta sheet
• In almost all cases the right handed motif
exists
R
L
Domains
• Domain ia a polypeptide chain or a part of a
polypeptide chain that can fold indepedently in a
stable tertiary structure with its own hydrophobic
core
• Domains can be formed from several simple
motifs and additional secondary structure elements
• Proteins can have anything from one to several
tens of domains
• In proteins with sevaral domains, most often each
domain is associated with a distinct biological
function
2xb hairpin + b
strand
16xb-a-b
2x Greek
key
• Domains are most often, but not always
continuous pieces of primary structure
N
C
N
C
N
C
Example of proteins with several
domains - lac repressor
hinge helix
Helix-turn-helix domain (binds to
DNA)
Core domain, containing two
subdomains, which in turn contain
several b-a-b motifs (binds ligand)
C-terminal helix (tetramerization)
Intact IgG contains 12 immunoglobulin-like domains
Each domain is made of two beta sheets with a
topology similar to two Greek key motifs
The quaternary structure
• Some proteins are biologically active as monomers.
For those proteins quaternary structure does not exist
• Other proteins, however, are active as homo- or
hetero- polymers
• The simplest case and by far the most common form
of quaternary structure is a homodimer
• The monomers in homopolymers are often arranged in
a symmetric fashion with one or several symmetry
axes going through the molecule or some sort of
helical arrangement
• Some biologically active units have a very
complicated quaternary structure –like ribosomes or
viral capsids
2-fold symmetry in Glutahione-S-transferase
9-fold symmetry in light-harvesting complex II
from Rhodopseudomonas acidophila.
222 symmetry in prealbumin
A simple icosahedral virus – 180
chemically identical subunits
Small subunit of ribosome: a lot of
different proteins, no symmetry