BCHS 6229 Protein Structure and Function · 2017. 11. 2. · 1 BCHS 6229 Protein Structure and...
Transcript of BCHS 6229 Protein Structure and Function · 2017. 11. 2. · 1 BCHS 6229 Protein Structure and...
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BCHS 6229
Protein Structure and FunctionProtein Structure and Function
Lecture 3 (October 18, 2011)
Protein Folding:
Forces, Mechanisms & Characterization
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The folding problem
• One of the greatest unsolved problems of Science
• The folding problem - Why so important?
• The mechanism(s) of protein folding
What is meant by mechanism?
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DNA
Sequence space
ProteinStructure space
The folding problem• Prediction of the 3D structure of a protein from
its amino acid sequence
• Translate “Linear” DNA Sequence data to spatial
information
a connection between the genome (sequence) and what
the proteins actually do (their function).
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Why solve the folding problem?
• Acquisition of sequence data relatively quick
• Acquisition of experimental structural information
slow
• Limited to proteins that crystallize or stable in
solution for NMR
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Chemical Stability of the covalent structure
Covalent changes, Irreversible
Conformational Stability of the folded state
Subject of our discussion
Measuring the conformational stability
Protein Stability
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Forces that stabilize protein structure: 1, 2, 3, etc..
1. The Hydrophobic Effect
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Zinc finger:
Nucleic acid-binding proteins
2. Electrostatic Interactions
3. Chemical Cross-links
Ion pair (salt bridge) of
myoglobinAsp 60
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Key Concepts:
• Electrostatics, hydrogen bonds and van der Waalsforces hold a protein together.
• Hydrophobic effects force global protein conformation.
• Peptide chains can be cross-linked by disulfides, Zinc,heme or other ligand compounds. Zinc has a completed orbital, one stable oxidation state and forms ligandswith sulfur, nitrogen and oxygen.
• Proteins refold very rapidly and generally in only onestable conformation.
Protein folding dynamics
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Uses of Potential Energy Function:
Energy minimizationBrings a total energy of a molecular conformation to a low
energy well of the potential energy surface
Molecular DynamicsSimulation: atoms and molecules are allowed to interact for a period of time
by approximations of known physics, giving a view of the motion of the
atoms
Monte Carlo SimulationsSimulation of a randomly distributed ensemble of trajectories (repeated
random sampling)
Vibrational AnalysisX-ray refinementConstraint/Restraint modelingFree Energy Differences …
(molecular trajectory)
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Protein denaturation and renaturation
An observation by Afinsen and colleagues
refolding exp
by making the polar and non-polarresidues have similar solubility
Chaotropic agents:
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Anfinsen’s Dogma
Anfinsen C, Haber E, Sela M, White F jr. (1961) PNAS 47:1309-14.
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Levinthal paradoxConsider a 100 residue protein. If each residue is
considered to have just 3 possible conformationsthe total number of conformations of the protein is3100. Conformational changes occur on a time scaleof 10-13 seconds i.e. the time required to sample allpossible conformations would be 3100 x 10-13 secondswhich is about 1027 years. Longer than the age ofthe universe!
Therefore, proteins must fold in “pre-arrangedpathways” and in a cooperative manner.
Levinthal, C (1968). "Are there pathways for protein folding?". Journal de
Chimie Physique et de Physico-Chimie Biologique 65: 44–45
Zwanzig R, et al. (1992). "Levinthal's paradox". PNAS 89 (1): 20–22.
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Mechanisms of the Protein Folding Reaction
1. Levinthal random sampling (paradox)
2. Sequential Folding Model
3. Nucleation/Growth Model
4. Diffusion-Collision-Adhesion
5. Framework Model
6. Hydrophobic Collapse Model
7. Jigsaw Puzzle Model
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Cooperativity in protein-folding
How a globally optimal state can be found without a
global search?
Dill et al., PNAS (1993) 90:1942-1946
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Coil-Helix transition
Paradigm for cooperativity in biopolymers
nucleation of -helix
Initiation of a helical turn is much harder than appending
another residue to a helical segment, due to higher entropic
penalty.
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Cooperative transition has a sigmoidal profile
Melting curve of a coiled-coil protein in aqueous medium
Cooperativity results roughly in a two-state system
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Folding funnel
Energy-entropy
relationship for
protein folding
The free energy of protein folding
From Alan Fersht. “Structure and
Mechanism in Protein Science”
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Hydrophobic interaction: entropic
S > 0
More Hydrocarbon-Water
Interfacial Area,
More Water ordered
Less Hydrocarbon-Water
Interfacial Area,
Less Water ordered
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Hydrogen Bonds: enthalpic
H-bonds
H-bonds within protein
&
H-bonds with water (hydration)HH-bonds = -(4~20) kJ•mol-1
Other Enthalpic interactions?
-
-
-
-
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Dissecting the free energy of protein folding
Unfolded Folded
G = H - T S G =~ -50 kJ/mol
Heating makesH less negative
Near freezing T,entropy of H2O aroundnon-polar residues isless different fromthose around polarresidues
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Protein folding is a balance of forces
• Proteins are only marginally stable
• Free energies of unfolding ~5-15 kcal/mol
• The protein fold depends on the summation of allinteraction energies between any two individualatoms in the native state
Denatured (D) Native (N)
GD-N = HD-N - T SD-N
• Also depends on interactions that individual atomsmake with water in the denatured state
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Thermodynamics of unfolding
•Denatured state has a high configurational entropy
S = kB ln W
Where W is the number of accessible states
kB is the Boltzmann constant
•Native state conformationally restricted
•Loss of entropy balanced by a gain in enthalpy
Define Entropy from a Probability Distributions•Number of way of arranging N particles in ni groups:
•Natural log is chosen because kBln(W1W2) = kBln(W1) + kBln(W2)
W =N!
n1!n2!n3!n4!LnN!
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Measuring the conformational stability
For a two-state unfolding
Folded (F) Unfolded (U)
Native (N) Denatured (D)
Selecting a technique to follow unfoldingUV difference spectroscopy
Fluorescence and Circular dichroism (CD)
Biological activity
Optical rotatory dispersion (ORD)
Nuclear Magnetic Resonance (NMR)
Viscosity and other hydrodynamic methods, etc…
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Measuring thermal denaturation
F or A
versus T
q versus T
Buffe
r profile
Protein
endo
therm
Cp
Corrected calorimetricprofile
fraction folded vs.
fraction unfolded
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Example: Spectra of folded and unfolded RNase T1
Intrinsic fluorescence emission
spectra upon excitation at 278 nmORD spectra
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Determining unfolding curves
Urea- and heat-induced unfolding transitions of RNase T1
y: physical parameters
pre-transition region
transition region
post-transition region
Equilibrium and reversibility
Urea and GdmCl / Thermal unfolding
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Analyzing unfolding curves
Two-state folding mechanism
Folded (F) Unfolded (U) (1)
ƒU = (yF - y) /(yF - yU) (2)
where (ƒF+ ƒU = 1, y = yFƒF + yUƒU )
K = ƒU/(1 - ƒU) = ƒU/ƒF = (yF - y)/(y - yu) (3)
G = -RT lnK = -RT ln [(yF - y)/(y - yu)] (4)
G = G(H2O) - m[denaturant] (5)
Many small single domain proteins are closely approximated bythe two state model and have few intermediates.
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Analyzing unfolding curves
Urea and GdmCl unfolding
Urea (M)
Fra
ctio
n u
nfo
lde
d
G (
kca
l/m
ole
)
Urea (M)
Fraction of RNase T1 unfolded, ƒU,
as a function of [Urea]
G for RNase T1 unfolding,
as a function of [Urea]
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Analyzing unfolding curves
Thermal unfolding
d(ln K)/d(1/T) = - H/R (6)
van’t Hoff equ.
van’t Hoff plot (lnK vs. 1/T) of protein unfolding transitions are found to be non-
linear --- indicates that H varies with T
d( H)/d(T) = Cp(U) - Cp(F) = Cp (7)
Recall
G(T) = Hm(1-T/Tm) - Cp[(Tm - T)] + T ln (T/Tm)] (8)
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Specific Heats
• Property defined by the amount of heat required per unit mass toraise the temperature by one degree.
• 1) Constant Volume Cv
• 2) Constant Pressure Cp
vvv
vT
u
T
U
mT
Q
mC ===
11
ppp
pT
h
T
H
mT
Q
mC ===
11
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Differential Scanning Calorimetry (DSC)
Constant pressure
(versus bomb-calorimeter)
Measures H
Cp=dq/dT
Cp=dH/dT
direct measurement of Tm, H, Cp from increased inheat transfer occurring with unfolding as a F(T)
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Melting Points
Knowing G=0 at equilibrium:
So now at any Temperature:
Change in Cp
For reversible two
state model
Note that CD > CN
Gibbs Free Energy at the melting point is:
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Enthalpy and Fractional Unfolding
Parallel to the van’t Hoff equation:
So change in enthalpy is related to the
fractions of native and denatured forms of the
protein.:
HvH is determined from the temperature dependence of Keq
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The van’t Hoff Relationship
• Methodology of finding dH and dS from experimental data.
lnKeq =G°
RT=
H° T S°
RT
lnKeq =1R
S°H°
T
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High Cp changes enthalpy significantly with T
• For a two state reversible transition N D
HD-N(T2) = HD-N(T1) + Cp(T2 – T1)
• When T2 > T1, & Cp (CD -CN) is positive and the enthalpybecomes more positive
• i.e. HD-N favors the native state
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High Cp changes entropy with temperature
• For a two state reversible transition N D
SD-N(T2) = SD-N(T1) + Cpln[T2 / T1]
• When T2 > T1, & Cp (CD -CN) is positive the entropybecomes more positive
• i.e. SD-N favors the denatured state
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Free energy of unfolding
• For
GD-N = HD-N - T SD-N
• Gives
GD-N(T2) = HD-N(T1) + Cp(T2 – T1)- T2( SD-N(T1) + Cpln[T2 / T1])
• As temperature increases T SD-N increases and causes the protein to
unfold and midpoint is where GD-N = 0
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Calculating the Equilibrium Mid Point
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Cold unfolding
• Due to the high value of Cp
• Lowering the temperature lowers the enthalpy
decreases
Tc = T2m / (Tm + 2( HD-N / Cp))
i.e. Tm ~ 2 ( HD-N ) / Cp
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•Guanidinium chloride (GdmCl) H2N+=C(NH2)2•Cl-
•Urea H2NCONH2
•Solubilize all constitutive parts of a protein
•Free energy transfer from water to denaturant solutions islinearly dependent on the concentration of the denaturant
•Thus free energy is given by
GD-N = GH2OD-N - mD-N [denaturant]
Solvent denaturation
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Two state transitions in multi-state reactions&
Rate determining steps
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Thermal stability curves.
a, Unfolding transitions of wild type Bc-
Csp and three destabilized variants.
b, Unfolding transitions of wild type Bs-
CspB and three stabilized variants in 100
mM Na-cacodylate/HCl, pH 7.0, at
protein concentrations of 4 muM.
The fractions of native protein obtained
after a two-state analysis of the data are
shown as a function of temperature. The
continuous lines show the results of the
analysis.
Perl D et al.(2000) Nat. Struct. Biol. 7, 380-3
Transition midpoint temperatures, Tm, define thermal unfoldingcurves but are not useful estimates of conformationalstability (why?). However, the Tm remains useful forcomparing closely related proteins (e.g. homologs, single sitemutants)
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Protein folding research
•Static: 3-D prediction (theoretical approach)
•Dynamic: protein folding (misfolding and
refolding) mechanism
– Experimental approach: in vivo and in vitro
(most studies)
– Theoretical approach (in vitro): molecular
dynamics, Monte Carlo simulation
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Protein misfolding and refolding
Most proteins except membrane proteins are soluble inin-vivo and in-vitro aqueous systems
In vivo and in vitro experimentInclusion body: protein aggregateMedical and industrial implications: loss of biological
functionsIncluding misfolding, aggregation, unexpectedmultimerization
Misfolded proteins can be refolded to regain theirbiological function
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Protein disulfide Isomerase
Proteins in vivo fold faster than in vitro…
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Molecular chaperons
GroEL GroES
Molecular chaperones:
(1) Hsp70 proteins function as monomer
(2) Chaperonins, large multisubunit proteins (Hsp60 & Hsp10)
(3) Hsp90 proteins for the folding of proteins involved with signaltransduction
(4) Trigger factor
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ATP binding and Hydrolysis coordinate the conformationalchanges in GroEL/ES
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Reaction cycle of the GroEL/ES cycle
1. GroEL ring binding 7 ATP and a
substrate (improperly folded protein).
Then it binds a GroES cap to become
the cis ring.
2. The cis ring catalyzes the
hydrolysis of its 7 ATP.
3. A 2nd substrate binds to the trans
ring followed by 7 ATP.
4. The binding of substrate and ATP
to the trans ring conformationally
induces the cis ring to release its
bound GroES, 7 ADP, and the better
folded substrate.The trans ring
becomes the cis ring.
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in-vitro protein folding
• Biopharmaceutical is a typical example, (e.g. insulin)
• Prevent protein misfolding (e.g. amino acid effect on
protein folding)
• Refold misfolded protein to regain its biological
function
Application
How long does protein folding take?
• s, ms, s, min…Depending on protein size, temp, etc.
Monitor protein folding
• Spectrofluorometer, Circular dichroism, Stop flow
system, PAGE (SDS gel and native gel), etc.