Download Lectrure 9 - Columbus Labs

MCAT Question
•
Covalent bonds are the strongest
chemical bonds contributing to the
protein structure A peptide bond is
formed between with of the following?
A.
B.
C.
D.
Carboxylic group and amino group
Two carboxylic groups
Two amino groups
Ester group and ammonium group
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But some amino acids have
multiple properties
• Y
• K
OH? Hydrogen bond donor or acceptor
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Quiz
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The Peptide Bond
• is usually found in the
trans conformation
• has partial (40%) double
bond character
• is about 0.133 nm long shorter than a typical
single bond but longer
than a double bond
• Due to the double bond
character, the six atoms of
the peptide bond group are
always planar!
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Characteristics of the Peptide Bond
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Proteins - Large and Small
• Insulin - A chain of 21
residues, B chain of 30
residues -total mol. wt. of
5,733
• Glutamine synthetase 12 subunits of 468
residues each - total mol.
wt. of 600,000
• Connectin proteins alpha - MW 2.8 million!
• beta connectin - MW of
2.1 million, with a length
of 1000 nm -it can stretch
to 3000 nm!
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The sequence of ribonuclease A
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The Sequence of Amino Acids
in a Protein
• is a unique characteristic of every protein
• is encoded by the nucleotide sequence of
DNA
• is read from the amino terminus to the
carboxyl terminus
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Architecture of Proteins
• Shape - globular or fibrous
• The levels of protein structure
- Primary - sequence
- Secondary - local structures - H-bonds
- Tertiary - overall 3-dimensional shape
- Quaternary - subunit organization
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“Protein”
One or more polypeptide chains
• One polypeptide chain - a monomeric protein
•
•
•
•
•
More than one - multimeric protein
Homomultimer - one kind of chain
Heteromultimer - two or more different chains
Hemoglobin, for example, is a heterotetramer
It has two alpha chains and two beta chains
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What forces determine the
structure?
• Primary structure - determined by
covalent bonds
• Secondary, Tertiary, Quaternary structures determined by weak forces and disulfide
bonds
• Weak forces - H-bonds, ionic interactions,
van der Waals interactions, hydrophobic
interactions
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Other Chemical Groups in
Proteins
Proteins may be "conjugated" with other
chemical groups
• If the non-amino acid part of the protein is
important to its function, it is called a
prosthetic group.
• glycoprotein, lipoprotein, nucleoprotein,
phosphoprotein, metalloprotein, hemoprotein,
flavoprotein.
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Prosthetic Groups
Heme
Metal centers
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Sequence Determination
Frederick Sanger was the first - in 1953,
he sequenced the two chains of
insulin.
• Sanger's results established that all
of the molecules of a given protein
have the same sequence.
• Proteins can be sequenced in two
ways:
- amino acid sequencing
- sequencing the corresponding
DNA in the gene
The sequence shown is
that of bovine insulin.
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Determining the Sequence
An Eight Step Strategy
•
•
•
•
•
1. Cleave (reduce) disulfide bridges
2. If more than one polypeptide chain, separate.
3. Determine composition of each chain
4. Determine N- and C-terminal residues
5. Cleave each chain into smaller fragments
and determine the sequence of each chain
• 6. Repeat step 5, using a different cleavage
procedure to generate a different set of
fragments.
• 7. Reconstruct the sequence of the protein from
the sequences of overlapping fragments
• 8. Determine the positions of the disulfide
crosslinks
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Step 1:
Cleavage of Disulfide bridges
• Performic acid oxidation
• Sulfhydryl reducing agents
- mercaptoethanol
- dithiothreitol or dithioerythritol
- to prevent recombination, follow with an
alkylating agent like iodoacetate
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Step 2:
Separation of chains
• Subunit interactions depend on weak forces
• Separation is achieved with:
- extreme pH
- 8M urea
- 6M guanidine HCl
- high salt concentration (usually
ammonium sulfate)
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Step 3:
Determine Amino Acid Composition
• The complex amino acid mixture in the
hydrolysate obtained after digestion of a protein
in 6 N HCl can be separated into the component
amino acids by either ion exchange
chromatography (separation by charge) or
reverse-phase chromatography (separation by
polarity)
• Both of these methods of separation and
analysis are fully automated in instruments
called amino acid analyzers. Analysis of the
amino acid composition of a 30-kD protein by
these methods requires less than 1 hour and
only 6 mg (0.2 nmol) of the protein.
• results often yield ideas for fragmentation of the
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polypeptide chains (Step 5, 6)
Problems with Acid hydrolysis aa
composition quantification
• S and T degrade, but the data from
different time points can be extrapolated to
determine the composition (Fig. 5.12)
• Asn and Gln are converted to Asp and
Glu, respectively, because the amide
linkages are acid labile – results are
usually reported for Asx and Glx
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Step 4:
•
Identify N- and C-terminal residues
N-terminal analysis:
– Edman's reagent
– phenylisothiocyanate
– derivatives are phenylthiohydantions
– or PTH derivatives
Efficiency of reaction
cycles between 10-50
(an amino acid each
cycle) depending on
protein properties (e.g
molecular weight)
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Step 4:
Identify N- and C-terminal residues
• C-terminal analysis
– Enzymatic analysis (carboxypeptidase)
– Carboxypeptidase A cleaves any residue except
Pro, Arg, and Lys
– Carboxypeptidase B (hog pancreas) only works
on Arg and Lys
– Carboxypeptidase C and Y cleave any residue
– Exopeptidases cleave from the termini
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Steps 5 and 6:
Fragmentation of the chains
• Enzymatic fragmentation
–
–
–
–
Trypsin - cleavage on the C-terminal side of Lys, Arg
Chymotrypsin - C-terminal side of Phe, Tyr, Trp
Clostripain - like trypsin, but attacks Arg more than Lys
Staphylococcal protease
• C-terminal side of Glu, Asp in phosphate buffer
• specific for Glu in acetate or bicarbonate buffer
• Endopeptidases cleave within the protein sequence – some are
non-specific such as pepsin and papain
• Chemical fragmentation - cyanogen bromide (CNBr)
– acts only on methionine residues
– is useful because proteins usually have only a few Met residues
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Enzymatic Cleavage e.g. Trypsin
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Mechanism of CNBr
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Step 7:
Reconstructing the Sequence
• Use two or more fragmentation agents in
separate fragmentation experiments
• Sequence all the peptides produced
(usually by Edman degradation)
• Compare and align overlapping peptide
sequences to learn the sequence of the
original polypeptide chain
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Reconstructing the Sequence
Compare cleavage by trypsin and
staphylococcal protease on a hypothetical
peptide:
• Trypsin cleavage:
A-E-F-S-G-I-T-P-K
L-V-G-K
• Staphylococcal protease:
F-S-G-I-T-P-K
L-V-G-K-A-E
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Reconstructing the Sequence
• The correct overlap of fragments:
L-V-G-K A-E-F-S-G-I-T-P-K
L-V-G-K-A-E F-S-G-I-T-P-K
• Correct sequence:
L-V-G-K-A-E-F-S-G-I-T-P-K
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Sequence analysis of catrocollastatin-C, a 23.6 kD
protein from the venom of Crotalus atrox
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MCAT Question
•
Pepsin, trypsin, and chymotrypsin cleave
polypeptides into fragments at a specific
point in the middle of the chain. These
enzymes are properly characterized as:
A.
B.
C.
D.
endopeptidases
zymogens
ligases
exopeptidases
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Nature of Protein Sequences
• Sequences and
composition reflect
the function of the
protein
• Membrane proteins
have more
hydrophobic
residues, whereas
fibrous proteins may
have atypical
sequences
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Frequencies of amino acids in proteins
Legend: gray = aliphatic, red = acidic,
green = small hydroxy, blue = basic,
black = aromatic, white = amide, yellow = sulfur
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Number of aa differences among cytochrome c sequences
Homologous proteins from different organisms have similar sequences
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Phylogeny of Cytochrome c
•
The number of amino acid
differences between two
cytochrome c sequences
is proportional to the
phylogenetic difference
between the species from
which they are derived
• This observation can be
used to build phylogenetic
trees of proteins
• This is the basis for
studies of molecular
evolution
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The Role of the Sequence in
Protein Structure
All of the information necessary for folding
the peptide chain into its "native” structure
is contained in the primary amino acid
structure of the peptide.
Interactions between amino acids and
backbone atoms stabilize protein structure
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The Weak Forces
What are they?
What are the relevant numbers?
• van der Waals: 0.4 - 4 kJ/mol
• hydrogen bonds: 12-30 kJ/mol
• ionic bonds: 20 kJ/mol
• hydrophobic interactions: <40 kJ/mol
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How do proteins recognize and
interpret the folding information?
• Certain loci along the chain may act as
nucleation points
• Protein chain must avoid local energy
minima
• Chaperones may help
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Consequences of the Amide Plane
Two degrees of freedom per
residue for the peptide chain
• Angle about the C(alpha)-N
bond is denoted phi Φ
• Angle about the C(alpha)-C
bond is denoted psi Ψ
• The entire path of the
peptide backbone is known
if all phi and psi angles are
specified
• Some values of phi and psi
are more likely than others.
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Steric Constraints on phi & psi
Unfavorable orbital overlap precludes some
combinations of phi and psi
• phi = 0, psi = 180 is unfavorable
• phi = 180, psi = 0 is unfavorable
• phi = 0, psi = 0 is unfavorable
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Steric Constraints on phi & psi
• G. N. Ramachandran
was the first to
demonstrate the
convenience of plotting
phi,psi combinations
from known protein
structures
• The sterically favorable
combinations are the
basis for preferred
secondary structures
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Classes of Secondary Structure
•
•
•
•
•
All these are local structures that are
stabilized by hydrogen bonds
Alpha helix
Other helices
Beta sheet (composed of "beta strands")
Tight turns (aka beta turns or beta bends)
Beta bulge
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The Alpha Helix
• First proposed by Linus Pauling and Robert Corey in 1951
• Identified in keratin by Max Perutz
• A ubiquitous component of proteins
• Stabilized by H-bonds
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The Alpha Helix
• Residues per turn: 3.6
• Rise per residue: 1.5
Angstroms
• Rise per turn (pitch):
3.6 x 1.5A = 5.4
Angstroms
• phi = -60 degrees
• psi = -45 degrees
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The Beta-Pleated
Sheet
Composed of beta strands
•
•
•
Also first postulated by Pauling and Corey, 1951
Strands may be parallel or antiparallel
Rise per residue:
– 3.47 Angstroms for antiparallel strands
– 3.25 Angstroms for parallel strands
– Periodicity of two residues
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The Beta Turn
•
•
•
(aka beta bend, tight turn)
allows the peptide chain to reverse direction
carbonyl C of one residue is H-bonded to the amide proton of a
residue three residues away
proline and glycine are prevalent in beta turns
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The β-bulge
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Amino acids have secondary structure propensities
Chou – Fasman Helix and Sheet Propensities (Pα and Pβ) of the Amino Acids
Amino Acid
Pα
Pβ
A Ala
1.42
0.83
C Cys
0.70
1.19
D Asp
1.01
0.54
E Glu
1.51
0.37
F Phe
1.130
1.38
G Gly
0.57
0.75
H His
1.00
0.87
I Ile
1.08
1.60
K Lys
1.16
0.74
L Leu
1.21
1.30
M Met
1.45
1.05
N Asn
0.67
0.89
P Pro
0.57
0.55
Q Gln
1.11
1.10
R Arg
0.98
0.93
S Ser
0.77
0.75
T Thr
0.83
1.19
V Val
1.06
1.70
W Trp
1.08
1.37
Y Tyr
0.69
1.47
Source: Chou, P. Y., and Fasman, G. D., 1978. Annual Review of Biochemistry 47:258.
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