Specific knowledge to get:

1. The types, structures and properties of amino acids

2. The 4 fundamental non-covalent forces of molecular biology

3. The structure of the peptide bond

4. Protein secondary, tertiary and quaternary structures

5. Protein ‘domains’, ‘motifs’, ‘folds’

6. Some examples in specific proteins

Proteins are covalently linked amino acids formed
according to the genetic code
DNA mRNA
transcription

tRNA

Protein amino acids

linked together
the peptide bond
 Proteins are polymers of amino acids

polyethylene: the most

abundant synthetic macromolecule

( )
R O
NH CH C
H H
polyamide proteins,
the most abundant natural
macromolecule. e.g. keratin
‘Condensation’ is the name of the reaction
between amino acids that creates a peptide
bond
Multiple peptide bonds make a peptide,
or polypeptide
Multiple peptide bonds make a peptide,
or polypeptide
 Resonance hybridization

 generates charge: partial (hydrogen bonding)

formal (electrostatic interactions)
 means the peptide backbone contributes to protein
structure and sometimes directly plays a part in catalysis
 The peptide bond is flexible, not rigid

• amino acids can rotate about the bonds C-C’ and N-C bonds

• C-C’ rotation is called psi (and the N- Cbond rotation is called phi ()

• rotation for each amino acid depends on if there is steric clash between the
amino acid R group and the main backbone

• psi and phi rotation, and steric clash influencing them give “backbone shape” to the
polypeptide
 Proline kinks the peptide bond
R R
O  O  N
C N C
C

C N C C N
 O   O R
R R R
Specific knowledge to get:

1. The types, structures and properties of amino acids

2. The 4 fundamental non-covalent forces of molecular biology

3. The structure of the peptide bond

4. Protein secondary, tertiary and quaternary structures

5. Protein ‘domains’, ‘motifs’, ‘folds’

6. Some examples in specific proteins

 Protein folding:
chains of amino acids in peptide bonds fold up into
proteins by non-covalent forces

http://youtu.be/sD6vyfTtE4U

polar versus hydrophobic

hydrophilic solvation shell
non-
covalent forces

hydrophobic core basis of protein folding

into 3-D structure
Proteins fold into a conformation of lowest energy
The four forces in proteins combine to fold up proteins into the correct conformation
Proteins fold into a conformation of lowest energy
The four forces in proteins combine to fold up proteins into the correct conformation
amino acid sequence is enough for protein folding,
but...

A single, isolated protein can have the forces abolished by denaturing agents such as
urea (shown in B), unfolding the protein. If the urea is removed the protein snaps back
into the original, folded conformation.

Amino acid sequence contains all information required to fold a protein

 Discovery of protein chaperones
virus virus

E. coli E. coli chap-

Viruses hijack cells. When this virus

virus virus
(called phage lambda) infects a bacterial
cell it’s genes are transcribed and translated

viral DNA viral DNA

The virus proteins fold by hijacking the
virus bacterial cells’ chaperones. In bacterial cells
virus
that were resistant to the virus it was noticed
that they lacked the chaperones (chap-)
 Protein structure heirarchy

Secondary &
Super-
 Protein structure hierarchy
Primary structure (1o): amino acid sequence and motifs
Secondary structure (2o): 3-D elements formed from folding
of discrete peptides/polypeptides

helix: 31% of 2o structure

chain/sheet: 28% of 2o structure

turns: 30% of 2o structure

Tertiary structure (3o): the overall protein fold and

shape
Amino acid composition in protein structures
protein secondary structures from when a chain of amino acids take on an
energetically favourable conformation based on the protein/peptide backbone
 1. Alpha () helix
• most commonly occurring protein secondary structure

• energetically very favourable for any amino acid sequence

because it involves peptide backbone interactions,
not R-groups

• the helix is right-handed: recall handedness

• discovered in -keratin, the major protein of hair

Linus Pauling (1951)

-helix hydrogen bonds are key to structure
O
- R H C
O
C N N
H
C N C
+ O O
R H R
C
N
H
 H-bonds between peptide backbone parts of residues
 R-groups are not involved
 H-bond between backbone carbonyl (C=O) and every 4th
amino acid nitrogen
 3.6 amino acids per complete turn of the helix
intramolecular H bonds in the
-helix

N
the H-bond dipoles of the -helix all point the same
way, making the helix itself a mega-dipole

+ve -ve

N
H
O
C
N
H

O
C
R-groups: helical wheel diagrams
http://cti.itc.Virginia.edu/~cmg/Demo/wheel/wheelApp.html
-helix amino acid composition depends on the
position of the helix in the protein
this again takes us back to:
• hydrophobic/hydrophilic parts of proteins
• the chemical nature of amino acid R-groups, polar, charged, hydrophobic?

1. citrate synthase 2. alcohol dehydrogenase 3.troponin C

 2. Beta () strand

• -strands rely on association with other -strands for stablity

• multiple -strands form -sheets

• discovered in the major protein of silk, fibroin

3 x 1 strand:
sheet

Bombyx mori
silk
Hydrogen bonds are also key to the -sheet
inter-molecular hydrogen bonds between strands give -sheets.
they associate as parallel or anti-parallel sheets

Note that as for -helix:

• H-bonds form between N-H
and C-O (though inter-
molecular)

• R-groups are not involved

 3. Protein turns and loops
the bits that join helices and strands together

 they are defined as not H-bonding within the main body of the
secondary structure

 often as few as 3-5 amino acid residues linking, for e.g., two helices

turn
 e.g. helix-turn-helix motif

recognition positioning or
helix structural helix
How to use -helix, -sheet and turn in figures
in essays etc.

loop

-1 2 -1 2 1 -1

N
N N mixed 

• Proteins have an N-terminus (NH2) and a C-terminus (COOH)

• Strands and helices are numbered in turn from the N-terminus

Super-secondary protein structure:
 coiled coil: alpha helical
Helical wheel coiled-coil
 Example of coiled coil function
the leucine zipper often occurs in DNA binding proteins

coiled-coil

e.g. DNA binding

 Super-secondary structure:
• two stranded beta sheet

• a small beta sheet lies across the DNA

major groove

• the beta strands are hydrogen bonded to

one another by peptide backbone interactions

• this leaves amino acid R-groups free to

interact with the DNA backbone and bases

bacterial met
repressor
Secondary &
Super-
Protein tertiary (3o) structures are arranged
in domains
• the fundamental unit of a protein’s overall tertiary structure is a domain
• domains are autonomously folding parts of a protein
• a protein may comprise one or several domains, each with different roles
• e.g. the DNA helicase UvrD, which we met them briefly in lecture 2

Hel308/HelQ helicase + DNA UvrD helicase + DNA

 Protein domains can be represented in
“cartoon”
UvrD helicase + DNA

1A 1B 2A 2B
N C
0 89 214 377 647

RecA domain
DNA helicases are characterised by having
at least one “RecA domain”

• the RecA domain of helicases is the motor of

the enzyme: it binds and hydrolyses ATP

• Helicase enzymes are a type of ATPase enzyme

• we saw in lecture 2 how the R-group of lysine

residues can make a crucial interaction with ATP

• this Lys-ATP interaction occurs in the RecA

domain of helicase enzymes

RecA domain

ATP
 some protein domains are common to many
proteins
e.g. RecA domain in any protein that hydrolyses ATP

RecA
RecA: DNA repair protein

UvrD: DNA repair helicase

GroEL: folding chaperone

Cas3: function unknown!!

RecA domain: ATP binding/ATPase powers helicase
 Protein domains contain common motifs

otein motifs:
Are based on conserved amino acid sequence that have the same specific function.

The motif sequence can be picked out in proteins from diverse organisms

The motif is part of a folded single domain, or a part of >1 domain

 Amino acid sequence motifs
• A short sequence of amino acids (e.g. 5-20 amino acids) that is recognizable in the
primary sequence of many proteins with the same function.

• In helicase enzymes (e.g. UvrD) the RecA domain (1A) binds ATP via a motif
that contains the Lysine residue we have spoken about. The motif is called a
Walker A box, and is found in most if not all helicases: GxGxGKS/T

Walker A box motif in UvrD is GAGSGKT

• remember those glycine residues too, from the Ramachandran plot...?

1A 1B 2A 2B
N C
0 89 214 377 647

30-GAGSGKT-36
 UvrD ATP binding

30-GAGSGKT-36

note: Walker A sometimes called “motif 1”

 Molecular detail of ATP binding by UvrD protein

GXXGXGKT/S
GAGSGKT (UvrD)

glutamine (Gln, Q)
(hydrogen bond
to OH group of tyrosine (Tyr, Y)
ATP sugar) (stacking 
electron interaction
with adenine ring)

Lysine (Lys, K)
(salt bridge to
gamma
phosphate of ATP)
 Lysine binding ATP in walker A motif in the RecA
domain of another DNA helicase, RecQ

RecQ structure can be downloaded at PDB as code 1OYY, and viewed using
freely downloadable software called PyMol.
I coloured RecA domain green, ATP blue and Walker A motif red (GxGGGKS)
 Lys-53

protein-
ATP
interactions
ATP

Tyr-23
 Structural motifs
these are distinguished from sequence motifs in that although they are
folded into the same secondary structure they can not often
be spotted in amino acid primary sequence

The Greek Key

Greek Key in Staphylococcus nuclease
 Helix-turn-helix motif

recognition positioning or
helix structural helix
the most common structural motif....
 Protein quaternary structure
when a functional protein is made of two or more interacting polypeptide chains

Secondary &
Super-
Quaternary structures can take many forms

DNA RNA Protein (in the form

of 1 polypeptide chain)

+
homomultimer
e.g. hexamer homodimer

heterodimer
heteromultimers
 a simple dimer
e.g. the essential integrase enzyme from the virus HIV-1. Interaction between
the monomers is by H-bonds and hydrophobic interactions.

More details on HIV enzymes in next lecture

a complex multimer: MCM hexamer as part of the
replisome complex
• MCM= Mini Chromosome Maintenance
• helicase is hexameric.
• it is found in archaea and in eukaryotes (analogue in bacteria is DnaB)
• MCM unwinds DNA to allow DNA replication by polymerases

MCM helicase

MCM
Specific knowledge to get:

1. The types, structures and properties of amino acids

2. The 4 fundamental non-covalent forces of molecular biology

3. The structure of the peptide bond

4. Protein secondary, tertiary and quaternary structures

5. Protein ‘domains’, ‘motifs’, ‘folds’

6. Some examples in specific proteins

 Proteins at work: DNA binding and unwinding

• DNA helicases
• HIV-1 enzymes
• A transcription factor
DNA substrate: major and minor grooves

DNA bases in the

major groove are easily
“read” by proteins
 less steric hindrance
to protein binding DNA in the major groove
e.g., DNA helicase binding to DNA
DNA helicases are enzymes that
catalyze unwinding of DNA using
energy from ATP hydrolysis.

They are protein motors

Double strand DNA Single strand DNA

ATP: Mg2+: H2O ADP + Pi

UvrD helicase binding to DNA

• in UvrD there are 4 HTHs that

make contact with DNA

• they do this by hydrogen bonding

• some proteins can use ionic bonds

to bond to the DNA backbone, but
UvrD does not
Cell, Volume 127, Issue 7, 2006, 1349 - 1360

http://dx.doi.org/10.1016/j.cell.2006.10.049
Examples of DNA-protein hydrogen bonds
at a HTH
HELIX 1

HELIX 2

• Arginine (Arg, R)
• Glycine (Gly, G): peptide bond!!: TURN
• Asparagine (Asn, N)
• Threonine (Thr, T)
This HtH uses hydrogen bonds/polar residues. It has a GIG motif
The R-groups for DNA hydrogen bonding

glycine
 Examples of DNA base-protein
hydrophobic/aromatic interactions

• DNA bases are exposed in ssDNA

• bases are aromatic and can be bound by

aromatic, hydrophobic amino acid R-groups

• an example is the ssDNA “anchor” of UvrD

• a clearer example is the tyrosine at DNA

unwinding interface
tryptophan
Transcription factors bind specific DNA sequence

e.g. Met Repressor

• structural motif: anti-parallel

beta-sheet.

• occupies major groove and

can bind to specific base
sequence by H-bonds

• different DNA/RNA base

sequences can present
a “hydrogen bond code”
to proteins that can be
“read” by amino acid
DNA sequence recognition code, by
hydrogen bonding
DNA-metal binding by UvrD helicase
ATP binding ATP hydrolysis: DExx box

• ATP + Magnesium ADP + Pi

The D and E of DEAD box hydrolyse ATP by
activating bound metal

DExx motif

walker A

with the cofactors water and magnesium

 2b. SARS-Cov2 polymerase active site

A common two-metal ion mechanism for

polymerases (seitz)
CRISPR-Cas9 gene editing: disrupt and replace DNA

the tracrRNA and crRNA molecules

are combined into a ‘single guide
RNA’ (sgRNA)
CRISPR-Cas9 gene editing:

Cas9 binds to DNA guided by an RNA and

cuts the DNA
Cas9 genome editing: improved targeting by reducing electrostatic DNA-
Protein interactions
Introduce K848A, K1003A, R1060A mutation into Cas9.
Previous study showed high fidelity Cas9 retaining wtCas9 efficiency on target
DNA cleavage while reducing off-target effect.

Taken from: Rationally engineered Cas9 nucleases with improved

specificity. Science, 351(6268), pp. 84-88