AMINO ACIDS & PROTEINS

INTRODUCTORY BIOCHEMISTRY
 AMINO ACIDS
General Structure
• Proteins are the most abundant organic molecules in cells, constituting 50% or more
of their dry weight
• There are many different kinds of proteins, each specialized for a different biological
function. And most of the genetic information is expressed by proteins
• Proteins are made up of 20 common α-amino acids

2
 AMINO ACIDS
General Structure
• Under normal cellular conditions amino acids are zwitterions (dipolar ions):
vAmino group = -NH3+
vCarboxyl group = -COO-

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 AMINO ACIDS
General Structure
• 19 of the 20 common amino acids have a chiral α-carbon atom (Gly does not)
vChiral carbons - have four different groups attached

Glycine Alanine

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 AMINO ACIDS
General Structure
• Threonine and isoleucine have 2 chiral carbons each (4 possible stereoisomers each)
v Stereoisomers - compounds that have the same molecular formula but differ in
the arrangement of atoms in space

Threonine Isoleucine
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 AMINO ACIDS
General Structure
• Mirror image pairs of amino acids are designated L (levo) and D (dextro) and are called
enantiomers
• Proteins are assembled from L-amino acids (a few D-amino acids occur in nature)

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AMINO ACIDS
Abbreviations

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 CLASSIFCATION OF AMINO ACIDS
Based on the Polarity of R Groups

① Nonpolar (hydrophobic) R groups Ala, Val, Leu, Ile, Pro, Trp, Phe, Met

② Uncharged Polar R groups Gly, Ser, Thr, Cys, Tyr, Asn, Gln

③ Charged Polar R groups Acidic: Asp, Glu

Basic: Lys, Arg, His

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 CLASSIFICATION OF AMINO ACIDS
Nonpolar R Groups
Alanine [A] Valine [V] Leucine [L] Isoleucine [I] Proline [P]
(Ala) (Val) (Leu) (Ile) (Pro)

Tryptophan [W]
(Trp) Phenylalanine [F] Methionine [M]
(Phe) (Met)

• Ala is the least

hydrophobic
member of this class

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 CLASSIFICATION OF AMINO ACIDS
Uncharged Polar R Groups
Glycine [G] Serine [S] Threonine [T] Tyrosine [Y] Cysteine [C]
(Gly) (Ser) (Thr) (Tyr) (Cys)

Asparagine [N] Glutamine [Q]

(Asn) (Gln)

• The thiol of Cys and phenolic –OH group of Tyr

makes them the most polar in this group. They tend
to lose protons by ionization more readily that the R
groups of the remaining 5 aa
• Cysteine can be oxidized to form cystine

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CLASSIFICATION OF AMINO ACIDS
Acidic Amino Acids

Aspartic Acid [D] Glutamic Acid [E]

(Asp) (Glu)

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 CLASSIFICATION OF AMINO ACIDS
Basic Amino Acids

Lysine [K] Arginine [R] Histidine [H]

(Lys) (Arg) (His)

+
H

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 CLASSIFICATION OF AMINO ACIDS
Hydrophobicity of Amino Acid Side Chains
• Hydropathy: the relative hydrophobicity of each
amino acid
• The larger the hydropathy, the greater the
tendency of an amino acid to prefer a hydrophobic
environment
• Hydropathy affects protein folding:
– hydrophobic side chains tend to be in the interior
– hydrophilic residues tend to be on the surface
• The free-energy change is for transfer of an amino
acid from the interior of a lipid bilayer to water

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 CLASSIFICATION OF AMINO ACIDS
Based on the Chemical Composition of R Groups
① Aliphatic Gly, Ala, Val, Leu, Ile

② Imino Pro

③ Alcohol Ser, Thr

④ Sulfur-containing Cys, Met

⑤ Aromatic Phe, Tyr, Trp

⑥ Acidic Asp, Glu, Asn, Gln

⑦ Basic Lys, Arg, His

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 CLASSIFICATION OF AMINO ACIDS
Nonpolar, Aliphatic R Groups
Glycine [G] Alanine [A] Valine [V]
• R groups are hydrophobic (Gly) (Ala) (Val)

• Gly is the smallest and has little

hydrophobic character

• Ala, Val, Leu and Ile have

saturated side chains and tend to Leucine [L] Isoleucine [I]
cluster together within proteins (Leu) (Ile)

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 CLASSIFICATION OF AMINO ACIDS
Imino R Groups

• Pro has an aliphatic side chain and secondary amino Proline [P]
(Pro)
(imino) group.

• The heterocyclic pyrrolidine ring of Pro restricts the

geometry of polypeptides

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 CLASSIFICATION OF AMINO ACIDS
Side Chains with Alcohol Groups

• Ser and Thr have uncharged polar side chains

Serine [S] Threonine [T]

(Ser) (Thr)

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 CLASSIFICATION OF AMINO ACIDS
Sulfur-Containing R Groups

• Cys (-CH2SH) Cysteine [C] Methionine [M]

(Cys) (Met)
• Met (-CH2CH2SCH3)
• Two cysteine side chains can be
cross-linked by forming a disulfide
bridge (-CH2-S-S-CH2-)
• Disulfide bridges may stabilize the
three-dimensional structures of
proteins

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 CLASSIFICATION OF AMINO ACIDS
Sulfur-Containing R Groups
Formation of cystine

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 CLASSIFICATION OF AMINO ACIDS
Aromatic R Groups

• Relatively nonpolar (hydrophobic) Phenylalanine [F] Tyrosine [Y] Tryptophan [W]

(Phe) (Tyr) (Trp)
• Participate in hydrophobic
interactions
• The –OH group of Tyr can form
hydrogen bonds and is an
important functional group in
some enzymes
• Absorb UV light
• Trp and Tyr absorb at 280nm (whereas Phe absorbs at 260nm)
• This accounts for the characteristic strong absorbance of light by most proteins at a
wavelength of 280nm, a property used to estimate the concentration of proteins in
solution 20
 CLASSIFICATION OF AMINO ACIDS
Acidic R Groups
Aspartate [D] Asparagine [N]
(Asp) (Asn)
• Negatively charged at pH6.0
• Asp and Glu are dicarboxylic acids, and
are negatively charged at pH 7
• Asn and Gln are uncharged but highly
polar
Glutamate [E] Glutamine [Q]
(Glu) (Gln)

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 CLASSIFICATION OF AMINO ACIDS
Acidic R Groups
Amide Derivatives
• Asp and Glu form amide linkages to form Asn and Gln

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 CLASSIFICATION OF AMINO ACIDS
Basic R Groups
Lysine [K] Arginine [R] Histidine [H]
• Positively charged at pH6.0 (Lys) (Arg) (His)

• Lys: alkylamino group

• Arg: guanidino group
• His: imidazole group
+
H

• Side chains are nitrogenous bases which are substantially positively charged at pH 7
• His residues facilitate many enzyme-catalyzed reactions by serving as proton
donors/ acceptors
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 CLASSIFICATION OF AMINO ACIDS
Based on Human Nutrition
• Amino acids that cannot be synthesized by our bodies are essential
Nonessential Essential
Alanine Arginine*
Asparagine Histidine
Aspartate Isoleucine
Cysteine Leucine
Glutamate Lysine
Glutamine Methionine
Glycine Phenylalanine
Proline Threonine
Serine Tryptophan
Tyrosine Valine
*Essential in young, growing animals but not in adults 24
 CLASSIFICATION OF AMINO ACIDS
Based on Human Nutrition
• Humans take essential amino acids from their diet (plants) or from bacteria living in
their intestines
• Bacteria such as E. coli can synthesize the entire common 20 amino acids, whereas
humans can make half of them
• Essential amino acids should be of particular concern to:
– Pregnant women
– Adults caring for children
– Vegetarians
– Weight watchers

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 OTHER AMINO ACIDS AND DERIVATIVES
Rare Amino Acids of Proteins

4-Hydroxyproline
• Found in plant cell wall proteins (extensins)
• Found in the fibrous protein collagen

5-Hydroxylysine
• Found in collagen

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 OTHER AMINO ACIDS AND DERIVATIVES
Non-protein Amino Acids
• Over 200 different amino acids are found in nature

β-alanine CH2 – CH2 – COOH

• Component of pantothenic acid (vitB5)
NH2

Homocysteine
• Intermediates in aa metabolism HS – CH2 – CH2 – CH – COOH
NH2

Homoserine
• Intermediates in aa metabolism HO – CH2 – CH2 – CH – COOH
NH2
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 OTHER AMINO ACIDS AND DERIVATIVES
Non-protein Amino Acids

Ornithine
• Key intermediate in the biosynthesis of H2N – CH2 – CH2 – CH2 – CH – COOH
arginine and in the urea cycle NH2

Citrulline
• Key intermediate in the biosynthesis of H2N – C – N – CH2 – CH2 – CH2 – CH – COOH
arginine and in the urea cycle O H NH2

Canavanine H2N – C – NH – O – CH2 – CH2 – CH – COOH

• Found in fungi and higher plants
NH NH2
• Toxic to certain other forms of life

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 OTHER AMINO ACIDS AND DERIVATIVES
Additional Common Amino Acids
• N-formylmethionine, selenocysteine, and pyrrolysine are incorporated at specific codons
à additions to the standard repertoire of protein precursors

N-formylmethionine
• 21st amino acid residue
• Initial amino acid during protein synthesis
in bacteria

Selenocysteine
• 22nd amino acid (rare)
• Contains selenium rather than the sulfur of
cysteine
• Incorporated into a few proteins
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 OTHER AMINO ACIDS AND DERIVATIVES
Additional Common Amino Acids

Pyrrolysine
• 23rd amino acid
• Found in some species of archaebacteria

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 OTHER AMINO ACIDS AND DERIVATIVES
Compounds Derived from Common Amino Acids

γ-Aminobutyrate (GABA)
• Derived from glutamate
• Important neurotransmitter in mammalian
brains

Histamine
• Derived from histidine
• Controls constriction of blood vessel
and secretion of HCl by the stomach
• Involved in allergic reactions

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 OTHER AMINO ACIDS AND DERIVATIVES
Compounds Derived from Common Amino Acids

Epinephrine (adrenaline)
Norepinephrine (noradrenaline)
• Hormones that help regulate ( )
metabolism in mammals

Thyroxine / Triiodothyronine
• Derived from tyrosine
• Regulate metabolism

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 AMINO ACIDS
Ionization of Amino Acids
• Ionizable groups in amino acids function as weak acids and bases:
α-carboxyl
α-amino
some side chains

• An amino acid lacking an ionizable side chain

acts as a dipolar ion at neutral pH (zwitterion)
• Amphoteric electrolytes or ampholytes are
substances with dual (acid-base) nature

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 AMINO ACIDS
Ionization of Amino Acids
• Each ionizable group has a specific pKa
AH B + H+
• For a solution pH below the pKa, the
protonated form predominates (AH)
• For a solution pH above the pKa, the
unprotonated form predominates (B)

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 AMINO ACIDS
Ionization of alanine
Titration curve for alanine
• Titration curves are used to determine
pKa values
pK1 = 2.4
pK2 = 9.9
pIAla = isoelectric point

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 AMINO ACIDS
Ionization of histidine
Titration curve of histidine
pK1 = 1.8
pK2 = 6.0
pK3 = 9.3

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 AMINO ACIDS
Ionization of histidine
Deprotonation of imidazolium ring

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 PEPTIDES
Peptide Bonds
• Peptide bond - linkage between amino acids is a secondary amide bond
• Formed by condensation of the α-carboxyl of one amino acid with the α-amino of
another amino acid (loss of H2O molecule)

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 PEPTIDES
Polypeptide chain nomenclature
• Amino acid residues compose peptide chains:
àFormation of peptide bonds eliminates the ionizable α-carboxyl and α-amino
groups of the free amino acids
• Peptide chains consist of a backbone of repeating units that differ in the R groups
• Peptide chains are numbered from the N (amino) terminus to the C (carboxyl)
terminus

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 PEPTIDES
Polypeptide chain nomenclature
A pentapeptide:
à Serylglycyltyrosylalanylleucine
à Ser-Gly-Tyr-Ala-Leu
à SGYAL

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 PEPTIDES
Important Biological Compounds
• Insulin (regulates glucose metabolism)
• Endorphins (naturally occurring molecules that modulate pain in vertebrates)
• Useful food additives (Aspartame; artificial
sweetener)
- commercially synthesized dipeptide methyl
ester - aspartylphenylalanine methyl ester
- about 200 times sweeter than table sugar)
- Used in diet drinks

Aspartame
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 PEPTIDES
Peptides of Non-protein Origins
• Glutathione (γ-glutamylcysteinylglycine)

O O
HOOC – CH – CH2 – CH2 – C – NH – CH – C – NH – CH2 – COOH
NH2 CH2
SH

• Antibiotics: penicillin, puromycin

• Hormones: oxytocin, vasopressin

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 PROTEINS
Three-Dimensional Structure and Function

• Proteomics - study of large sets of proteins, such as the entire complement of

proteins produced by a cell

• E. coli has about 4000 different polypeptides (average size 300 amino acids, Mr
33,000)

• Fruit fly (Drosophila melanogaster) about 16,000, humans, other mammals about
40,000 different polypeptides

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 PROTEINS
Three-Dimensional Structure and Function
• Proteins from E. coli cells are separated by Escherichia coli proteins.
two-dimensional gel electrophoresis

• In the first dimension, the proteins are

separated by a pH gradient where each
protein migrates to its isoelectric point

• The second dimension separates proteins

by size on an SDS–polyacrylamide gel. Each
spot corresponds to a single polypeptide

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 PROTEINS
Three-Dimensional Structure and Function

• Conformation - three dimensional shape

• Native conformation - each protein folds into a single stable shape (physiological
conditions)

• Biological function of a protein depends completely on its native conformation

• A protein may be a single polypeptide chain or composed of several chains

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 PROTEINS
Levels of Protein Structure
• Primary structure - linear sequence of amino acids in a polypeptide or protein
àdetermined by the number, kind and sequence of amino acids
àDevoid of function or any biological activity
• Secondary structure - regions of regularly repeating conformations of the peptide
chain, such as α-helices and β-sheets
• Tertiary structure - describes the shape of the fully folded polypeptide chain
• Quaternary structure - arrangement of two or more polypeptide chains into
multisubunit molecule

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 PROTEINS
Levels of Protein Structure

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 PROTEINS
Methods for Determining Protein Structure
• X-ray crystallography is used to determine the three-dimensional conformation of
proteins

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 PROTEINS
Methods for Determining Protein Structure
Bovine (Bos taurus) ribonuclease A
• Ribonuclease A is a secreted enzyme that hydrolyzes RNA during digestion

Space-filling model Cartoon ribbon model Substrate-binding

(bound substrate analog black) (shows secondary structure) site view

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 PROTEINS
Methods for Determining Protein Structure
• NMR (nuclear magnetic resonance) is used to analyze protein structure in solution
• The basis of NMR is the observation that an atomic nucleus such as a proton (a
hydrogen nucleus) resonates in an applied magnetic field in a way that is sensitive to
its electronic environment and its interactions with nearby nuclei

Ribonuclease A determined by NMR

(polypeptide chain backbone)

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 PROTEINS
Structural Bioinformatics
• Bioinformatics deals with information related to molecular sequences and structures
• Structural bioinformatics is a branch of bioinformatics concerned with how
macromolecular structures are displayed and compared (see tools on next slide)
• The atomic coordinates of nearly 50,000 macromolecular structures, including
proteins, nucleic acids and carbohydrates are archived in the Protein Data Bank
(PDB)

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 PROTEINS
Structural Bioinformatics
Structural Bioinformatics Internet Addresses (1/2)
Structural Databases
Protein Data Bank (PDB): http://www.rcsb.org/pdb/
Nucleic Acid Database: http://ndbserver.rutgers.edu
Molecular Modeling Database (MMDB): http://www.ncbi.nlm.nih.gov/Structure/index.shtml
Most Representative NMR Structure in an Ensemble: http://pqss.ebi.ac.uk/pqs-nmr.html
PQS Protein Quaternary Structure Query Form at the EBI: http://pqs.ebi.ac.uk
Molecular Genetics Programs
Cn3D: http://www.ncbi.nhlm.nih.gov/Structure/CN3D/cn3d.shtml
Jmol: http://jmol.sourceforge.net/
KiNG: http://kinemage.biochem.duke.edu
FirstGlance: http://molvis.sdsc.edu/fgij/index.htm
Swiss-PDB Viewer (Deep View): http://us.expasy.org/spdbv

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 PROTEINS
Structural Bioinformatics
Structural Bioinformatics Internet Addresses (2/2)
Structural Classification Algorithms
CATH (Class, Architecture, Topology, and Homologous superfamily): http://www.cathdb.info/latest/index/html

CE (Combinatorial Extension of optimal pathways): http://cl/sdsc.edu

FSSP (Family of Structurally Similar Proteins): http://scop.mrc-lmb.cam.ac.uk/scop
VAST (Vector Alignment Search Tool): http://www.ncbi.nlm.nih.gov/Structure/VAST/vast.shtml

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 PROTEINS
The Conformation of the Peptide Group
• The peptide group consists of 6 atoms
• Peptide bonds have some double bond properties
so that their conformation is restricted to either
Trans
trans or cis
• Nearly all peptide groups in proteins are in the
trans conformation
• Cis conformation is less favorable than trans due
to steric interference of α-carbon side chains
• Cis-trans isomerases can catalyze the
interconversion of cis and trans conformations

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 PROTEINS
The Conformation of the Peptide Group
Resonance structure of the peptide bond

Peptide bond shown Peptide bond shown Actual structure is a

as a C-N single bond as a double bond hybrid of the two
resonance forms.
Electrons are delocalized
over three atoms: O, C, N

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 PROTEINS
The Conformation of the Peptide Group
• Rotation around the C—N bond is restricted due to the double-bond nature of the
resonance hybrid form
• Peptide groups (blue planes) are therefore planar

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 PROTEINS
The Conformation of the Peptide Group
• The backbone of a protein (main chain) can be drawn as a linked sequence of rigid
planar peptide groups (repeating N—Cα—C backbone)

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 PROTEINS
Rotation of Atoms in a Peptide Group
• The rotation of the backbone can be (a) Peptide groups in an extended
conformation
described by the rotation angles around the
Cα—N bond (f) and the Cα—C bond (Y) of
each residue
• Rotation is restricted by steric interference
between main-chain and side-chain atoms
(b) Peptide groups in an unstable
conformation
• Rotation of the N—Cα bond in proline is
restricted because of the pyrrolidine ring
structure

58
 PROTEINS
Ramachandran plots
• The Ramachandran plot indicates allowed conformations of polypeptides
• Conformation of a polypeptide chain can be solely described by f and Y angles
• Ramachandran plots of f and Y show permissible angles for polypeptide chains
• Some f and Y angles are not allowed because of steric hindrance (f and Y values
that would bring atoms closer than the corresponding van der Waals distance - the
distance of closest contact between non-bonded atoms)

59
 PROTEINS
Ramachandran plots
• Conformations of several types of secondary structures fall within permissible areas

Sterically
allowed f and Y
angles for all aa Orange circles
except Gly and represent
Pro conformational angles
of secondary structures
(α) α-helix
(αL) left handed α-helix
(⥣) parallel β- sheet
More crowded (⥮) antiparallel β sheet
(outer limit) f (C) Collagen helix
and Y angles

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 PROTEIN SECONDARY STRUCTURE
The α Helix
• The simplest arrangement the polypeptide chain can C-terminus
assume is a helical structure
• The α helix forms more readily than any other
conformation
• Helix is stabilized by many hydrogen bonds (which are
nearly parallel to long axis of the helix)
• Each C=O (residue n) forms a hydrogen bond with the
amide hydrogen of residue n+4
• All C=O groups point toward the C-terminus (entire
helix is a dipole with (+) N, (-) C-termini)
N-terminus

61
PROTEIN SECONDARY STRUCTURE
The α Helix
C-terminus The φ and ψ angles of each
residue are similar:
near -57 (φ) and near -47 (ψ)

3.6 amino acid

residues per turn

N-terminus 62
 PROTEIN SECONDARY STRUCTURE
The α Helix
• The length of a helix in a protein can range from 4 or 5 residues to more than 40 – the
average is about 12
• The helix is amphipathic – hydrophilic amino acids on the face of the helix cylinder
and hydrophobic residues on the opposite face
α helix in horse liver alcohol dehydrogenase

• Highly hydrophobic residues

are blue
• Less hydrophobic residues are
green
Helical wheel diagram
• Highly hydrophilic residues
are red

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 PROTEIN SECONDARY STRUCTURE
The α Helix

α helix in horse liver alcohol dehydrogenase

• Amphipathic α helix (blue ribbon)
• Hydrophobic residues (blue) directed inward,
hydrophilic (red) outward

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 PROTEIN SECONDARY STRUCTURE
The α Helix

Leucine zipper of yeast

• A common structure in DNA-binding proteins
• The DNA-binding region consists of two
amphipathic helices, one from each of the
two subunits of the protein.
• The side chains of leucine residues are shown
in a darker blue than the ribbon. Only the
leucine zipper region of the protein is shown

65
 PROTEIN SECONDARY STRUCTURE
β Strands and β Sheets

• β Strands - polypeptide chains that are almost fully extended (0.32 – 0.34 Å)
• β Sheets - multiple β strands arranged side-by-side
• Each strand has an average of 6 amino acid residues and each sheet is made of 2 – 15
strands
• β Strands are stabilized by hydrogen bonds between C=O and -NH on adjacent
strands
• The adjacent polypeptide chains in a β sheet can be either parallel or antiparallel
(more stable)

66
 PROTEIN SECONDARY STRUCTURE
β Strands and β Sheets
Parallel β sheet Antiparallel β sheet

Arrows indicate the N- to C-terminal direction of the peptide chain 67

 PROTEIN SECONDARY STRUCTURE
β Strands and β Sheets

View of two strands of an antiparallel b sheet from

influenza virus A neuraminidase
• Only the side chains of the front b strand are shown
• The side chains alternate from one side of the b
strand to the other side
• β strands twist in a right-handed direction

68
 PROTEIN SECONDARY STRUCTURE
Interactions of β Sheets

• β Sheet side chains project alternately above and below the plane of the β strands
• One surface of a β sheet may consist of hydrophobic side chains that can interact
with other hydrophobic residues in protein interior
• Amphipathic α helices have hydrophobic side chains projecting outward that can
interact with hydrophobic faces of β sheets or other helices

69
 PROTEIN SECONDARY STRUCTURE
Interactions of β Sheets
Structure of PHL P2 from Timothy grass (Phleum pratense) pollen
Antiparallel β sheets within a protein Stereo view of the β sandwich

• Polar residues (red) and hydrophobic residues (blue)

• A number of hydrophobic interactions connect the two β sheets
70
 PROTEIN SECONDARY STRUCTURE
Loops and Turns
• Loops and turns connect α helices and β strands and allow a peptide chain to fold
back on itself to make a compact structure. They cause directional change in the
polypeptide
• Loops - often contain hydrophilic residues and are found on protein surfaces
• Turns - loops containing 5 residues or less
• β Turns (reverse turns) - connect different antiparallel β strands

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 PROTEIN SECONDARY STRUCTURE
Loops and Turns
Type I β Turn Type II β Turn

Stabilized by a hydrogen bond between the Stabilized by a hydrogen bond between

carbonyl oxygen of the first N-terminal the carbonyl oxygen of the first N-
residue (Phe) and the amide hydrogen of terminal residue (Val) and the amide
the fourth residue hydrogen of the fourth residue (Asn)

72
 PROTEIN TERTIARY STRUCTURE

• Tertiary structure results from the folding of a polypeptide chain into a closely-
packed three-dimensional structure
• Amino acids far apart in the primary structure may be brought together
• Stabilized primarily by noncovalent interactions (e.g. hydrophobic effects, van der
Waals and charge-charge interactions, and hydrogen bonding) between side chains
• Disulfide bridges also part of tertiary structure

73
 NONCOVALENT INTERACTIONS

THE HYDROPHOBIC EFFECT

• Nonpolar side chains associate with each other causing a polypeptide chain to
collapse to a molten globule
• The driving force for protein folding is the large increase in entropy from water
released to bulk solvent
• Nonpolar side chains are in the interior
• Polar and charged side chains remain on the surface facing water
• Hydrophobic collapse of a polypeptide occurs at the same time as formation of
secondary structures

74
 NONCOVALENT INTERACTIONS

HYDROGEN BONDING Examples of hydrogen bonds in proteins

• Contributes to cooperativity of folding

• Helps stabilize tertiary structures and native
conformation

75
 NONCOVALENT INTERACTIONS

VAN DER WAALS AND CHARGE-CHARGE INTERACTIONS

• VDW contacts occur between nonpolar side chains and contribute to the stability of
proteins
• Charge-charge interactions between oppositely charged side chains in the interior of a
protein also may stabilize protein structure

76
 PROTEIN TERTIARY STRUCTURE
Supersecondary Structures
• Certain combinations of secondary structures form motifs
• Grouping of secondary structural elements, called supersecondary structures (motifs), occur
in many unrelated globular proteins

Helix-loop-helix • two helices connected by a turn

Coiled-coil • two amphipathic a helices that interact in

parallel through their hydrophobic edges

Helix bundle • several α helices that associate in an

antiparallel manner to form a bundle

77
 PROTEIN TERTIARY STRUCTURE
Supersecondary Structures
βαβ unit • two parallel β strands linked to an
intervening a helix by two loops
• most common form of motifs

Hairpin • Two adjacent antiparallel β strands

connected by a β turn

β meander • An antiparallel sheet composed of

sequential β strands connected by loops or
turns

Greek key • 4 antiparallel strands (strands 1,2 in the

middle, 3 and 4 on the outer edges)

β sandwich • Stacked β strands or sheets

78
 PROTEIN TERTIARY STRUCTURE
Domains
• Independently folded, compact units in proteins (~1400 different protein domains)
• Domain size: ~25 to ~300 amino acid residues
• Each domain is a distinct compact unit consisting of various elements of secondary
structure
• Domains are connected to each other by loops, bound by weak interactions between
side chains

79
 PROTEIN TERTIARY STRUCTURE
Domains
• A single domain may have a particular function (e.g. binding small molecules,
catalyzing a single reaction)
• Interfaces between two separate domains provide crevices, grooves, and pockets on
the surface of a protein for binding or catalytic sites
• In multifunctional enzymes, each catalytic activity can be on one of several domains

80
 PROTEIN TERTIARY STRUCTURE
Domains
Four categories of protein domains

All α • Domains consist almost entirely of α helices and loops

All β • All domains contain only β sheets and non-repetitive

structures that link the β strands

Mixed α/β • Contain supersecondary structures such as the αβα

motif, where regions of α helix and β strand alternate

α+β • Domains consist of local clusters of α helices and β sheet

in separate, contiguous regions of the polypeptide chain
81
 PROTEIN TERTIARY STRUCTURE
Domains
• Domains illustrate the evolutionary conservation of protein structure. It is the
fundamental unit of protein evolution
• Essential structural and functional elements of proteins, rather than their amino acid
residues are conserved during evolution

Conservation of cyt c structure

(a) Tuna (+heme)
(b) Tuna
(c) Rice
(d) Yeast
(e) Bacteria

82
 PROTEIN TERTIARY STRUCTURE
Domains
• Within each of the four main structural categories, domains can be classified by
characteristic folds
• A fold is a combination of secondary structures that form the core of a domain
• Some domains have simple folds, others have more complex folds (~200 different
folding patterns)

Parallel twisted
sheet β barrel α/β barrel β helix

83
PROTEIN TERTIARY STRUCTURE
Examples

84
 PROTEIN QUATERNARY STRUCTURE

• Refers to the organization of subunits in a protein with multiple subunits (an

oligomer )
• Subunits (may be identical or different) have a defined stoichiometry and
arrangement
• Subunits are held together by many weak, noncovalent interactions (hydrophobic,
electrostatic)
• The variety of multisubunit proteins ranges from simple homodimers (triose
phosphate isomerase) to large complexes (photosystems in plants and bacteria)

85
 PROTEIN QUATERNARY STRUCTURE

• The fact that a large proportion of proteins consist of multiple subunits is probably
related to several factors:
Oligomers are more stable
Active sites are formed by residues from adjacent polypeptide chains
Binding of ligands changes the 3D structure of many oligomeric proteins –
important in regulation of biological activity
Different proteins can share the same subunits – evolution
A multisubunit protein may bring together two sequential enzymatic steps

86
 PROTEIN QUATERNARY STRUCTURE
Natural occurrence of oligomeric proteins in Escherichia coli
Oligomeric Number of Number of
state homooligomers heterooligomers Percent
Monomer 72 19.4
Dimer 115 27 38.2
Trimer 15 5 5.4
Tetramer 62 16 21.0
Pentamer 1 1 0.1
Hexamer 20 1 5.6
Heptamer 1 1 0.1
Octamer 3 6 2.4
Nonamer 0 0 0.0
Decamer 1 0 0.0
Undecamer 0 1 0.0
Dodecamer 4 2 1.6
Higher oligomers 8 2.2
Polymers 10 2.7 87
PROTEIN QUATERNARY STRUCTURE
Examples

88
 PROTEIN FOLDING AND STABILITY

• Folded proteins occupy a low-energy well that makes the native structure most stable
• Many proteins can fold spontaneously to this low-energy conformation
• Proteins are thought to fold cooperatively … the first few interactions assist
subsequent alignment and folding
• Folding is extremely rapid, the native conformation is generally reached < 1 second

89
 PROTEIN FOLDING AND STABILITY

• During folding the polypeptide collapses in upon itself due to the hydrophobic effect
• An intermediate “molten globule” forms with elements of secondary structure
• The backbone is rearranged to achieve a stable native conformation

90
 PROTEIN FOLDING AND STABILITY
Energy well of protein folding
(a)
• The funnels represent the free-energy
potential of folding proteins
(a) A simplified funnel showing two
possible pathways to the low-energy native
protein. In path B the polypeptide enters a
local low-energy minimum as it folds

(b) A more realistic version of the possible (b)

free energy forms of a folding protein with
many local peaks and dips

91
 PROTEIN FOLDING AND STABILITY

Hypothetical protein-folding pathways

• The initially extended polypeptide

chains form partial secondary
structures, then approximate tertiary
structures, and finally the unique
native conformations
• The arrows within the structures
indicate the direction from the N- to
the C-terminus.

92
 PROTEIN FOLDING AND STABILITY
Chaperones

• Molecular chaperones increase rate of correct folding and prevent the formation of
incorrectly folded intermediates
• Chaperones can bind to unassembled protein subunits to prevent incorrect
aggregation before they are assembled into a multisubunit protein
• Most chaperones are heat shock proteins (synthesized as temperature increases)

93
 PROTEIN FOLDING AND STABILITY
Chaperones

• Hydrolysis of several ATP molecules is required

94
 PROTEIN DENATURATION AND RENATURATION

• Denaturation - disruption of native conformation of a protein, with loss of biological

activity
• Energy required is small, perhaps only equivalent to 3-4 hydrogen bonds
• Proteins can be denatured by:
à heat
à extremes of pH
à chaotropic agents (urea, guanidine hydrochloride)
à detergents (SDS)
• Some proteins can be refolded or renatured

95
 PROTEIN DENATURATION AND RENATURATION
Thermal Denaturation

• Heat has a complex effect on the weak Heat denaturation of ribonuclease A

and apomyoglobin
interactions in a protein (primarily
hydrogen bonds)
• Denaturation takes place over a small
range of temperature
• Unfolding is a cooperative process Tm Tm

• Melting temperature (Tm) corresponds

to the temperature at the midpoint of
transition between native and
denatured forms

96
 PROTEIN DENATURATION AND RENATURATION
Chemical Denaturation

Organic solvents and detergents

• Chaotropic agents (urea and guanidinium chloride) and the hydrophobic tail of
detergents (SDS) penetrate protein interior and disrupt hydrophobic interactions

97
 PROTEIN DENATURATION AND RENATURATION
Denaturation and Renaturation of Ribonuclease A

• 2-mercaptoethanol unfolds the

protein and disrupts disulfide bonds
to produce reduced, reversibly
denatured ribonuclease A
• When proteins fold inside a cell,
they occasionally adopt a non-native
conformation and form
inappropriate disulfide bridges. An
enzyme called Protein disulfide
isomerase (PDI) catalyzes the
reduction of incorrect bonds

98
 PROTEIN PURIFICATION TECHNIQUES
Column Chromatography
• Preparation of a solution of proteins - Most techniques are performed at 0-4oC
Breaking open tissue cells and releasing their proteins into a solution (crude
extract)
Precipitation of proteins by addition of certain salts (ammonium sulfate)
Removal of small solutes and salts by dialysis

• Column chromatography is the most useful method to to fractionate the mixture of

proteins - Takes advantage of differences in protein charge, size, binding affinity, and
other properties

99
 PROTEIN PURIFICATION TECHNIQUES
Column Chromatography
• A cylindrical column is filled with an
insoluble material (cellulose or synthetic
beads), protein mixture is applied to the
column and washed through the matrix of
insoluble material by the addition of
solvent
• As solvent flows through the column, the
eluate (liquid emerging for the bottom of
the column) is collected in many fractions.
• Absorbance is measured at 280nm
• May be performed under high pressure
(HPLC)
100
 PROTEIN PURIFICATION TECHNIQUES
Ion-Exchange Chromatography
• Separation based upon the overall charge
of molecules
• The matrix carries positive charges
(anion-exchange resins) or negative
charges (cation-exchange resins)
• Anion-exchange matrices bind negatively
charged proteins, retaining them in the
matrix for subsequent elution (and
cation-exchange resins bind positively
charged proteins)
• Bound proteins can be serially eluted by
gradually increasing the salt
concentration in the solvent 101
 PROTEIN PURIFICATION TECHNIQUES
Gel-Filtration Chromatography
• Also called size-exclusion chromatography
• Separation based upon molecular size
• The gel is a matrix a porous beads
• Proteins that are smaller than average pore
size penetrate much of the internal volume
of the beads and are therefore retarded by
the matrix as the buffer solution flows
through the column
• The smaller the protein, the later it elutes
from the column

102
 PROTEIN PURIFICATION TECHNIQUES
Affinity Chromatography
• Relies on specific binding interactions
between the target protein and some other
molecule (an antibody that recognizes the
target protein or another protein) that is
covalently bound to the matrix of the column
• While all the proteins pass through the
column, only the target protein binds to the
matrix
• The target protein is eluted by washing the
column with a solvent containing a high
concentration of salt that disrupts the
interaction between the protein and the
matrix 103
 PROTEIN PURIFICATION TECHNIQUES
Electrophoresis

• Polyacrylamide gel electrophoresis (PAGE) Separates molecules on a

polyacrylamide gel matrix when an electric field is applied
• SDS-PAGE. Sodium dodecyl sulfate (SDS) coats proteins with negative charges.
Coated polypeptide chains then separate by molecular mass (method to determine
molecular weight)

104
PROTEIN PURIFICATION TECHNIQUES
Electrophoresis

105
 AMINO ACID SEQUENCING OF PROTEINS
Importance

Determining the 3D structure of a protein and understanding its molecular

mechanism of action

Sequence comparisons among analogous proteins from different species gives

insights into protein function and evolutionary relationships

Clinical application – many inherited diseases are caused by mutations that result
in an amino acid change in a protein
- Sickle cell anemia: results from the replacement of glutamate residue (6) in the
β chains of normal adults with a valine residue

106
 AMINO ACID SEQUENCING OF PROTEINS
Step 1 – Separating Subunits

• Chaotropic agents (8M urea and guanidinium chloride) and detergents (SDS) are used
• The subunits are separated by ion-exchange chromatography or gel filtration
• Disulfide bonds must be cleaved:
à To permit the separation of polypeptide chains
à To prevent the native protein conformation

107
 AMINO ACID SEQUENCING OF PROTEINS
Step 1 – Separating Subunits
Breaking disulfide bonds by performic acid (1/3)
• Performic acid converts all cysteine residues whether linked by S-S bridges or not to
cysteic acid residues
• Cysteic acid is stable in both acidic and basic solutions so the total cysteine content of
a protein may be determined as cysteic acid
• Disadvantages of using performic acid:
• Oxidizes methionine residues to methionine sulfone (BrCN cannot be used in
sequencing)
• Partially destroys the indole side chain of tryptophan

108
 AMINO ACID SEQUENCING OF PROTEINS
Step 1 – Separating Subunits
Breaking disulfide bonds by performic acid (2/3)

+
Performic acid

Cysteine Cysteic acid

109
 AMINO ACID SEQUENCING OF PROTEINS
Step 1 – Separating Subunits
Breaking disulfide bonds by performic acid (3/3)

Methionine Performic acid Methionine sulfone

110
 AMINO ACID SEQUENCING OF PROTEINS
Step 1 – Separating Subunits
Breaking disulfide bonds by 2-Mercaptoethanol
2-Mercaptoethanol is used in
the reduction of a cystine
residue

Treating the reduced protein

with the alkylating agent
iodoacetate converts all free
cysteine residues to stable S-
carboxymethylcysteine
residues, thus preventing the
re-formation of disulfide bonds
in the presence of oxygen
111
 AMINO ACID SEQUENCING OF PROTEINS
Step 2 - Determining Amino Acid Composition
• Polypeptide chains are hydrolyzed by heating in 6M HCl at 110ºC for 24-72hrs?

112
 AMINO ACID SEQUENCING OF PROTEINS
Step 2 - Determining Amino Acid Composition
• The amino acid composition is quantitatively determined using an automated amino
acid analyzer, which separates amino acids by ion-exchange chromatography
• Amino acids are identified as peak on a chromatogram – both kinds and amounts of
amino acids are detected
• The analysis of a protein digest can be done in <1h and can detect as little as 1 ρmol
of each amino acid
• Phenylisothiocyanate (PITC) used to derivatize the amino acids prior to HPLC analysis

113
 AMINO ACID SEQUENCING OF PROTEINS
Step 2 - Determining Amino Acid Composition
Amino acid treated with Chromatogram obtained from HPLC
phenylisothiocyanate (PITC) separation of PTC–amino acids

Absorbance at 254 nm
B = totals of asparagine + aspartate
Z = glutamine + glutamate

114
 AMINO ACID SEQUENCING OF PROTEINS
Step 3 - Edman Degradation
N-TERMINAL IDENTIFICATION
Treat peptide with phyenylisothiocyanate (PITC; also known as Edman’s reagent)
which reacts with the N-terminus to form a phenylthiocarbamyl (PTC) polypeptide
Treat with anhydrous trifluoroacetic acid (TFA) or hydrofluoric acid (HF) to selectively
cleave the N-terminal peptide bond as a thiazolinone derivative
Separate N-terminal derivative from peptide into an organic solvent
Convert derivative to the more stable phenylthiohydantoin (PTH) amino acid
derivative by treatment with aqueous acid
The PTH-amino acid may be identified by comparing it with known standards using
TLC, HPLC, GLC, or electrophoresis
115
 AMINO ACID SEQUENCING OF PROTEINS
Step 3 - Edman Degradation
Edman degradation procedure (1/2)

116
 AMINO ACID SEQUENCING OF PROTEINS
Step 3 - Edman Degradation
Edman degradation procedure (2/2)

• Edman degradation remains the

most useful method
• Exopeptidases (amino peptidases
or carboxypeptidases) can be used
to sequence amino acids
117
 AMINO ACID SEQUENCING OF PROTEINS
Step 4 - Cleaving the Polypeptide Chain
• Polypeptides that are longer than 40 to 100 residues cannot be directly sequenced
• Chemical agents (such as cyanogen bromide BrCN) and Proteases (enzymes cleaving
peptide bonds) are used to selectively cleave the protein into smaller fragments

118
 AMINO ACID SEQUENCING OF PROTEINS
Step 4 - Cleaving the Polypeptide Chain

Protein cleavage by cyanogen bromide (BrCN)

• Cyanogen bromide cleaves polypeptide chains at the C-terminal side of methionine
residues to form peptidyl homoserine lactone

0.1 N HCl
or
70% formic acid
Peptidyl homoserine lactone
+
BrCN

Aminoacyl peptide
119
 AMINO ACID SEQUENCING OF PROTEINS
Step 4 - Cleaving the Polypeptide Chain
Protease enzymes cleave specific peptide bonds
• Trypsin: C-side of basic residues (Lys, Arg), provided the next residue is not Pro

Trypsin
+
H2O

120
 AMINO ACID SEQUENCING OF PROTEINS
Step 4 - Cleaving the Polypeptide Chain
Protease enzymes cleave specific peptide bonds
• Staphylococcus aureus V8 protease: C-side of negatively charged residues (Glu, Asp)
• Chymotrypsin: C-side of aromatic or bulky noncharged aliphatic residues (e.g. Phe, Tyr,
Trp, Leu)

121
AMINO ACID SEQUENCING OF PROTEINS
Step 4 - Cleaving the Polypeptide Chain

122
 AMINO ACID SEQUENCING OF PROTEINS
Step 5 – Overlap Peptides
Cleavage and sequencing of an oligopeptide

123
 AMINO ACID SEQUENCING OF PROTEINS
Sequences of DNA and protein

• Protein amino acid sequences can be deduced from the sequence of nucleotides in
the corresponding gene
• A sequence of three nucleotides specifies one amino acid (A,C,G,T are DNA residues )

124
125
 TYPES OF PROTEINS
Classification based on their Conformation
Fibrous proteins
- Physically tough and insoluble in water
- Often assembled into large cables or threads
- Provide mechanical support and are basic structural elements in the connective
tissue of higher animals
- α-Keratins: major components of hair and nails
- Collagen: major component of tendons, skin, bones and teeth

126
 TYPES OF PROTEINS
Classification based on their Conformation
Globular proteins
- Usually water soluble, compact, roughly spherical
- Hydrophobic interior, hydrophilic surface
- Globular proteins include enzymes, antibodies, regulatory and carrier proteins
(serum albumin and hemoglobin)

• Some proteins fall between fibrous and globular proteins (myosin and fibrinogen)

127
 TYPES OF PROTEINS
Classification based on their Composition
Simple Proteins
- Those which on hydrolysis yield only amino acids and no other major organic or
inorganic products

Conjugated proteins
- Those which on hydrolysis yield not only amino acids but also a prosthetic group
made up of organic or inorganic components

128
 TYPES OF PROTEINS
Classification based on their Composition
Conjugated Proteins
Class Prosthetic group(s) Example
Nucleoproteins DNA, RNA Chromosomes, ribosomes
Lipoproteins Lipids β1-Lipoprotein of blood
Glycoproteins Carbohydrates Immunoglobin G
Phosphoproteins Phosphate groups Casein of milk
Hemoproteins Heme (iron porphyrin) Hemoglobin
Flavoproteins Flavin nucleotides Succinate dehydrogenase
Metalloproteins Iron Ferritin
Zinc Alcohol dehydrogenase
Calcium Calmodulin
Molybdenum Nitrogenase
Copper Cytochrome oxidase

129
 TYPES OF PROTEINS
Glycoproteins
• Proteins that contain covalently-bound oligosaccharides
• Oligosaccharide chains exhibit great variability in sugar sequence and composition. They
include 1 to over 30 sugar residues and may account for 80% of the mass of the protein
• 9 sugars predominate in eukaryotic glycoproteins
• Hexoses: L-fucose, D-galactose, D-glucose, D-mannose
• Pentoses: D-xylose, L-arabinose
• Amino sugars: N-acetyl-D-galactosamine, N-acetyl-D-glucosamine and sialic
acid (N-acetlyneuraminic acid)
• Glycoforms - proteins with identical amino acid sequences but different oligosaccharide
chain composition
• A single glycoprotein molecule might contain up to four branches of oligosaccharides,
which might be O-glycosidic or N-glycosidic or both
130
 TYPES OF PROTEINS
Glycoproteins
• Two major types of carbohydrate-peptide linkages (O-glycosidic and N-glycosidic)

O-GLYCOSIDIC BOND
• From N-acetylglalactosamine to the –OH of serine or threonine

131
 TYPES OF PROTEINS
Glycoproteins
Four subclasses of O-glycosidic linkages

GalNAc-Ser/Thr
(most common)

5-Hydroxylysine (Hyl) to D-galactose

(unique to collagen)

Gal-Gal-Xyl-Ser-core protein
(found in certain proteoglycans)

GlcNAc to a single serine or threonine

(found in some proteins)

132
 TYPES OF PROTEINS
Glycoproteins
N-GLYCOSIDIC BOND
• From N-acetylglucosamine to the amide nitrogen of asparagine
• Most N-linked oligosaccharides can be divided into three subclasses: high mannose,
complex, and hybrid

133
 TYPES OF PROTEINS
Glycoproteins
Structures of N-linked oligosaccharides

High-mannose
chain

Complex
chain

Hybrid chain

134
 TYPES OF PROTEINS
Glycoproteins
Some Functions of Glycoproteins
• Included in nearly all proteins of the blood serum:
• Enzymes (ribonuclease B, glucose oxidase)
• Hormones (human chorionic gonadotropin hCG)
• Blood group proteins of the erythrocyte membrane
• Interferon and ovalbumin
• Only a handful of sugar-free serum proteins are known: insulin and albumin

135
 PROTEINS
Functional Diversity of Proteins

Function Description Examples

1. Enzymes - Largest group Hexokinases, lactate
- All are globular proteins dehydrogenase, DNA polymerase

2. Storage proteins - Store amino acids as nutrients Ovalbumin, casein

- Building blocks for the growing embryo

3. Transport proteins - Capable of binding and transporting specific Hemoglobin, myoglobin, serum
types of molecule via the blood albumin, lipoproteins

4. Contractile proteins - Generate force for muscle contraction Actin, myosin

136
 PROTEINS
Functional Diversity of Proteins

Function Description Examples

5. Protective proteins - In vertebrate blood Blot clot proteins (Thrombin,
- Defensive function fibrinogen) antibodies (immune
globulins)

6. Hormones - Signaling molecules targeting distant organs Growth hormone, insulin

to regulate physiology and behavior

7. Toxins - Mainly in bacteria and snake venoms Clostrodium botulinum toxin,

ricin, snake venoms

137
 PROTEINS
Functional Diversity of Proteins

Function Description Examples

8. Structural proteins - form structural framework of various organs
and connective tissues (hairs, nails, bones,
cartilage, skin, ligaments, blood vessels,
uterus, lung, etc.)
- provide strength and mechanical support
Viral-coat proteins, glycoproteins
in cell coats and walls, α-keratin,
fibroin, collagen, mucoproteins

9. Antifreeze proteins - Prevents blood from freezing in animals Antifreeze protein

living in arctic areas

138