Transcript Protein notes


Amino acids link together through covalent bonds to form proteins
(18.7)
proteins are polymers of amino acids linked head to tail
short polymers of amino acids (short proteins) are called peptides
peptide bond - the covalent bond that links amino acids together
carboxylic acid from one aa and amino group from another
water is released
residue - an individual amino acid in a protein
H
H
H
N
C
H
R1
O
H
C
O
H
H
N
C
H
R2
O
C
O
H2O
H
H
H
O
N
C
C
H
R1
H
N
C
H
R2
O
C
O
peptide bond
1

Peptides and proteins are directional
amino terminus - end with free amino group
carboxyl terminus - end with free carboxylic acid group
proteins are synthesized and specified from the amino to carboxyl terminus
H
H
H
O
N
C
C
H
R1
H
O
N
C
H
R2
R1R2
C
O
amino terminus
carboxyl terminus
H
H
H
O
N
C
C
H
R2
H
R2R1
O
N
C
C
H
R1
O
when writing peptide names using full aa names, drop the “ine” from each aa except the
carboxyl end and replace with “yl”
H
H
H
O
N
C
C
H
R1
H
O
N
C
C
H
R2
H
O
N
C
C
H
R3
H
O
N
C
C
H
R4
O
2
Threonine and valine can combine to form two different dipeptides. Draw them and write
their names using full names, three letter and one letter abbreviations.
3
Identify the amino acids in the following dipeptide and tripeptide, and write their names
using full names, three letter and one letter abbreviations.
4

The peptide bond has partial double bond character
the electrons in the double bond here, are shared a lot with this bond (the C-N bond)
H
H
H
O
N
C
C
H
R1
H
N
C
H
R2
O
C
O
you cannot rotate around the C=O or C-N bonds.
causes the peptide bond and protein backbone to be planar (flat)
side chains alternate above and below plane

The peptide bond is trans
the carboxyl oxygen and amide hydrogen are on opposite sides to minimize steric
interactions of the oxygen and hydrogen (overlapping of e- clouds = repulsion)
5
Four Tiers of Protein Structure (18.8-18.11)
 Primary structure (1° structure)
amino acid sequence of the protein written from amino to carboxyl terminus
The amino acid sequence determines its 3D structure (which then determines its
properties.)
depends solely on covalent bonds
Why determine primary structure?
1. Determining primary structure is the first step in characterizing a protein - can help
determine the function of a protein by comparison to other protein sequences
2. primary structure determines 3D structure (which then determines its properties.)
3. changes in 1° can make protein fold differently or cause disease (sickle cell anemia
results from a change of 1 amino acid in hemoglobin glu to val)
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1° Structure determines 3D shape and therefore protein function (or dysfunction)
Experimental determination of 1° structure
STEP 5
STEP 6
7
Experimental determination of 1° structure
Split the protein into several tubes
Step 1 - use for amino acid constituent analysis
Determine which amino acids are present and in what proportions
use high acid concentration and high temps (100-110 C) for 12-36 hours
hydrolyzes peptide bonds
determines number of possible cleavage sites for steps 3 and 4.
resulting solution put through an amino acid analyzer to aa present and ratios
only identifies which amino acids are present and ratios- not their order
Step 2 - use for end group analysis
Identify N-terminal and C-terminal amino acids
can be used to determine the number of polypeptide chains - process is automated
N-terminal
Edman degradation
reliable for
polypeptide
chains up
to 40-60
amino acids
amino acid
derivative
can be easily
identified
+
H3N
1
2
3
4
5
COO -
n
Original peptide
Phenyl isothiocyanate
(Edman’s reagent)
N
C
S
S
H
N
C
1
NH
2
3
4
5
COO -
n
Modified peptide
Can be used in another
round as in steps 3 and 4
O
C
CH
N
C
S
NH
R1
+
+
H3N
2
3
4
5
n
COO -
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Step 2 (cont.)
“ase” is the
ending given
to denote an
enzyme
C-terminal
Enzymatic analysis with carboxypeptidases
Carboxy means it
works on the
carboxyl end
of the protein
Peptid because
they cleave
peptide bonds
carboxypeptidases are exopeptidase (cleave end bonds)
Four carboxypeptidases are used since not one enzyme will use each aa as substrate
+
H3N
1
2
3
4
n-1
5
n
COO -
Original peptide
Treat with carboxypeptidases
+
H3N
1
2
3
H2O
4
5
n-1
COO -
+
Rn
H3N
n
CH
COO amino acid is easily identified
COO -
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Steps 3 and 4- Fragmentation of the Polypeptide Chain
goal is to produce the correct size polypeptide fragments for sequencing through
Edman degradation (Step 4 below)
automated Edman is reliable for polypeptide chains up to 40-60 amino acids
most proteins are >100 aa so the protein must be broken into fragments
break up large protein into smaller polypeptides using endopeptidases & cyanogen bromide
Site Specific peptidases - enzymes that cleave peptide bonds at very specific sites in
the protein
Most commonly used endopeptidases:
Enzyme name
Trypsin
Chymotrypsin
Peptide bond cleavage Specificity
cleaves after R or K amino acids
cleaves after Y, W, and F
Commonly used chemical:
cyanogen bromide (CN-Br) - cleaves after M
will see all fragments end in M except 1 and that is the carboxyl end of the protein
chymotrypsin
N-ala-ser-phe-pro-lys-gly-gly-met-arg-trp-asp-met-gly-tyr-lys-ala-cys-C
trypsin
CN-Br
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chymotrypsin
ala-ser-phe-pro-lys-gly-gly-met-arg-trp-asp-met-gly-tyr-lys-ala-cys
trypsin
CN-Br
Chymotrypsin digestion yields 4 fragments (remember, their sequences are unknowon at this time):
ala-ser-phe
pro-lys-gly-gly-met-arg-trp
asp-met-gly-tyr
Trypsin digestion yields 4 fragments also:
ala-ser-phe-pro-lys
gly-gly-met-arg
trp-asp-met-gly-tyr-lys
Cyanogen bromide digestion yields 3 fragments:
ala-ser-phe-pro-lys-gly-gly-met
lys-ala-cys
arg-trp-asp-met
ala-cys
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gly-tyr-lys-ala-cys
What fragments are produced by trypsin, chymotrypsin, and CNBr digestion of the following peptides?
1) gly-met-lys-gly-ala-cys-met-asp-trp-arg-met-val-tyr-iso-ala-cys-met-phe-leu
Trypsin fragments
gly-met-lys
gly-ala-cys-met-asp-trp-arg
Chymotrypsin fragments
gly-met-lys-gly-ala-cys-met-asp-trp
CNBr fragments
gly-met
lys-gly-ala-cys-met
met-val-tyr-iso-ala-cys-met-phe-leu
arg-met-val-tyr
asp-trp-arg-met
iso-ala-cys-met-phe
val-tyr-iso-ala-cys-met
leu
phe-leu
1) VGCMAWGYLEMTSRGGF
Trypsin fragments
VGCMAWGYLEMTSR
GGF
Chymotrypsin fragments
VGCMAW
GY
CNBr fragments
VGCM
SWGYLEM
LEMTSRGGF
TSRGGF
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Step 5- Sequence determination of each polypeptide from steps 3 and 4
step 4 produces polypeptides whose sequences are still unknown. So, each polypeptide
produced from digestion with trypsin, chymotrypsin, or cyanogen bromide treatment
undergoes Edman degradation to determine its sequence.
Step 6 - Fragment alignment
The sequences of the fragments determined in step 5 are put together like a puzzle to
produce the sequence of the original protein.
Example:
A solution of a small protein of unknown sequence was divided into two samples. One
sample was treated with trypsin and the other with chymotrypsin. The smaller peptides
obtained by trypsin treatment had the following sequences:
Leu-Ser-Tyr-Ala-Ile-Arg
Asp-Gly-Met-Phe-Val-Lys
The smaller peptides obtained by chymotrypsin treatment had the following sequences:
Val-Lys-Leu-Ser-Tyr
Ala-Ile-Arg
Asp-Gly-Met-Phe
Deduce the sequence of the original protein.
Trypsin treatment indicates 2 basic aa in the peptide, one of which must be the c-terminal aa - otherwise another fragment would
13 have
been produced that lacked a basic residue at the c end - like in chymotrypsin, there is a piece that does not have an aromatic at the end.

Secondary structure (2° structure)
common structures in proteins that result from H- bonding of the backbone atoms only
(the carbonyl and amide nitrogen)
The a-Helix (alpha-helix)
polypeptide backbone coils around an imaginary pole (like a telephone cord)
in the figure on the left, green lines denote hydrogen bonds between
the oxygen of C=O and the hydrogen of N-H
In this figure, the side chains of the amino acids
are shown in green and purple. Note how they
stick out of the helix perpendicular to the
direction of the helix.
Most common 2° structure found in proteins.
If you combine all of the amino acids from
all proteins whose structures are known,
1/3 of the aa are found in an helix 14
Some proteins are composed entirely of a-helices
There are 7 a-helices in this polypeptide.
This polypeptide is part of hemoglobin.
Hemoglobin has four of these polypeptides
(called “subunits”) that interact non-covalently.
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What holds an alpha-helix together?
Hydrogen bonds between carbonyl and amide nitrogen four aa away
hydrogen bonds shown by dotted lines
H-bonds are
between the
oxygen of a
carbonyl and
the amide
hydrogen 4
amino acids
away
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The b-Sheet (beta-sheet)
polypeptide backbone is stretched out like an extended accordion
There are two kinds of b-sheets
parallel
anti-parallel
17
What holds a beta-sheet together?
Hydrogen bonds between carbonyl and amide nitrogen
Antiparallel b-sheet with 5 strands
H-bonds are
between the
oxygen of a
carbonyl and
the amide
hydrogen
on another
strand
Strand 1
Strand 3
Strand 2
Strand 5
Strand 4
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Some proteins are composed almost entirely of b-sheets
neuraminidase from influenza virus
(pdb code 1EUU)
Retinol binding protein
(pdb code 1ggl)
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Some proteins have both a-helices and b-sheets
Carboxypeptidase (digestion protease)
(pdb code 5CPA)
Hexokinase (glycolysis)
(pdb code 5CPA)
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
Tertiary structure (3° structure)
overall 3D shape of all of the atoms in the protein (not just those involved in
a-helices or b-sheets)
proteins have different 3D shape compared to each other (though same family are
similar) but each carboxypeptidase folds the same as every other carboxypeptidase
secondary structure - interactions of backbone atoms
tertiary structure - mainly results from interaction of side chains far apart in
primary sequence or side chain-backbone interactions - residues far apart in
primary sequence can be close together in space
hydrophobic residues usually buried interior and hydrophilic on exterior
Shape determining interactions in proteins
1. Hydrophobic interactions between non-polar side chains
2. H-bonds between side chains
3. H-bonds between backbone atoms
4. H-bonds between a side chain and a backbone atom
5. Salt bridge (ionic attraction between charged side chains)
6. Disulfide bonds (covalent bond between cysteine residues)
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Shape-Determining Interactions in Proteins (18.8)
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
Quaternary structure (4° structure)
some proteins have more than one amino acid chain (called subunits) that interact
non-covalently. The arrangement of these subunits with each other is 4° structure.
dimer - protein with 2 subunits (2 non-covalently linked polypeptide chains)
trimer - protein with 3 subunits
tetramer - protein with 4 subunits
Examples: Hemoglobin - tetramer
ATP Synthase - trimer
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Disease and Protein Structure

Sickle Cell Diseases
Group of diseases affecting shape and oxygen carrying
capacity of red blood cells.
mainly affects persons of African or Mediterranean
descent (Italian, Spanish)
Inherited diseases - not contagious
Diseases are diseases of the protein hemoglobin
result from mutations in hemoglobin genes or lack of production of hemoglobin
autosomal recessive diseases
beta
chain
(146 aa)
alpha
chain (141 aa)
beta
chain (146 aa)
alpha
chain
(141 aa)
Hemoglobin is a tetramer
Heme
oxygen binds here
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Hemoglobin genes
two sets of hemoglobin genes - one set from mother and one set from father
each set contains 2 alpha genes (chromosome 16) and 1 beta gene (chromo 11)
gives a total of 4 alpha genes and 2 beta genes
in normal hemoglobin production, the amt. of alpha produced=amt of beta
alpha and beta chains have only 20% sequence homology but virtually identical 3D
Types of hemoglobin:
Hemoglobin A - normal hemoglobin - normal alpha and beta chains
Hemoglobin F - fetal hemoglobin - has higher affinity for oxy than Hgb A
- production declines by 6 months of age
- normal alpha chains; beta replaced by gamma chains
Hemoglobin S - normal alpha chains, mutated beta chain where Glu-6 is
replaced by Val - sickle cell
Hemoglobin C genes - normal alpha genes, mutated beta genes where Glu-6 is
replaced by lys
beta-thalassemia - normal genes but less beta produced
alpha - thalassemia - normal genes but less alpha produced
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Sickle Cell Disease is not
necessarily Sickle Cell Anemia
Sickle Cell Trait Pedigree
AA
Sickle Cell Anemia Pedigree
AS
AS
AS
Common Types of Hemoglobin proteins
1) Hemoglobin AA - normal, healthy hemoglobin
AA AA AS AS
AA
AA AS AS SS
2) Sickle Cell Trait (AS) - alpha genes normal - one normal beta gene, one beta sickle
- healthy and will have no symptoms associated with sickle cell disease.
- both normal and sickle hemoglobin produced; mostly normal since a binds to b better
3) Sickle Cell Anemia (SS) - alpha genes normal - two sickle beta genes
4) Sickle C Disease (SC) - alpha genes normal - one sickle beta gene - one hemo C disease
5) Sickle beta-thalassemia (S/b-thalassemia) - alpha genes normal - one sickle and thala
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normal red blood cells
sickle red blood cells
Sickle hemoglobin has same affinity for oxygen as normal hemoglobin but in low oxygen
areas (veins and venous capillaries), hemoglobin molecules clump together
clumping causes together causing red blood cells to become sickle in shape and become
hard
sickle, hard red blood cells don’t flow through capillaries as easily - get caught and
block capillaries blocking blood and oxygen flow to tissues
shortness of breath, stokes, fatigue, infections, jaundice, lung blockage, leg ulcers,
bone damage, kidney damage, eye damage, and obviously low rbc counts.
Normal rbc survive about 120 days but sickle rbc usually last 20 days - sickle cell patients
have less rbc and so less hemoglobin
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Hemoglobin Clumping in Sickle Cell Anemia:
Hemoglobin molecules clump under low oxygen concentrations (venous blood) due to
hydrophobic interactions between different hemoglobin molecules
Hbg S
- Glutamic acid at position 6 of the
beta chains are mutated to valine
- Hydrophobic patch on the beta chain
of another hemoglobin molecule
- val-6 and hydrophobic patch (ala, phe, leu)
interact
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Diagnosing Sickle Cell Disease
Electrophoresis - uses a gel to separate molecules based on size, charge and/or shape
- At the pI of a specific protein - the protein molecule carries no net charge
and does not migrate in an electric field.
- At pH above the pI - the protein has a net negative charge and migrates
towards the anode (the positive end).
- At pH below the pI - the protein obtains a net positive charge on its
surface and migrates towards the cathode (the negative end).
For proteins of same size, migration depends on net charge
- the more negatively charged, the faster the migration towards anode
cathode
anode
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Hemoglobin electrophoresis
Test that determines the types of hemoglobin in a patient
different forms of hemoglobin will migrate in an electric field differently because of
different charges:
Hemoglobin A
Hemoglobin S - changes glutamic acid (neg) to valine (neutral) - one less negative than A
per beta chain so 2 less neg total
Hemoglobin C - changes glutamic acid (neg) to lysine (pos) - two less negative than A
and one less than S per chain - so, four less than A and two less than S
origin
Hbg C
Cathode
(-)
Hbg S
Power Supply
Hbg A
Anode
(+)
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Control
Patient 6
Patient 5
Patient 4
Control
Patient 3
Patient 2
-
Patient 1
Determine the type of hemoglobin disease (if present) in patients
whose hemoglobin electrophoresis patterns are as follows:
origin
Patient Disease
1
2
C
3
4
S
5
F
6
A
+
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Disease and Protein Structure (Application on pg.

Prions -
“proteinaceous infectious particles”
In 1997, Stanley Prusiner (a neurologist) won the Nobel Prize for Physiology and
Medicine for discovering prions
prions are naked proteins that infect and destroy brain tissue
first time something other than organisms containing nucleic acid could replicate and
cause disease - met w/ high level of criticism - eventually most accepted hypothesis
Spongiform Encephalopathy - general category of diseases caused by prions
Takes the form Greek for brain
of a sponge
Greek for disease
Symptoms:
dementia, Involuntary and irregular jerking movements,
loss of vision, speech, coordination, depression,
withdrawal
Brains infected with prions look like a sponge.
The holes are areas of brain cells that have
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died as a result of prion infection.
Prion diseases affect many species
can be inherited or acquired:
Prion Disease
Other Names
Species Infected
Bovine Spongiform Encephalopathy
Mad Cow Disease or BSE Cows
variant Creutzfeldt-Jakob Disease
nvCJD or vCJD
acquired; humans from eating cattle with BSE
Kuru
infectious; cannabilistic humans in Papua, New Guinea
Scrapie
Sheep
Feline Spongiform Encephalopathy
FSE
Cats
Transmissible Mink Encephalopathy
TME
Mink
Chronic Wasting Disease
Elk/Deer
Gerstmann-Sträussler-Scheinker disease GSS
inherited disease of humans
Fatal Familial Insomnia
FFI
inherited disease of humans
Creutzfeldt-Jakob Disease
CJD
inherited disease of humans
Prion diseases can be a genetic disorder or can be acquired through:
1)
2)
3)
4)
5)
Eating infected animal parts (brain/spinal cord) - cross species
Brain surgery - prions remain infectious on surgical instruments after sterilization
Corneal transplant
Contaminated growth hormone from pituitary glands
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spontaneous mutation
Prions are Molecular Transformers
Key players:
PrPc - protein normally attached outside nerve cells in brain
- mainly a-helical (“c” for cellular)
Prpsc - identical in sequence to Prpc but is
- mainly b-sheet (“sc” for scrapie)
- over-abundant in brain tissue of scrapie sheep
Prion theory: Prpsc has identical primary structure to Prpc but has a different 3D shape.
Prpsc causes Prpc to change shape into more Prpsc which clog cells because normal cellular
enzymes that destroy proteins are ineffective against Prpsc. Prpsc eventually causes
infected cells to lyse which releases Prpsc into adjacent brain tissue to recruit even more
Prpc to change shape - cycle continues.
Nothing added - nothing
deleted - just a change in
shape!
PrPc
PrPsc
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Evidence that Prions are Proteins
 When PrPsc is purified, scrapie infectivity is also purified
 The amount of PrPsc directly coincides with
infectivity levels
 Procedures known to destroy nucleic acids do not
destroy prion infectivity
 PrP gene mutations that lead to inherited prion disease
also produce PrPsc
 Over-production of PrP increases rate of prion disease
 PrP knock-out mice did not get prion disease after inoculation with prion material
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Destroying Protein Structure (Section 18.12)

Protein denaturation
Protein hydrolysis - hydrolysis of peptide bonds to produce amino acids
destroys all levels of protein structure - even primary
Protein denaturation - protein unfolding - primary structure still intact
primary structure intact
Review:
What forces hold proteins in a specific 3-D shape?
Hydrogen bonding
van der Waals
salt bridges
hydrophobic
dipole-dipole
36

Methods of protein denaturation
pH changes - cause amino acids to alter their protonation state so can destroy
hydrogen bonding and salt bridges
ionic concentration - high salt out-competes amino acids for each other
temperature - increased thermal motions break energy barriers - supplies enough
energy to overcome the forces holding a molecule together
mechanical agitation - beating of egg whites causes proteins to denature
detergents - makes solution more hydrophobic so protein unfolds
organic compounds - polar solvents like acetone and ethanol - make solution more
hydrophobic and also compete for hydrogen bonding with amino acid side chains
reducing/oxidizing agents - break/form disulfide bonds
most denaturation is reversible but there are many cases where the protein can
renature when the reason for its denaturation is removed.
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