Last Lecture: Protein 3 Structure, Folding

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Last Lecture Protein 3 Structure,
Folding Today Structure Determination, Heme
Proteins Mid-term to end of Mondays lecture

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Problems 1. Draw a possible hydrogen bonding
interaction-containing two hydrogen bonds for the
following pairs of amino acid side chains
Asn/Gln, Asp/Glu (protonated). 2. Consider a
folded protein in which two Glu side chains are
positioned close to one another in space. What
is the effect of this on the pKa representing
the ionization of the second side chain? 3.
Would you expect to find positively or negatively
charged side chains positioned near the C
terminus of an a helix? 4. Name the most
conservative mutation for the following amino
acids given the choices in brackets (a) Glu (F,
A, E, V) (b) N (Asp, Met, Pro, His) (c) Phe (Y,
P, E, V)
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Structure Determination
NMR -solution structure -dynamics -size
limitations
X-ray -crystal structure -high resolution
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1D-1H NMR Spectrum
CH3CH2Cl
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X-ray Diffraction
Structure
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Crystallization -"Hanging Drop"
Buffer A Protein
Buffer A buffer salts additives
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Crystallization -"Hanging Drop"
Buffer A Protein
water vapour diffusion
Buffer A buffer salts additives
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Crystallization -"Hanging Drop"
Buffer A Protein
water vapour diffusion
Buffer A buffer salts additives
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High Throughput Crystallization
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X-ray Diffraction
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Electron Density to Structure
Tyr or Phe
Pro
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BRCT Domain - BRCA1
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BRCT Domain - BRCA1
A1708
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1D-1H NMR Spectrum
CH3CH2Cl
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1D 1H Spectrum of Protein
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2D-NMR Spectrum (1H-1H NMR)
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2D-NMR Spectrum (15N- 1H NMR)
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NMR
-connectivity (linear sequence) -coordinates
(through space)
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Nuclear Overhauser Effect (NOE)
-relaxation by through space transfer
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Detail of 2D Spectrum
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Set of NMR Derived Structures
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Protein Function
Structure cellular architecture Carriers
transport Enzymes Catalysis
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Oxygen Transport and Storage Hemoglobin and
Myoglobin
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Hemoglobin and Human Disease
-Sickle Cell b chain E6 to V6
-b thalassemia imbalance in a to b ratio
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Binding of Oxygen to Myoglobin
1 torr 133 Pa 0.132 atm
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Structural Features of Hb
1. Hb is a tetramer (4 subunits) a2b2 2. One
heme bound per subunit 3. Each heme binds one
O2 4. Hb exists in 2 states T deoxy Hb R
oxy Hb 5. T state is stabilized by intrasubunit
and intersubunit salt bridges 6. Conversion T
R involves breaking salt bridges 7. T R is
also characterized by changes in a1 b2
(a2 b1)contacts subunits move closer
together to expel 2,3 BPG 8. More extensive
contacts remain unchanged a1 b2 a2 b1

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Oxygen Binding Curves
hyperbolic (My) and sigmoidal (Hb)
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Four Factors Affect O2 Binding to Hb
Hb stripped of these factors has high O2
affinity, P50 12 torr little cooperativity
(hyperbolic O2 binding curve) H ions bind to
deoxyHb and stabilize it Cl- ions also
stabilize deoxyHb BPG (2,3-bisphosphoglycerate)
binds to deoxyHb preferentially CO2
binds to deoxyHb preferentially All 4 of these
factors stimulate Hb to release its bound O2 in
the capillaries by reducing the O2 binding
affinity
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Oxygen Transfer Between Hemoglobins
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Myoglobin
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Structure of DeoxyHb
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Structure of OxyHb
a2
b1
a1
b2
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T to R Transition in Hemoglobin
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(oxy)
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Specific Binding of Bisphosphoglycerate to
Hemoglobin