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Biochemistry
Sixth Edition
‫ביוכימיה היא מדע החוקר את התכונות הכימיות‬
‫המאפיינות מולקולות המיוצרות בתאים חיים‬
.‫ ומאפשרות להם לבצע את תפקידם‬,‫וייחודיות להם‬
Berg • Tymoczko • Stryer
Chapter 2
Protein Composition and Structure
Copyright © 2007 by W. H. Freeman and Company
Functions of some Proteins
Catalysis (enzymes)
Structural (collagen)
Contractile (muscle)
Transport (hemoglobin)
Storage
(myoglobin)
Electron transport (cytochromes)
Hormones (insulin)
Growth factor (EGF)
DNA binding (histones)
Ribosomal proteins
Toxins and venoms (cholera & melittin)
Vision (opsins)
Immunoglobins
Levels of Protein Structure
• Primary structure (1o) – sequence of amino acids
starting from the N-terminus of the peptide.
• Secondary structure (2o) – conformations of the
peptide chain from rotation about the a-Cs,
e.g. a-helices and b-sheets, etc.
• Tertiary structure (3o) – three dimensional shape
of the fully folded polypeptide chain.
• Quaternary structure (4o) - arrangement of two or
more protein chains into multisubunit molecule
Levels of Protein Structure
Bonding:
1o = covalent
2o = H-bond
3o = covalent &
noncovalent
4o = noncovalent
Amino Acids
The 20 common are those used in making
protein on a ribosome using mRNA and
tRNA.
These are called a-amino acids since each
has a carboxyl group and an amino group
attached to an a-carbon atom. They differ
by the sidechain or “R” group.
a
+NH -CH-COOH
3
l
R
Amino Acid Classification
Classification is made using the structure of the
side chain, R. (* = essential)
1. None (hydrogen):
2. Non-polar:
Aliphatic:
Alicyclic:
3. Aromatic:
4. Polar uncharged:
5. Thiol:
6. Acidic:
7. Basic:
Gly
Ala, Val*, Leu*, Ile*, Met*
Pro
Phe*, Tyr, Trp*
Ser, Thr*, Asn, Gln
Cys
Asp, Glu
Lys*, His*, Arg*
ball & stick
model
wedge
Fischer
projection
Amino Acid Names & Codes
All are Chiral at the a-carbon
atom except Gly
D, L Assignments
The convention for making D, L assignments is
to draw a Fischer projection with the
carboxyl at the top and the R at the bottom.
+NH
3
to the left = L
to the right = D
COO H
+
NH3
H
CH 2-OH
COO H
H
NH3+
CH 2-OH
L stereochemistry = S, for this case
Acids and Bases, pH, pKa
• pH is defined as the negative logarithm
of the concentration of H+
pH = - log [H+] = log 1/ [H+]
• pKa is defined as the negative logarithm
of an acid ionization constant Ka.
pKa = - log [Ka] = log 1/ [Ka]
Acetic Acid is a Weak Acid
• Weak acids and bases do not ionize
(dissociate) completely in H2O.
CH3COOH <==> CH3COO- + H+
acetic acid
acetate anion
conjugate acid
form
conjugate base
form
Acid Ionization Constant
[CH3COO-][H+]
Ka = -----------------[CH3COOH]
[CH3COO-]
-log Ka = -log [H+] + -log ---------------[CH3COOH]
[CH3COO-]
pH = pKa + log ---------------[CH3COOH]
Henderson-Hasselbalch
Written in a more general form this expression
is called the Henderson-Hassebalch equation.
This relates the pH of a solution to the pKa of
the weak acid in solution and the ratio of its
conjugate base/conjugate acid forms.
[A-]
pH = pKa + log -----[HA]
Buffers Resist Change in pH
• Buffer capacity is the ability of a solution to resist
changes in pH.
• Most effective buffering occurs where:
solution pH = buffer pKa
• At this point: [conjugate acid] = [conjugate base]
• Effective buffering range is usually at pH values
equal to the pKa ± 1 pH unit
Amino Acid Ionization, pKas
Each of the 20 common a-amino acids has two
pKa values, for the carboxyl group and the
amino group attached to the a-carbon.
+NH
+NH -CH -COO- + H+
-CH
-COOH
<
===
>
3
2
3
2
+NH
- < === > NH -CH -COO- + H+
-CH
-COO
3
2
2
2
Seven of the 20 have an ionizable sidechain
and therefore have a third pKa value.
Amino Acid pKas
Gly
Ala
Val
Leu
Ile
Met
Pro
Phe
Trp
Ser
Thr
Asn
Gln
Cys
Asp
Glu
Tyr
Lys
His
Arg
2.18
a-COOH
2.34
2.34
2.32
2.36
2.36
2.28
1.99
1.83
2.83
2.21
2.63
2.02
2.17
1.71
2.09
2.19
2.20
8.95
1.82
2.17
a-+NH3
9.60
9.69
9.62
9.60
9.60
9.21
10.60
9.13
9.39
9.15
10.43
8.80
9.13
10.78
9.82
9.67
9.11
R (sidechain)
8.33
3.86
4.25
10.07
10.79
9.17
9.04
6.00
12.48
Peptide
3.86 D, 4.25 E
Peptide
10.0
Effect of Change in pH
The Structure of an Amino Acid
An amino acid can never exist as an uncharged compound.
© 2014 Pearson Education, Inc.
The pI of Alanine
The isoelectric point (pI) of an amino acid is the
pH at which it has no net charge.
© 2014 Pearson Education, Inc.
The pI of Lysine
© 2014 Pearson Education, Inc.
The pI of Glutamic Acid
© 2014 Pearson Education, Inc.
Formation of a Peptide
Two amino acids are joined together to form a
peptide (amide) bond with a loss of HOH.
After becoming part of a peptide or protein these
are called “residues” due to loss of HOH.
Primary Structure (1o)
The sequence of amino acids (N-term to C-term)
in a peptide or protein is its primary structure.
A pentapeptide
Disulfide Bond Formation
Disulfide Bridges
Disulfide bridges contribute to the overall shape of the protein.
© 2014 Pearson Education, Inc.
Straight and Curly Hair
© 2014 Pearson Education, Inc.
Insulin
Insulin has
•two interchain disulfide bridges and
•one intrachain disulfide bridge.
© 2014 Pearson Education, Inc.
Peptide Bond Resonance
Due to resonance participation of the unshared
pair of electrons on N, amides are neutral.
..
Peptide Bond Planarity
6 atoms are coplanar
Peptide Bond Structures
Less crowded in the favored trans arrangement
Rotation Sites, y and f
The rotational arrangements about a-carbons of a
peptide or protein gives its secondary (2o) structure.
f
View from
N-term
to a-carbon
y
View from
a-carbon to
C-term
Ramachandran Plot
Secondary Structure (2o)
The two most common secondary structures
are the a-helix and the b-sheet.
Each of these 2o structures have fairly specific
y and f angles.
All other rotational angles represent “random”
secondary structure.
Secondary structure is maintained by
hydrogen bonding.
a-helix by intramolecular H-bonds
b-sheet by intermolecular H-bonds
a-helix in a Ramachandran Plot
y = -47
f = -57
The a-helix, a 3.613 helix
The a-helix
Hydrogen Bond Contacts
a-helix in a Protein
b-sheet in a Ramachandran Plot
y = +135
f = -139
A b-sheet strand
Anti-parallel b-sheet
Parallel b-sheet
Anti-parallel b-sheet
b-sheet in a
Protein
b-turn or hairpin turn
A turn is 4-5
aa residues.
A b-turn
(hairpin) is
4 aa residues.
Loops are
usually larger
than turns.
>5 aa residues
a-Keratin, a fibrous protein,
forms an a coiled coil
Each strand is a modified a-helix, 3.5 residues/turn.
Right-handed helices form a left-handed supercoil.
Collagen, a triple helix
Gly every third residue; Gly-Pro-HPro is frequent.
Interstrand H-bond at Gly.
No Cys, so, no -S-S-
Tertiary Structure (3o)
The three dimensional folding of a polypeptide
is its tertiary structure.
Both the a-helix and b-sheet may exist within
the tertiary structure.
Generally the distribution of amino acid
sidechains in a globular protein finds mostly
nonpolar residues in the interior of the
protein and polar residues on the surface.
Tertiary structure is maintained by noncovalent
interactions and disulfide bonding.
Myoglobin, a globular protein
Myoglobin
Surface
Blue = charged
Cross-section
Yellow = hydrophobic
Porin, a membrane spanning
protein
Domain
A domain is a discrete globular area within protein.
There are four general types:
All a
All b
Mixed a/b
a+b
only a- helices and loops
only b- sheet & loops or turns
alternating abab
cluster of a then cluster of b
Domain
Multiple domains exist in the protein below.
Quaternary Structure (4o)
is an assembly of 3o structures
(two or more subunits).
A dimer
Hemoglobin, a tetramer
Quaternary structure is maintained by
noncovalent interactions
Denaturing Proteins
Denaturing agents destroy the protein 3o structure
(causes the protein to unravel).
Methods:
Heat;
Extremes of pH;
Detergents;
Mechanical agitation;
Mercaptoethanol – breaks -S-S- bonds;
6M guanidine HCl or 10M urea:
these are chaotropic agents that break
up noncovalent interactions.
Denaturing agents
Disulfide oxidation-reduction
Oxidized
Reduced
Ribonuclease
4 disulfide
bonds.
-S-S-
Then removing urea and
ME permits reoxidation.
Reoxidation reforms
–S-S- but not
necessarily in the
correct place.
A trace of ME allows
reduction/oxidation
to occur until the low
free energy form is
found and >98% of
activity is restored.
Sharp Transition
suggests an all or nothing effect in denaturing.
Lysozyme
Ribbon diagrams
3o structure
Active site & -S-S-
Protein Cleavage
Protein sequencing is most manageable with
small polypeptides.
Therefore, in order to sequence a large protein,
it must be cleaved into smaller pieces.
Cleavage is conducted using either chemical or
enzymatic methods.
The pieces must be separated and purified
before sequencing.
Chemical and Enzymatic Cleavage
CNBr Cleavage at Met
Enzymatic cleavage by Trypsin
‫שאלה‪ :‬לפניך תוצרי הביקוע של פפטיד באמצעות טריפסין וכימוטריפסין‪:‬‬
‫כימוטריפסין‪:‬‬
‫‪Val-Ser-Arg-Gly-Trp‬‬
‫‪Met-Asn-Lys-Phe‬‬
‫טריפסין‪:‬‬
‫‪Phe-Val-Ser-Arg‬‬
‫‪Met-Asn-Lys‬‬
‫‪Gly-Trp‬‬
‫הערות‪ :‬טריפסין חותך בצד הקרבוקסילי של ‪Lys, Arg :‬‬
‫וכימוטריפסין חותך בצד הקרבוקסילי של ‪. Phe,Trp,Tyr :‬‬
‫מהו רצף החומצות האמיניות בפפטיד‪.‬‬
‫תשובה‪:‬‬
‫‪Met-Asn-Lys-Phe-Val-Ser-Arg-Gly-Trp‬‬
‫‪82‬‬
An Example, Peptide overlap