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DBT2117: Biochemistry (I)
Lecture 12
Protein folding
Quaternary structure
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Disruption of Protein structures – denaturation
• Under harsh conditions, a protein loses its functional 3‐D structure.
• This process is called denaturation.
• Denaturing conditions include:
• Increased temperature
• pH becomes extremely acidic or alkaline
• Organic solvents or urea (chaotropic agents: disrupting H‐bonding between water, reducing hydrophobic effect) *note urea can solubilize denatured proteins
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Protein folding
How do proteins know what secondary/tertiary structures to form?
• Most of the information for determining the 3‐D structure of a protein is carried in the
amino acid sequence of that protein
• Chris Anginsen RNase A refolding experiment showed that denaturation and refolding
processes can be reversible. (Nobel prize in 1972)
Incorrectly folded protein due to random disulfide bonds –
only 1% of these molecules are active (because 8 Cys can have 105 possible different combinations)
Here urea is used to denature and solubilize the protein
Denaturation of proteins can be monitored
Thermal denaturation of RNase A:
• Differences between the native and denatured
conformations can be detected by several
spectroscopic methods.
• This graph shows the fraction of protein that is
denatured as measured by:
• increase in solution viscosity
• change in optical rotation at 365 nm
• change in UV absorbance at 287 nm
• All 3 techniques indicate the same fraction
unfolding
as a function of increasing temperature.
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Folding of globular protein is thermodynamically favorable
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The folding of a globular protein is clearly a thermodynamically favorable process under
physiological conditions.
The overall free energy change for folding must be negative.
This negative free energy change is achieved by a balance of several thermodynamic factors:
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Conformational Entropy – entropy decreases when proteins fold
random coil (higher entropy)  folded protein (lower entropy)
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Charge–Charge Interactions – stabilizing when opposite charges are near
when pH is too high or too low, charges and attractions are lost – denature.
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Internal Hydrogen Bonds – stabilizing H‐bonds form between polar R‐groups
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van der Waals Interactions – stabilizing attraction as globular proteins are well
packed.
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Hydrophobic effect
Overall balance in thermodynamics for protein folding
The stability of the folded structure of a globular protein depends on the interplay of three factors:
• The unfavorable conformational entropy change, which favors the unfolded state.
• The favorable enthalpy contribution arising from intramolecular interactions.
• The favorable entropy change arising from the burying of hydrophobic groups within
the molecule.
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Disulfide bond
Disulfide bonds are not essential for refolding (as demonstrated in the RNAse experiment), however they provide stability to the structure once folded.
• Some folded proteins are stabilized by internal disulfide bonds, in addition to noncovalent
forces.
• Disulfide bonds bonds are relatively rare
and are found primarily in proteins that are
exported from cells, such as ribonuclease,
BPTI, and insulin.
• One explanation is that the environment
inside most cells is reducing and tends to
keep sulfhydryl groups in the reduced state.
• External environments, for the most part,
are oxidizing and stabilize bridges.
Thermal denaturation of BPTI: • The percent denaturation as a function of temperature at pH 2.1 is indicated for the native protein and for the protein in which the Cys 14–Cys 38 disulfide bond has been reduced and carboxymethylated.
Kinetics of protein folding
•
The folding of globular proteins from their denatured conformations is a remarkably rapid
process, often complete in less than a second. (example: E. coli can make 100 amino acid
residue polypeptide in about 5 seconds at 37 C)
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Protein folding is not a completely random search through a vast conformational space.
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Rapid kinetic studies, using a variety of physical techniques to monitor different aspects of
protein structure, show that folding takes place through a series of intermediate states.
A simplified representation of the folding pathway for a protein: •“U” is the unfolded or denatured state.
•“F” is the folded or native state. •“I” “on‐pathway” are intermediate states. •Off‐pathway states include aggregates and other
non‐native states that may be kinetic or thermodynamic “dead‐ends” •In fact, not all pathways leading to such states are
irreversible.
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Off pathway states
•
Folding can be delayed by trapping of molecules in “off‐pathway” states.
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One of the most common of folding errors results from the incorrect cis–trans
isomerization of the amide bond adjacent to a proline residue:
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Trans conformation is more stable than cis (4:1)
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Conversion of cis to trans by an enzyme call prolyl isomerase speeds up folding in vivo
Folding funnel model
As folding protein follows a “downhill” trajectory on the energy landscape, the funnel
becomes narrower, restricting the number of conformations accessible to the protein.
Molten globule states
Partially folded states that have some native‐
like seoncdary
structures, but lacking defined tertiary
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“multistate” because of metastable intermediates (local minima)
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Protein topology and folding rates
• Different primary sequence can have
similar topologies.
• The two SH3 domains fold with similar
rates, as do ADAh2 and acyl phosphatase,
suggesting that protein topology is an
important factor in determining the rate
of protein folding.
• Examples of low and high contact order
(sequence separation between residues
that physically contact in the native
state).
•
Proteins with higher contact orders
tend to fold with slower rates.
Molecular chaperones
•
While many proteins can fold themselves, some proteins require the action of specialized
proteins called molecular chaperones to achieve proper folding.
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Molecular chaperones functions to keep the newly formed protein out of trouble.
• improper folding
• aggregation
• Improper folding results from intermolecular contacts (such as exposed hydrophobic groups
aggregating with other polypeptide strands)
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Molecular chaperone GroES and GroEL
The cavity of GroEL‐GroES complex (chaperonins) provides a favorable environment in which non‐native proteins can fold properly
Many chaperone proteins are also heat shock proteins
Which help refold denatured enzyme upon increased temperature
1. Unfolded protein line 2. ATP and GroES binds
up with Hydrophobic and changes GroEL
residues
conformation
3. Conformation change
lead to hydrophilic/ negative surfaces
4. Folded protein
leaves GroEL‐ES
Protein misfolding
Protein misfolding is the basis for several diseases, including Alzheimer’s disease and Parkinson’s disease.
In human, most of these diseases are associated with the formation of highly ordered protein aggregates called amyloid fibrils or plaques
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Amyloid fibrils
Prion diseases
Prions – Proteinaceous infectious particles (proteins)
Prions cause neurodegenerative diseases.
(transmitted independent of DNA or RNA)
• Prion related protein (PrPC) is a normal protein
found in cells.
• PrPC has chances to spontaneously form PrPSC, the
infectious prion.
• PrPSC interacts with normal PrPC and turn them into
PrPSC.
• PrPSC is super stable – can even tolerate very high
temperatures
PrPC
PrPSC
• Prions cause mad cow disease, scrapie in sheep,
kuru in humans, etc
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Structure prediction
•
Protein secondary structure can now be predicted with good accuracy. Tertiary structure
prediction is not as accurate, but can also be done with good approximation. •
The more known structures we have, the more accurate we can design our software and
models to predict protein structures.
• Many computational algorithms
have been developed for protein
prediction.
• One simple approach is to look at
the frequency of amino acid
residues that favor particle
secondary structures.
Structure prediction – tertiary structures
In prediction of tertiary structures, algorithm seeks the global energy minimum
• this calculation is based on quantum mechanics and assumptions about solvent
interactions • as the program looks for global energy minimum. Its search method must be smart.
Otherwise, it will waste a lot of time looking at the vast possible conformation that a
protein can adopt
Rosetta – an algorithm designed to predict protein structure.
• First, fragments of the protein sequence are organized into local short structure motifs.
This is done by comparing sequences to a structural library.
• Then, these short fragments are
packed together in ways that give
low‐energy conformations, ignoring
side chain details (only look at charge
and polarity).
• Lastly, the details of the side chains
are added back and calculation
optimizes energy minimum
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Online prediction tools
http://robetta.bakerlab.org/
Quaternary structures
The four levels of protein structure: Homotetramer
Homotypic – association between identical polypeptide chains
Heterotypic – association between subunits of very different protein structures.
Multiple subunits join together:
Dimer, Trimer, Tetramer, Pentamer, Hexamer, Heptamer, Octamer…. So on 國立交通大學生物科技學系 蘭宜錚老師
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Quaternary structures
• The interactions between the folded polypeptide chains in multisubunit proteins
are of the same kinds that stabilize tertiary structure
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salt bridges
hydrogen bonding
van der Waals forces
the hydrophobic effect
disulfide bonding
• These interactions provide the energy to stabilize the multisubunit structure.
• Association of polypeptide chains to form specific multisubunit structures is the
quaternary level of protein organization.
Quaternary structures ‐ interactions
Heterologous interaction – the interacting groups lie in different regions of the subunit Isologous interaction – symmetrical and identical interactions between the subunits
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Quaternary structures ‐ symmetry
• Although composed of
asymmetric polypeptides,
proteins adopt many
symmetrical patterns in forming
quaternary structures.
• Most proteins assemble into
point group symmetry (shown
on the right).
• n‐fold axis of symmetry
corresponds to a rotation of
360/n to its neighbor
• Some proteins assemble into
helical structures
• Additional isologous
interactions can give rise to
more levels of symmetry (such
as the Dn symmetry, cubic,
icosahedral)
Quaternary structures – asymmetrical Heterotypic Protein–Protein Interactions:
•A dimer without symmetry.
o Two subunits can associate by
heterologous interactions and still not
yield an indefinite chain.
o The interaction sites (A, B, C, D) are
blocked from reaction with more
monomers by the close fit of the
surfaces.
Interaction of BPTI with trypsin: • The BPTI molecule fits snugly onto the
surface of the trypsin molecule (blue), blocking the active site of trypsin. 國立交通大學生物科技學系 蘭宜錚老師
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Quaternary structures
Beside each electron micrograph
is a diagrammatic representation of the helical aggregate structure. (a)Actin
(b)Tobacco mosaic virus. •
In the virus, protein subunits form a
helical array about a helically coiled
RNA.
Viral capsid proteins are often icosahedral As it maximize internal space.
Protein folding
https://www.youtube.com/v/q7sloonEE5k
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