5p2-1 chapter 5: outline amino acids amino acid classesstereoisomers bioactive aatitration of aa...
TRANSCRIPT
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5P2-1
Chapter 5: OutlineAmino Acids
Amino acid classes Stereoisomers
Bioactive AA Titration of AA
Modified AA AA reactions
Peptides
Proteins (We are here)
Protein structure
Fibrous proteins
Globular proteins
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5P2-2
Protein Function1. Catalysis
2. Structure
3. Movement
4. Defense
5. Regulation
6. Transport
7. Storage
8. Stress Response
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5P2-3
Proteins by Shape-1Fibrous proteins exist as long stranded molecules: Eg. Silk, collagen, wool. A collagen segment in space-filling mode illustrates this point.
Red spheres represent oxygen, grey carbon, and blue nitrogen
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5P2-4
Proteins by Shape-2Globular proteins have somewhat spherical shapes. Most enzymes are globular. Eg. myoglobin, hemoglobin. Myoglobin in space-filling mode is the chosen example.
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5P2-5
Proteins by CompositionSimple
Contain only amino acids
Conjugated
simple protein (apoprotein)
prostetic group (nonprotein)
glycoproteins
lipoproteins
metaloproteins
etc.
Holo-protein
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5P2-6
Four Levels of Protein Structure
Primary, 1o
the amino acid sequenceSecondary, 2o
3-D arrangement of backbone atoms in space
Tertiary, 3o
3-D arrangement of all the atoms in space
Quaternary, 4o
3-D arrangement of subunit chains
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5P2-7
Determining Primary Structure1. Hydrolyze protein with hot 6M HCl.
Identify AA and % of each.
Usually done by chromatography
2. Identify the N-term and C-term AAs
C-term via carboxypeptidase
N-term via Sanger’s Reagent, DNFB
2,4-dinitrofluorobenzene
Often step 2 can be skipped today.
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5P2-8
Det. Primary Structure: 23. Selectively fragment large proteins
into smaller ones.
Eg. Tripsin: cleave to leave Arg or Lys as C-term AA
Eg. Chymotrypsin: cleave to leave Tyr or Trp or Phe as C-term AA
Eg. Cyanogen bromide cleaves at internal Met leaving Met as C-term homoserine lactone
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5P2-9
Det. Primary Structure: 34. Determine AA sequence of peptides
with AA sequencer using Edman’s reagent:
phenyl isothiocyanate which reacts with the N-term AA
See the next slide
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5P2-10
Det. Primary Structure: 3bNH3
+COO
-1 2 3
N C S
NH C
S
NH COO-
1 2 3
NH3
+COO
-2 3NH C
S
NCH
CO
R1
aqueous acid
+
N
CS
NH
CHCO
R1
RAR
protein
Edman’s reagent
Phenylthiohydantoin (PTH)derivative of N-term AA
Thiazoline derivative
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5P2-11
Det. Primary Structure: 45. Reassemble peptide fragments from
step 3 to give protein.
An example follows on the next slide.
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5P2-12
Det. Primary Structure: 4b
A twelve AA peptide was hydrolyzed.
Trypsin hydrolysis:
Leu-Ser-Tyr-Gly-Ile-Arg
Thr-Ala-Met-Phe-Val-Lys
Chymotrypsin hydrolysis
Val-Lys-Leu-Ser-Tyr
Gly-Ile-Arg
Thr-Ala-Met-Phe
Deduce the AA sequence
One isC-term
Lys is internal!
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5P2-13
Det. Primary Structure: 4c
Tr Leu-Ser-Tyr-Gly-Ile-Arg
Ct Gly-Ile-Arg
Ct Val-Lys-Leu-Ser-Tyr
Tr Thr-Ala-Met-Phe-Val-Lys
Ct Thr-Ala-Met-Phe
The complete sequence is:
Thr-Ala-Met-Phe-Val-Lys-Leu-Ser-Tyr-Gly-Ile-Arg
Keeping in mind the N-term AA and overlaping the sequences properly gives:
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5P2-14
Secondary Structure
The two very important secondary structures of proteins are:
-helix
-pleated sheet
Both depend on hydrogen bonding between the amide H and the carbonyl O further down the chain or on a parallel chain.
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5P2-15
Helix: Peptide w Hbonds
First six C=O to N hydrogen bonds shown
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5P2-16
Sheet: stick form Protein G
Chain 1
H bonds in dotted red-blue
Chainsegment 1
Seg 2
Seg 3
Seg 4
H bonds shown in dotted red-blue
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5P2-17
B Sheet: Lewis Structure
CH
C
N
CH
C
N
CH
C
N
O
H
H
O
H
O
N-term
C-term C-term
N-term
CH
C
N
CH
C
N
CH
C
N
O
H
H
O
H
O
Parallel sheet
CH
C
N
CH
C
N
CH
C
N
O
H
H
O
H
O
N-term
C-term
C-term
N-term
CH
C
N
CH
C
N
CH
C
N
O
H
H
O
H
O
Antiparallel sheet
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5P2-18
Supersecondary StructureReverse turns in a protein chain allow
helices and sheets to align side-by-side
Common AA found at turns are:
glycine: small size allows a turn
proline: geometry favors a turn
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5P2-19
Supersecondary Structure: 2
Combinations of helix and sheet.
meander
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5P2-20
Tertiary StructureThe configuration of all the atoms in the
protein chain:
side chains
prosthetic groups
helical and pleated sheet regions
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5P2-21
Tertiary Structure: 2
Protein folding attractions:
1. Noncovalent forces
a. Inter and intrachain H bonding
b. Hydrophobic interactions
c. Electrostatic attractions
+ to - ionic attraction
d. Complexation with metal ions
e. Ion-dipole
2. Covalent disulfide bridges
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5P2-22
Tertiary interactions: diag.
NH3
+
Polypeptide Chain
COO CH2 S
CH2S
O HC
O O
CH3CH3
CH2OHCH2 OH
CHCH3 CH3CHCH3 CH3 CO
ONH3
+
Mg2+
ionic
disulfide
hydrophobicH bondsor dipoleIon-dipole
metal coord’n
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5P2-23
DomainsDomains are common structural units
within the protein that bind an ion or small molecule.
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5P2-24
Quaternary Structure-1Quaternary structure is the result of
noncovalent interactions between two or more protein chains.
Oligomers are multisubunit proteins with all or some identical subunits.
The subunits are called protomers.
two subunits are called dimers
four subunits are called tetramers
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5P2-25
Quaternary Structure-2If a change in structure on one chain
causes changes in structure at another site, the protein is said to be allosteric.
Many enzymes exhibit allosteric control features.
Hemoglobin is a classic example of an allosteric protein.
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5P2-26
Denaturation-loss of protein structure, 2o 4o, but not
1o.1. Strong acid or base2. Organic solvents3. Detergents4. Reducing agents5. Salt concentrations6. Heavy metal ions7. Temperature changes8. Mechanical stress
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5P2-27
Denaturation-2
Denaturing destroys the physiological function of the protein.
Function may be restored if the correct conditions for the protein function are restored.
But! Cooling a hardboiled egg does not restore protein function!!
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5P2-28
Fibrous ProteinsFibrous proteins have a high
concentration of -helix or -sheet. Most are structural proteins.
Examples include:
a-keratin
collagen
silk fibroin
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5P2-29
Globular ProteinsUsually bind substrates within a
hydrophobic cleft in the structure.
Myoglobin and hemoglobin are typical examples of globular proteins.
Both are hemoproteins and each is involved in oxygen metabolism.
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5P2-30
Myoglobin: 2o and 3o aspectsGlobular myoglobin has 153 AA arranged in eight -helical regions labeled A-H.
The prosthetic heme group is necessary for its function, oxygen storage in mammalian muscle tissue.
His E7 and F8 are important for locating the heme group within the protein and for binding oxygen.
A representation of myoglobin follows with the helical regions shown as ribbons.
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5P2-31
Some helical regions
Myoglobin: 2o and 3o aspects
Heme group with iron (orange)at the center
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5P2-32
The Heme Group
NN
NN
CH2CH2COO-
CH2CH2-OOC
CH3 CH3
CH
CH2
CH3
CH3 CH CH2
Fe(II)Pyrrole ring
N of HisF8 bindstofifth site onthe iron.
His E7 actsas a ”gate” for oxygen.
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5P2-33
Binding Site for Heme
Lower His bonds covalently to iron(II)
Oxygen coordinates to sixth site on iron and the upper His acts as a “gate” for the oxygen.
NN
H
O
O
N
N
N
N N
N
Fe
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5P2-34
HemoglobinA tetrameric protein
two -chains (141 AA)
two -chains (146 AA)
four heme units, one in each chain
Oxygen binds to heme in hemoglobin cooperatively: as one O2 is bound, it
becomes easier for the next to bind.
Lengthy segments of the and chains homologous to myoglobin.
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5P2-35
Hemoglobin: ribbons + hemesEach chain is in ribbon form and color coded.
The heme groups are in space filling form
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5P2-36
Oxygen Binding CurvesOxygen bonds differently to hemoglobin
and myoglobin.
Myoglobin shows normal behavior while hemoglobinn shows cooperative behavior. Each oxygen added to a heme makes additon of the next one easier.
The myoglobin curve is hyperbolic.
The hemoglobin curve is sigmoidal.
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5P2-37
Oxygen Binding Curves-2
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5P2-38
The Bohr Effect (H+ and Hb)
Lungs:
pH higher than in actively metabolizing tissue. (Low H+). Hb binds oxygen and releases H+.
Muscle at Work:
pH lower (H+ product of metabolism). Hb releases oxygen and binds H+.
HbO2 + H+ + CO2
metabolism O2 + H+-Hb-CO2
in lungs