Module 1 Building Blocks of Biochemistry 6 of 6

Proteins 3:
2°& 3°Structure &
F ldi and
Folding d Stability
St bilit
Shoba Ranganathan
Dept. of Molecular Sciences
4WWally’s
ll ’ W
Walk
lk (B
(Building
ildi F7B)
F7B), Room
R 121
T: 02 9850 6262; E: shoba.ranganathan@mq.edu.au
Protein structure – definition of terms
• Zeroth level of protein structure: aa composition
(historical)
• Primary structure
 Amino acid sequence (last lecture)
• Secondary structure
 Structural elements in proteins that are primarily
formed throughg p
peptide
p backbone interactions
• Tertiary structure
 The overall three dimensional structure of a protein
p
• Quaternary Structure
 Arrangement
g of subunits within a multisubunit p
protein
Module 1: Building Blocks of Biochemistry 2
Most commonly found
secondary
d structures
t t
and their hydrogen
b di patterns
bonding tt

 -helices and
-sheets are
stabilized by
backbone
hydrogen
y g bonds
 -strands are held
together by
hydrogen bonds to
form -sheets

Module 1: Building Blocks of Biochemistry 3

Hydrogen bonds in an -helix:
helix: from
residue i to residue i+4: 1-5, 2-6, etc.

Proline residues do not have this free H

atom, because of the ring structure of its
side chain bonding back to the -N –
th therefore
they th f b k -helices!
break h li !
Module 1: Building Blocks of Biochemistry 4
Hydrogen
b d patterns
bond tt
in anti-parallel
andd parallel
ll l
-sheets
 -strands:
 opposite direction
– anti-parallel
ti ll l or
 same direction –
parallel
• Look at protein
chain direction:
N- to C- for each
amino acid

Module 1: Building Blocks of Biochemistry 5

 Connecting Adjacent β Strands
b loops
by l or turns
t

• Turns are short hydrogen

bonded segments
• Loops
L and
d tturns ffor lilinks
k
between:
• helices;
• strands; and
• helices and strands.
Module 1: Building Blocks of Biochemistry 6
 Sequence Affects 2º
Structure
Unhappy in -helix:
Pro, Gly, Asn, Tyr, Ser, Cys

Unhappy in -sheet:
Glu, Asp, Pro

Gly prefers loops or turns

Thus, the aa composition and primary

structure of a protein determine its
secondary structure.

Module 1: Building Blocks of Biochemistry 7

Collagen: Triple Helix

• Most abundant vertebrate (insoluble) protein: in

connective tissue: cartilage, bone, teeth, tendon
and fibrous matrices of skin and blood vessels
vessels, with
46 different proteins.
• Gly and Pro rich proteins.
• L ft h d d helix
Left-handed h li with
ith 3 residues
id per tturn and
d
assembled into a trimer: collagen helix.
• Collagen diseases are caused by lack of Vitamin C.

Module 1: Building Blocks of Biochemistry 8

Protein Secondary Structure - summary
• The planar nature of the peptide group limits the
conformational flexibility of the polypeptide chain.
• The α-helix and the β-sheet allow the polypeptide chain
to adopt favorable φ and ψ angles and to form
hydrogen bonds.
• Fibrous proteins contain long stretches of regular
secondary structure, such as the “coiled coils” (α-
helices twisted together)
g ) in α-keratin and the
polyproline triple helix in collagen.
• Not all polypeptide segments form regular secondary
structure such as α helices or β sheets.

VVP5: Chapter 6
6.1
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Module 1: Building Blocks of Biochemistry 9
 Point of Origin
ER Season 5 Episode 18

Module 1: Building Blocks of Biochemistry 10

 Proteins have a defined 3
3-D
D structure
• Many proteins can be crystallized
crystallized, from which their
3D structure can be determined.
• An experiment performed in the 1960’s
1960 s by White
and Anfinsen appeared to show
 the primary structure determines the 3D structure and
 that proteins can fold unaided into their 3D structure.
• We are able currently to computationally predict
upto tertiary structure from the primary structure (i.e.
sequence) for many new protein sequences
sequences.

Module 1: Building Blocks of Biochemistry 11

Protein tertiary structure determination

• Thousands of protein 3D structures have

b
been d
determined
t i d using:
i
 NMR
 X-Ray crystallography
 Electron diffraction
 Electron microscopy

Module 1: Building Blocks of Biochemistry 12

Protein Crystals to structure

NMR Spectrum
Spectr m to str
structure
ct re

Module 1: Building Blocks of Biochemistry 13

Where do the side chains go?
• Based on polarity
 Nonpolar (Val, Leu, Ile, Met, Phe) prefer the hydrophobic
interior
 Charged polar residues (Asp, Glu, His, Lys, Arg) are usually
in contact with aqueous solvent and thus on the surface.
 Uncharged polar groups (Ser
(Ser, Thr,
Thr Asn,
Asn Gln,
Gln Tyr) are usually
on the surface but when buried, form hydrogen bonds.
 Amino acid mutations may disturb this distribution, and hence
affect
ff t the
th stability
t bilit and
d the
th 3D structure.
t t

• Hydrophobicity
y y is the main driving
g force for protein
tertiary structure.

Module 1: Building Blocks of Biochemistry 14

Side Chain Distribution in Cytochrome

Hydrophilic Hydrophobic

Module 1: Building Blocks of Biochemistry 15

 Protein Classification: α, β, or α/β

Cytochrome b562
Human immunoglobulin
g fragment
g Dogfish
g lactate dehydrogenase
y g
PDBid 256B PDBid 6LDH
PDBid 7FAB
Module 1: Building Blocks of Biochemistry 16
 2-Domain Protein : GAPDH
Glyceraldehyde-3-phosphate
y y p p
dehydrogenase
Long polypeptides form PDBid 1GD1

do a s
domains
• >200 aa
• Residues appear
“ l t d” iinto
“clustered” t llobes
b
• Another dehydrogenase
shown here
• Common domains have
same function: e.g.
Rossman fold binds
NAD+
• Ligand binding sites are
usually located in clefts
between the domains

Module 1: Building Blocks of Biochemistry 17

 Structure is More Conserved
Than Sequence
c-type cytochromes from different species

• Sequences are varied but structures are very similar.

• The major structural differences are in the surface loops
• Essential structural and functional elements of proteins are
conserved during g evolution.

Module 1: Building Blocks of Biochemistry 18

Internet Bioinformatics Tools
(Practical 3)

Module 1: Building Blocks of Biochemistry 19

 Tertiary Structure - summary

• 3D protein structures are determined by X-Ray crystallography

and NMR spectroscopy.
spectroscopy
• A protein’s tertiary structure consists of secondary structural
elements that combine to form motifs and domains.
• Nonpolar residues tend to occur in the protein interior and polar
residues on the exterior. Hydrophobicity is the main driving force.
• Over
O ti
time, a protein’s
t i ’ structure
t t iis more hi
highly
hl conserved
d th
than itits
sequence. Only essential residues need to be conserved.
g to amino acid sequences
• Changes q could lead to changes
g in 3D
structure, with possible stimulation of immune response.
• Bioinformatics databases store macromolecular structure
coordinates. Software programs help to visualize proteins and
compare their structural features.
VVP5: Chapter 6 6.2
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Module 1: Building Blocks of Biochemistry 20

4º Structure of Hemoglobin

• 4 subunits
assemble to
make the
functional
hemoglobin
protein
• 2  chains
h i and
d
2 β chains,
coloured up
• 4 heme groups
are in red
Deoxyhemoglobin
PDBid 2DHB

Module 1: Building Blocks of Biochemistry 21

 Stability of proteins
• Proteins are only marginally stable under
physiological conditions!
• The different noncovalent forces acting on a protein
appear to be in a delicate balance:
1. Hydrophobic effects;
1
2. Electrostatic interactions; and
3 Hydrogen bonds
3. bonds.

Module 1: Building Blocks of Biochemistry 22

Hydrophobic effect affects
protein stability the most
• Consequence
q of hydrophobic
y p
effect:
 burial of hydrophobic side
chains in the interior of the protein;
 surface exposure of hydrophilic
residues.
• Hydropathy
H d th values
l
 Larger the value, the residue tends
to be buried in the interior
 Smaller the value (i.e. more
negative), the residue prefers a
surface location, i.e. exposed to
aqueous solvent.

Module 1: Building Blocks of Biochemistry 23

 Hydropathy Plot:
Bovine Chymotrypsinogen
From a protein
protein’s
s primary structure (sequence),
(sequence) we can get
an idea which parts are buried or exposed.

Buried

Exposed

Module 1: Building Blocks of Biochemistry 24

Electrostatic interactions and stability:
Ion Pairs in Hemoglobin

• Important for buried ionic

residues - usually paired
 Called ion pairs or
 “salt bridges”

• Result:
R l buried
b i d charged
h d are
compensated, leading to
stability in the hydrophobic
interior or “core” of the
protein.

Module 1: Building Blocks of Biochemistry 25

 Small proteins are stabilized by
metal ions or disulfide bonds
• These pproteins are too
small to have a “core” for
burying hydrophobic
residues.
 Metal ions stabilize some
small protein domains
 E.g.
E g zinc fingers: 25-
25
60 residues long.
 This domain is not

stable
t bl iin th
the absence
b off
zinc ions.
 Disulfide bonds are
formed between two
DNA-binding protein Zif268 cysteine (Cys) residues.
PDBid 1ZAA

Module 1: Building Blocks of Biochemistry 26

Proteins can undergo denaturation and
renaturation
t ti
• Denaturation leads to loss of function.
• Proteins can be denatured by:
 Heating: the entire polypeptide unfolds or “melts”
melts .
Most proteins melt below 100oC.
 pH: changes charge distributions and hydrogen
bonding patterns
 Detergents:
g stabilize hydrophobic
y p side chains and
can invert a folded protein.
 Chaotropic
p agents
g

Module 1: Building Blocks of Biochemistry 27

Denatured (unfolded) proteins can be
renatured (folded): Anfinsen 1957
• Ribonuclease A (RNase A) A), a 124-residue single-
chain protein, was denatured by urea.
• When the urea was removed
removed, the protein
spontaneously renatured (i.e. refolded)!
• The four
f disulphide bonds re-formed
f correctly,
giving a functional protein.
• Proteins fold spontaneously into their native
confirmations under physiological conditions
• Implies the protein’s primary structure determines
its 3D structure!
Module 1: Building Blocks of Biochemistry 28
Process Diagram: Denaturation and
Renaturation of RNase A

Denaturation and
disulphide bond
cleavage
l
-urea
-mercaptoethanol

-urea
+mercaptoethanol
mercaptoethanol

Module 1: Building Blocks of Biochemistry 29

Hypothetical Protein Folding Pathway
• A protein folds from from
high energy and high
entropy
t to
t low
l energy andd
low entropy.
• Proteins need to be
properly folded for
biological activity
• Misfolded proteins are
shredded by proteases.
• Proper folding of misfolded
proteins is possible with
help from large proteins
(chaperonins).
• Misfolding can lead to
diseases.
Module 1: Building Blocks of Biochemistry 30
Protein Misfolding & Disease

Module 1: Building Blocks of Biochemistry 31

Alzheimer’s Disease: Amyloid Plaques

Module 1: Building Blocks of Biochemistry 32

 Protein Stability and Folding - summary

• P
Protein
t i stability
t bilit iis mainly
i l ddetermined
t i db by th
the
hydrophobic effect.
• Protein
P t i ffolding
ldi follows
f ll a pathway
th ffrom hi
highh energy
and high entropy to low energy and low entropy.
• Amyloid diseases result from protein misfolding.
misfolding

VVP5 Ch
VVP5: Chapter
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