Biological Chemistry – Lecture 20 Protein Structure 1

Biological Chemistry - Lecture 20 Lecture overview

1) structure and chemistry of amino acids

2) how amino acids are linked together through

Protein Structure 1 peptide bonds to form a polypeptide chain

3) sequencing a protein

4) how the polypeptide chain folds in 3D

Peter Brick (p.brick@imperial.ac.uk) - secondary structure elements (-helix and -sheet)

1 2

Proteins Structure of amino acids

-carbon At neutral pH:
Proteins are linear chains side chain
of amino acids. (one of 20) (or C) NH2 is a base NH3+
COOH is an acid COO-
Protein chains fold in 3D due to the FOLDING
non-covalent interactions between amino carboxyl
regions of the linear sequence group group

In proteins there are 20 different C is a chiral centre zwitterion

types of amino acid, each with (dipolar form)
mirror
different physicochemical
properties.
CORN rule: looking
down the H-C bond
- Function depends on 3D structure for an L amino acid
- 3D structure depends on sequence we read the groups
L-amino acid D-amino acid
- Sequence is determined genetically CO-R-N clockwise
all amino acids in proteins are L!!
3 4

The 20 amino acids The peptide bond

non-polar aromatic
hydrophobic

H
H CH3 CH2 CH2 N C The amino acids of a protein are joined together through a covalent bond between
CH
Gly
Ala CH CH2 H2C CH2 H2C CH3 CH2 CH2 the carboxyl group of one aa and the amino group of the next aa (peptide bond).
H3C CH3 C CH3
S H2
CH2 Leu
CH CH3 Ile HN
SH H3C CH3 Pro
Cys Met
Val Phe Trp
ed
polar uncharge

CH2 CH2 CH2 CH CH2

H3C OH
C CH2 OH
O NH2
C
Tyr A chain of amino acids which is asymmetric: at one end there is a free NH2
Ser Thr
Asn O NH2 group (N terminus) and at the other end a free COOH (C terminus).
Gln
hydrophilic

OH

H O H O H O H A peptide/protein sequence
CH2 N H2N C C NH C C NH C C NH C COOH C is always given from the N to
CH2
CH2 CH2
CH2
CH2
R R' R'' R'''
the C terminus (here RAFG).
CH2
basic

C CH2 His
CH2 Arg N
CH2
O O C
Lys NH
Arg Ala Phe Gly
Asp O O CH2 NH
Glu
NH3 C NH2 R A F G
acidic NH2 5 6

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Biological Chemistry – Lecture 20 Protein Structure 1

Planarity of the peptide bond Conformation of the peptide bond

The peptide bond is a resonance hybrid between two contributing structures
and has double bond character.
The peptide bond can assume a trans
O O or a cis conformation: the trans form is
favoured 1000:1.
C N C N
H H steric clash

R1 H O R2 C
R1 C O
C N C For prolines the trans form is
C C C N
C H only favoured 15:1
O R2
O

Delocalisation of the  electrons trans cis

over the entire peptide bond, Partial double bond character of the
rather than simply over the C=O N-C bond leads to restricted
bond. rotation - the region NH-CO is
planar. 7 8

Sequencing peptides Sequencing peptides

Edman degradation Edman degradation
1. Attach peptide to a solid surface
N 1 2 3 4 5 6 7 C Peptide attached to solid surface
2. Remove one residue from the N-terminus
3. Identify removed residue by chromatography
PTC N-terminus labelled using phenyl isothiocyanate (PTC)

Limitations
1 2 3 4 5 6 7
Limited to max of ~50 aa (efficiency of 98%)

Cannot sequence if N-term modified (acetylated of N-term is common Labelled amino acid released under acid conditions
in eukaryotes)
1 2 3 4 5 6 7

9 Labelled amino acid identified by chromatography 10

Edman degradation Sequencing longer polypeptides

R1 O R2 O

Phenylisothiocyanate N C S H2N CH C NH CH C ... Polypeptide

• Chop longer polypeptides up into fragments
mildly alkaline conditions
using a specific protease
R1 O R2 O
NH C NH CH C NH CH C ... • Separate the fragments
S
Phenylthiocarbamoyl derivative • Sequence each fragment using Edman
anhydrous F3CCOOH
d
degradation
d ti
R1 R2 O
N
CH
NH C + NH3 CH C ... To determine the order of the peptides the procedure is
C
S O repeated with a different protease
aqueous acid

S
Instead of using a proteases one can use cyanogen
C
bromide (N≡C-Br) that cleaves specifically after a Met
NH
N identified using chromatography (HPLC)
CH
C R1
11 12
O

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Biological Chemistry – Lecture 20 Protein Structure 1

Sequencing longer polypeptides Levels of structure in proteins

Primary Secondary Tertiary Quaternary
structure structure structure structure
Cleavage by protease 1
Number and relative position of
Local organisation Packing of the the subunits in a multimeric
of the polypeptide secondary elements protein.
chain. to give a 3D structure.
N C

Cleavage by protease 2

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Conformation of the main chain Proline and Glycine

Residues contain a single bond on either side of the C Proline No rotation about this bond

H O H O H O H O O
H
HN C C N C C
N C C N C C N C C H2C CH2
R
C C
H R1 H R2 H R3 H2

N cannot form H-bonds

Glycine
Different conformations of the main chain are possible by rotation H O H O
around these single bonds. HN C C N C C
H
R H
Lack of side chain permits
greater rotational freedom of
15 main chain 16

Main-chain hydrogen bonding Secondary structure

Local organisation of the polypeptide chain:
+ -helix
C -sheet
carbonyl group acts as a
- O hydrogen-bond acceptor
Linus Pauling and Robert Corey
• carried out X-ray diffraction studies of the structure of amino acids and
+ H small peptides to obtain a set of standard bond lengths and bond
amine group acts as a angles
N hydrogen-bond donor
- • using these data by modelling they proposed two periodic structures:
the -helix and the -sheet (1951). Some years later their predictions
would be confirmed by experiments.
Hydrogen bonds are dipole-dipole interactions.
Hydrogen bonds are weaker and longer than covalent bonds

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Biological Chemistry – Lecture 20 Protein Structure 1

-helix Helical wheel

Polyalanine C 3.6 amino acids per turn: 100 degree per residue
Right-handed helix
Looking along axis of an -helix:
Hydrogen bond

All main-chain CO and NH are bonded

Side chains Each ball represents
extending a C atom
Red – oxygen 100°
outwards
Blue – nitrogen
5.4 Å pitch
Grey – carbon
White – hydrogen

3.6 amino acids per turn

1.5 Å rise per amino acid Right-handed helix (whichever end you look at it)
N 19 20

H-bonding in an -helix Pro and -helix

O O O
All amino acids accept Pro occur in -helices
Ri H Ri+2 H Ri+4 H
H H H
C N C C N C C N C
N C C N C C N C C H O O
H H H H
O Ri+1 H O Ri+3 H O Ri+5 H HN C C N C C
R H2C CH2
C
H2

N cannot form H-bonds

Main-chain hydrogen bonds between the carbonyl of

the (i)th residue and the nitrogen of the (i+4)th residue.

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-strand The -sheet

anything between 2 and few
hundreds amino acids

Side chains -strand

-sheet
-strand
(zig-zag)
3.4 Å

N C The polypeptide is almost fully extended (3.4 Å per residue)

hydrophilic
OUTSIDE

Side chains points alternatively up and down INSIDE

hydrophobic

Main chain in an extended conformation Stabilised by main-chain:main-chain NH/CO hydrogen bonds between
adjacent strands.
Side chains alternately UP and DOWN Unlike -helices the H bonds between NH/CO groups are far apart in the
amino-acid sequence.
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Biological Chemistry – Lecture 20 Protein Structure 1

-sheet Larger sheets

Side chains in green
Strands can come together to form sheets. The strands can be
pure parallel, purely antiparallel or mixed.
N C
Parallel strands

N C

Hydrogen bonding between strands Side chains alternately up then down

C N
Antiparallel strands
N C
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Conformation of the main chain Dihedral or torsion angle

H O H O H O
Residues contain a single bond on either side of the C
N C C N C C N C C
H O H O H O H R1 H R2 H R3

N C C N C C N C C  

H R1 H R2 H R3
Looking down the N-C bond Looking down the C-C bond

  Cn-1
Nn


Cn Nn+1
 denotes the rotation around the N-C bond N C
 denotes the rotation around the C-C bond

 and  are called torsion angles or dihedral angles

27
Newman projections 28

Torsion angles and  Ramachandran plot

R3 Because of steric clashes, only certain combinations of torsion angles are
C allowed: we can plot these allowed combinations in the () plane - this is
1 C
called the Ramachandran plot.
O N H
H C
2

omega () = rotation around C-N bond
not allowed because of resonance,
therefore =180 (for trans)
planar region
phi ()= rotation around N - C bond
psi ()= rotation around C-C bond
R1
N  R2


C 
C O
The main-chain conformation is
defined by the sequence of the favorable regions for all aa
() angles: a list of the () for
each amino acid specifies the fold allowed regions for all aa
of the polypeptide chain, i.e. the 3D
structure of the protein


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Biological Chemistry – Lecture 20 Protein Structure 1

Ramachandran plot: Ramachandran plot: glycine residues

secondary structure elements
side chain=H
The main chain conformation is (very small,
collagen helix hardly any steric
defined by the sequence of the
() angles: the list of the () hindrance)
for each amino acid specifies the
fold of the polypeptide chain,
antiparallel parallel i.e. the 3D structure of the protein
 sheet
-sheet -sheet
left handed
helix
Secondary structure elements
are associated with specific
values of  and therefore with
-helix
specific regions of the
Ramachandran plot.

regions allowed only for glyine

extended chain
regions allowed only for all aa

END

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