Professional Documents
Culture Documents
1. Primary structure
Amino acid sequence
Edman degradation, MS, deduce from DNA
2. Secondary structure
Recurring structural pattern
Circular dichroism (CD, )
3. Tertiary structure
3D folding of a polypeptide chain
X-ray crystallography, NMR
4. Quaternary structure
Fig 5-16 Subunits arrangement within a protein
1
The 3-D structure of proteins
Folded (native)
Lowest free energy
Stabilized by disulfide bond (covalent) and weak (non-covalent)
interactions:
9 Weak interactions
9 Van der Waals interaction
9 H-bond
9 Hydrophobic
9 Ionic
Anti-parallel
-sheet
Parallel
-sheet
O O
H3 N+ H3N+ C O-
C O-
CH CH2 CH
H CH2 CH2
+
7
Electric dipole of an -helix
-
Peptide bond dipole
Helix dipole
End residues and
helix stability
Fig 6-2a
+ Fig 6-6
8
-conformation
Zigzag, extended protein chain, with the R groups
alternating above and below the backbone.
Side by side -conformation -sheet
9 H-bonds between adjacent peptide chain (backbone).
9 Parallel or antiparallel orientations
Silk fibroin layers of -sheets
parallel antiparallel 9
-turn
A 180o turn involving 4 a.a.
H-bond between -CO of the 1st a.a. and the -NH of the
4th a.a.
Common a.a.
9 Gly (small and flexible, type II -turn)
9 Pro (peptide bonds involving the imino N in cis configuration)
3 3 (Gly)
4 4
Fig 6-8a
2 1 2 1 10
Occurrence in 2o structure
Relative probability of a.a.
Fig 6-10
11
Circular Dichroism Spectroscopy
Determine the content of 2o structure of a protein
http://www-structure.llnl.gov/cd/cdtutorial.htm 12
Membrane proteins Lehninger 4th ed.
13
Classification (p. 170)
Fibrous proteins (e.g. Table 6-1)
9 Long strands or sheets
9 Consist of a single type of 2o structure
9 Function in structure, support, protection
9 -keratin, collagen
Globular proteins (e.g. Table 6-2)
9 Spherical or globular shape
9 Contain several types of 2o structure
9 Function in regulation
9 Myoglobin, hemoglobin
14
Structure of hair
-keratin: hair, wool, nails, claws, quills, horns,
hooves, and the outer layer of skin
Fig 6-11, p. 171
Monomer
Dimmer
15
Collagen
Tendons, bone, cartilage, skin, and cornea
Primary sequence:
9 Gly-X-Pro (HyPro)
9 Repeating tripeptide unit collagen
Structure
9 Monomer ( chain)
9 Left-handed helix, 3 a.a. per turn
9 Trimer: coiled-coil (tensile strength). tropocollagen
9 Stabilized by H-bond
9 Crosslink between triple helixes
Genetic defect:
9 Osteogenesis imperfecta
9 Abnormal bone formation in babies
9 Ehlers-Danlos syndrome
16
9 Loose joint
More on Collagen Harpers 26th, p. 38-39.
17
Denature and unfolding
Loss of function due the structural disruption
9 Cooperative process
9 Denatured conformation: random but partially folded
No covalent bonds in the polypeptide are broken !!
Denaturing agent
9 Heat (H-bond)
9 Extreme pH (change ionic interaction)
9 Miscible organic solvent (hydrophobic interactions) No function
9 Alcohol, acetone
9 Certain solutes (hydrophobic interactions)
9 Urea, guanidino hydrochloride (Gdn HCl), detergent
Fully
functional
18
The prion disease
Spongiform encephalopathies
Disease caused by a protein (prion)
Proteinaceous infectious particle
Related diseases:
9 Mad cow disease
9 Kuru
9 Creutzfeldt-Jakob disease (human)
9 Scrapie (sheep)
Misfolded prion
PrPC PrPSC
(normal) (infectious)
19
Protein Function
20
O2 binding to Heme
Heme = organic ring (porphyrin) + Fe2+
Free heme Fe2+ (binds O2) vs. Fe3+ (does not bind)
O2 rich blood (bright red) vs. O2 depleted blood (dark purple)
CO, NO binds with higher affinity than O2
21
Protein-ligand interaction
p. 207
P+L PL
[PL]
Ka = Ka: association constant
[P] [L] (M-1)
[PL]
Ka [L] =
[P]
Hyperbola
[L]
=
[L] + Kd
Fig 7-4a
23
O2 binding of Mb
[L]
O2 binds tightly to Mb =
[L] + Kd
tissues lungs
0.26 kPa
Fig 7-4b
24
Structure affects Kd
Kd for O2 Kd for CO
Free heme 1x 1/20,000x
Heme in Mb 1x 1/200x
Steric hindrance
9 Distal His, (His64 of Mb)
Molecular motion (breathing)
9 O2 in/out buried cavity
25
Mb vs. Hb
O2 storage O2 transport
In muscle tissue Found in erythrocyte
Mb = monomer Hb = tetramer
9 4 x (polypeptide chain + heme)
9 1 polypeptide chain
(153 a.a.) + 1 heme Hb m.w. = 64.5 KDa
Mb m.w. = 16.7 kDa Interactions between
subunits (tetramer)
Fig 7-3
Fig 7-10 26
Hb has 2 conformations
T state R state
-O2 structure stable unstable
+O2 unstable stable
Kd (O2) large small
O2 binding to T triggers a conformational change to R
Fig 7-10
27
HbO2 binding curve
A sigmoid (S-shape) binding curve
Permit highly sensitive response to small change in
pO2 or [ L]
= 0.96
= 0.64
Fig 7-12
28
O2 binding to Hb
Cooperativity
9 One subunit binding of O2 affects Kd of the
adjacent subunits
9 4 x (subunit + O2)
9 1st O2 binds Hb (T) weakly, initiate T R
9 2nd O2 binds Hb (TR) with higher affinity
9 3rd O2 binds Hb (TR) with even higher affinity
9 4th O2 binds Hb (R) with highest affinity
9 S-shaped (sigmoid) binding curve multimer only
Allosteric protein
9 Homotropic: modulator = ligand (substrate)
9 e.g. O2, CO
9 Heterotropic: modulator ligand (substrate)
9 e.g. H+, CO2, BPG
29
Quantification
P+nL PLn Slope = n (Hill coefficient)
[PLn] n > 1, + Coop.
Ka = n = 1, no Coop.
[P] [L]n
n < 1, - Coop.
Binding sites occupied [L]n
= =
Total binding sites [L]n + Kd
[L]n
= log
1- Kd 1-
log = n log [L] log Kd
1-
Y = ax - b
log [L]
Hill equation
30
Hill plot of Mb vs. Hb
Mb: nH = 1
Hb: nH = 3
Fig 7-13
31
Hb also transports H+ and CO2
Bohr effect
pH and CO2 modulate the affinity of Hb for O2
9 Hb binds O2 and (H+ or CO2) with inverse affinity
9 Hb binds O2, H+, and CO2 at different sites
9 Tissues: pH and CO2, O2 affinity , Hb release O2
9 Lungs: pH and CO2 , O2 affinity , Hb binds more O2
In lung
In tissue
32
BPG (2,3-bisphosphoglycerate)
BPG binds to a.a. in the cavity between subunits in Hb (T state)
9 BPG stabilize T state O2 affinity
[BPG] at sea level vs. high altitude
Fetal Hb needs to have a higher O2 affinity than mothers Hb
9 Fetal Hb : 22
[BPG] , after storage, transfusion
People suffering from hypoxia, [BPG]
33
CO intoxication (Box 5-1)
CO has a higher affinity for Hb
9 Smoker has higher level of COHb (3~15%) vs. < 1%
9 Binding of CO to Hb increase the O2 affinity of Hb
9 O2 transport become less efficient (Fig 2)
Suspected CO intoxication
9 Rapid evacuation
9 Administer 100% O2
HbA
35