Transcript The Three-Dimensional Structure of Proteins

The Three-Dimensional
Structure of Proteins
Chapter 4
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Protein Structure
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Proteins are polymers of amino acids
linked by covalent peptide bonds – leads
to different conformations
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Protein’s native conformation – has
biological activity
Levels of protein structure
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Primary structure is the sequence of amino acids in a polypeptide
chain (covalently linked), from N-terminal end to C-terminal
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Secondary structure is the arrangement in the space of
atoms/localized regions of a polypeptide backbone chain
Repetitive interactions resulting from hydrogen bonding between
amide N-H and carbonyl groups of peptide backbone
Side chain conformations of amino acids are not part of secondary
structure
Independently folded portions of proteins – domains or super
secondary structure
e. g., α-helix and β-pleated sheet
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Levels of proteins structure
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Tertiary structure is the three-dimensional
arrangement of all atoms in the protein, including
those in side chains and in any prosthetic groups
Quaternary structure includes those proteins which
have multiple polypeptide chains called subunits
- Mediated by noncovalent interactions such as
hydrogen bonds, electrostatic attractions and
hydrophobic interactions
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Primary structure of proteins
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Primary structure of a protein determines its
other levels of structure
Determines its properties and functioning
A single amino acid substitution – sickle cell
anemia
Site directed mutagenesis – amino acid residue
can be replaced with another amino acid
Secondary structure of protein
2˚ of proteins is hydrogen-bonded arrangement of
backbone of the protein
• Two bonds have free rotation: Ramachandran
angles
1)
2)
Bond between -carbon and amino nitrogen in
residue - Ǿ (phi) angle
Bond between the -carbon and carboxyl carbon of
residue – Ψ (psi) angle
α-Helix
What are the two periodic structures in
protein backbones?
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Coil of the helix is clockwise or right-handed
3.6 amino acids per turn/helix
Repeat distance-Pitch- is 5.4Å
C=O of each peptide bond is hydrogen bonded to
the N-H of the fourth amino acid away
All R groups point outward from helix.
What are the factors disrupting α-helix?
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Proline has cyclic structure – does not fit into αhelical structure
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Rotation around the bond between the nitrogen
and the α-carbon is restricted
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α-amino group cannot participate in intrachain
hydrogen bonding
What are the factors disrupting α-helix?
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α-carbon is outside the helix – crowding of side
chains and bonding of carbon to other atom than
hydrogen – Valine, Isoleucine and Threonine
Strong electrostatic repulsion caused by the
proximity of several side chains of like charge,
e.g., Lys and Arg or Glu and Asp
-Pleated Sheet
Parallel
Antiparallel
β-sheet
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C=O----H-N hydrogen bonds are perpendicular to
direction of the sheet
R groups are alternating – first above and then
below the plane
Differences
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α-helix
Rodlike structure and
involves one
polypeptide chain
Hydrogen bonding is
parallel to α-helix
within backbone
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β-sheet
Pleated structure and
involves one or more
polypeptide chain
Hydrogen bonding is
perpendicular to
direction of protein
chain
-Pleated Sheet
-bulge- a common no repetitive irregular 2˚ motif
in anti-parallel structure
Why is Glycine frequently found in
reverse turns?
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Reverse turns make transition from secondary structure to
another form
Single hydrogen of side chain prevents crowding
Proline (cyclic structure) has perfect geometry
Schematic Diagrams of Supersecondary Structures
What are the Supersecondary structures
and domains?
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Combinations of α-helix and β-pleated sheet –
Supersecondary structures
βαβ unit: Two parallel strands of β-sheet are
connected by a stretch of α-helix
αα unit: Two antiparallel α-helices (helix-turnhelix)
β-meander: an antiparallel sheet formed by series
of tight reverse turns connecting stretches of
polypeptide chain
What are the Supersecondary structures
and domains?
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Greek key: an antiparallel sheet formed when
polypeptide chain doubles back on itself
β-barrel : when β-sheets are extensive enough to
fold back on themselves
β-meander or Greek key can be found in β-barrel
in tertiary structure of proteins (figure 4.10)
Motifs are repetitive supersecondary
structures (figure 4.9)
Collagen Triple Helix (figure 4.11)
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Consists of three polypeptide chains wrapped around each
other in a ropelike twist – triple helix – Tropocollagennot α helix
Each chain – repeating sequence of three amino acids, XPro-Gly or X-Hyp-Gly
30% of aa in each chain are Pro and Hyp. Hydroxylysine
is also found
Three strands held by hydrogen bonding –
hydroxyproline and hydroxylysine
Intramolecular and intermolecular linking by covalent
bonds – formed by reactions of lysine and histidine aa
Collagen Triple Helix
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Linking between lysine and histidine residues
increases with age – meat in older animals is
tougher than younger animals
Lack of hydroxylation of proline to
hydroxyproline – makes collagen less stable
Scurvy – result of fragile collagen
Vitamin C prescribed for Scurvy
Protein Conformation: Fibrous and
globular proteins
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Fibrous proteins
consist of parallel long
fibers or large sheets
mechanically strong
insoluble in water and
dilute salt solutions
play important structural
roles in nature
Examples: Keratin of hair
and wool, collagen etc
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Globular proteins
folded to more or less
spherical shape
All have α helices and β
sheets
Soluble in water and salt
solutions
Tertiary and quaternary
structures are complex
Forces involved in tertiary structure of
proteins
Noncovalent interactions
-Hydrogen bonding between polar side chains, eg: Serine and
threonine
-Non polar side chains – hydrophobic interactions. Eg: Valine
and Isoleucine
-Electrostatic attraction between oppositely charged groups.
Eg: Lysine and Glutamine
Covalent interactions – disulfide (-S-S) bonds between side
chains of cysteines
Myoglobin and hemoglobin – no disulfide bonds
Trypsin and chymotrypsin – have disulfide bonds
Forces That Stabilize Protein Structure
How can three dimensional structure of
protein be determined?
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X-ray crystallography determines tertiary structure
Perfect crystals of some proteins can be grown under
carefully controlled conditions
Crystal exposure to beam of X-rays – diffraction
pattern is produced on a photographic plate or a
radiation counter
Heavier atoms scatter more effectively than the other
Scattered X-rays from individual atoms can reinforce
or cancel each other – gives rise to characteristic
pattern for each type of molecule
X-ray crystallography
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Series of diffraction patterns
taken from several angles –
needed information to
determine tertiary structure
Extracted via Fourier series –
a mathematical analysis
Thousands of such calculations
– determine structure of
protein – performed by
computer
Nuclear magnetic resonance spectroscopy
(2D)
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Protein samples are
present in aqueous
solution (small quantities
in milligrams)
Determines protein
structure based on
distance between
hydrogen atoms
Computer analysisFourier series
Denaturation and Renaturation
Refolding–
recovery of
unfolded
protein
Unfolding denaturation
of protein
Denaturation of proteins
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Denaturation – heat
High or low extremes of
pH
Detergents (SDS) –
disrupt electrostatic
(hydrophobic) interactions
Urea or guanidine
disrupt hydrogen bonding
β-mercaptoethanol –
reduces disulfide
bridges/bonds
Quaternary structure of protein
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The association of polypeptide monomers into
multisubunit proteins
dimers, trimers and tetramers
Noncovalent interactions present – electrostatic,
hydrophobic interactions and hydrogen bonding
Many multisubunit proteins – allosteric effects
Differences between Myo and
Hemoglobin
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Myoglobin-single polypeptide chain with 153 aa, 8 α-helical
regions and prosthetic group-heme
Hemoglobin – two α (141 aa) and two β chains (146 aa)
One molecule of oxygen binds to one molecule of Myo.
Four molecules of oxygen bind to one molecule of hemo
Why does oxygen have imperfect
binding to heme group in Myoglobin?
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CO (25,000) and O2 can
bind to heme
Blocked by His E7
Heme must release O2
In absence of oxygen
carrying proteins – iron of
heme group can be oxidized
to Fe (III)-will not bind to
O2
No positive cooperativity
What is positive
cooperativity/cooperative binding?
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One oxygen binds – it makes subsequent binding
of oxygen molecules easy
Graph 4.22 – oxygen binding curve in myoglobin
is hyperbolic and in hemoglobin is sigmoidal
H+, CO2, Cl-, and 2,3-bisphosphoglycerate (BPG)
affect the ability of Hb to bind and transport
oxygen
How does Hemoglobin work?
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Affinity for oxygen is controlled by several
factors – oxygen pressure and pH
When pH drops or oxygen pressure is low –
hemoglobin tend to release more oxygen and
viceversa-binds to oxygen
Oxygenated and deoxygenated
hemoglobin
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Hemoglobin has different
quaternary structures in
unbound (deoxygenated)
and bound (oxygenated)
β-chains are closer in
oxygenated hemoglobin
than deoxygenated
hemoglobin
Different crystal
structures
Protein folding dynamics
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Prediction of tertiary
structure of protein –
possible.
Biochemistry +
Computers –
Bioinformatics
Prediction – sequence
homology
Hydrophillic and Hydrophobic
interactions
Why is correct folding of proteins
important?
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Correctly folded proteins – soluble in aqueous
environment and attached to membranes
Incorrectly folded proteins – form aggregates with
other proteins (figure 4.35)
Several neurodegenerative disorders –
Alzheimer’s, Parkinson’s and Huntington’s
diseases
Prion disease – mad cow disease
What are Chaperones?
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Chaperones are special proteins
Aid in timely and correct folding of proteins
Hsp 70 - first discovered chaperones in E.coli
Exist in all organisms – prokaryotes to humans
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