Proteins

Instructor: 黃啟清 助理教授

Office: 醫學系生化學科 , Room 411
Phone: 3121101 ext 2306 ext 15.
Email: cchwang@cc.kmu.edu.tw
Comparison of myoglobin and hemoglobin

A~H: α-helical regions

 David L. Nelson and Michael M. Cox

Lehninger Principles of
Biochemistry
Fourth Edition

Chapter 3:
Amino Acids, Peptides, and Proteins

Copyright © 2004 by W. H. Freeman & Company

• Proteins play an enormous variety of roles: carry out the
transport and storage of small molecules; make up a large part
of the structural framework of cells and tissues; muscle
contraction, the immune response, and blood clotting are
mediated by proteins.
• Enzymes-the catalysts that promote the tremendous variety of
reactions that channel metabolism into essential pathways.

• 1. Proteins are linear polymers built of monomer units called

amino acids.
• 2. Proteins contain a wide range of functional groups.
• 3. Proteins can interact with one another and with other
biological macromolecules to form complex assemblies.
• 4. Some proteins are quite rigid, whereas others display
limited flexibility
Some functions of proteins
 From gene to protein:
The DNA sequences of genes are transcribed into messenger RNA
molecules, which are in turn translated into proteins. Triplets of nucleotides
(codons) are used to code for each amino acid.

Relationships of DNA to mRNA to polypeptide chain.

 Amino Acid
• 1. Amino acids are organic acids containing an
amine group.
• 2. Only 20 L--amino acids are used to make
proteins
• 3. Amino acids can exist as zwitterions -
substances containing the positive and negative
charge - due to their carboxyl and amine groups.
• 4. Amino acids are built into proteins by the
process of translation using the genetic code.
General structure of an amino acid
 Stereoisomerism in α-amino acids
CHO CHO
L-Glyceraldehyde HO C H H C OH D-Glyceraldehyde

CH2OH CH2OH
The 20 common amino acids of proteins
 H
+
Absorption of ultraviolet light by
aromatic amino acids
Disulfide bonds between Cys residues
 (in prothrombin)

(in plant cell wall proteins and collagen)

(in collagen)
(in elastin)

(in myosin)
Some 300 additional AAs have been
found in cells.

(metabolites in the urea cycle)

Nonionic and zwitterionic forms of
amino acids
Amino acids have characteristic
titration curves
 HA H+ + A-
Ka = [H+][A-]/[HA]
Henderson-Hasselbach equation
pH = pKa + log ([A-]/[HA])
Titration of an amino acid

pI = (pK1 + pK2)/2

Isoelectric point or
Isoelectric pH: The
characteristic pH at
which the net electric
charge is zero
Titration curve for glutamate
pI = (pK1 + pKR)/2
Titration curve for histidine

pI = (pKR + pK2)/2
 Peptides and the Peptide Bond
Dipeptide contains 2 amino acids linked by a peptide bond
Oligopeptide contains a few amino acids joined by peptide bonds
Polypeptide contains many amino acids joined by peptide bonds
• Dipeptide
• Tripeptide
• Tetrapeptide
• Oligopeptide
• Polypeptide
• Protein
• oligomer
The formation of peptide bond
by condensation
Ser-Gly-Tyr-Ala-Leu
Alanylglutamylglycyllysine

The groups ionized at pH 7.0 are in red.

How long are the polypeptide chains in proteins
Polypeptides have characteristic amino acid compositions
Conjugated protein: protein contains permanently associated
chemical components in addition to amino acids.
Levels of structure in proteins
• Four levels of protein structure are commonly defined.

• A description of all covalent bonds (mainly peptide bonds

and disulfide bonds) linking amino acid residues in a
polypeptide chain is its primary structure. The most
important element of primary structure is the sequence of
amino acid residues.
• Secondary structure refers to particularly stable
arrangements of amino acid residues giving rise to
recurring structural patterns.
• Tertiary structure describes all aspects of the three-
dimensional folding of a polypeptide.
• When a protein has two or more polypeptide subunits,
their arrangement in space is referred to as quaternary
structure.
Separation and purification of
proteins
 Purification and Characterization of Proteins
• Proteins can be purified according to solubility, size, charge, and binding
affinity
• 1. Salting out. Most proteins are less soluble at high salt concentrations.
• 2. Dialysis. Proteins can be separated from small molecules by dialysis
through a semipermeable membrane, such as a cellulose membrane with
pores.
• 3. Gel-Filtration Chromatography. More discriminating separations
on the basis of size. Large molecules flow more rapidly through the
column and emerge first because a smaller volume is accessible to them.
• 4. Ion-exchange chromatography. Proteins can be separated on the
basis of their net charge.
• 5. Affinity chromatography. This technique takes advantage of the
high affinity of many proteins for specific chemical groups.
• 6. Determinations of purity and molecular weight of proteins by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Dialysis
Column chromatography
Ion exchange chromatography

The column matrix is a synthetic

polymer containing bound charged
groups; those with bound anionic
groups are called cation exchangers,
and those with bound cationic groups
are called anion exchangers.
Size-exclusion chromatography
Affinity chromatography
Electrophoresis
Estimating the molecular weight of a protein

( a)
Isoelectric focusing

IPG strips

IPGphor
Two-dimensional electrophoresis
The Amino Acid Sequences of
Proteins
AA sequence of
bovine insulin
 (Frederick Sanger)
The Nobel Prize in
Chemistry 1958:
"for his work on
the structure of
proteins,
especially that of
insulin"

The Nobel Prize in Chemistry 1980: "for their

contributions concerning the determination of
base sequences in nucleic acids"
Steps in sequencing a polypeptide
Breaking disulfide bonds in proteins
Cleaving proteins and sequencing and
ordering the peptide fragments
 Investigating proteins with mass
spectrometry
Ion source:
1. Matrix Assisted Laser Desorption Ionization (MALDI)
2. Electrospray Ionization (ESI)

Mass:
1. MALDI-TOF
2. LC-ESI-Q-TOF
 The Nobel Prize in Chemistry 2002
"for the development of methods for identification and
structure analyses of biological macromolecules"

John B. Fenn (USA) 1/4 of the prize

Koichi Tanaka (Japan) 1/4 of the prize
"for their development of soft desorption ionisation methods for
mass spectrometric analyses of biological macromolecules“

 Kurt Wüthrich (Switzerland)

"for his development of nuclear magnetic resonance
spectroscopy for determining the three-dimensional structure of
biological macromolecules in solution
 MALDI: Matrix Assisted Laser
Desorption Ionization
Lens Laser
Sample mixed with a strong
UV absorbing matrix
The matrix absorbs energy
MH+ from the laser causing the
“molecular ion” sample and matrix to be
+
Target
+
+
+ +
+
+
volatilized
Ionized matrix molecules
transfer a proton to the
sample
Grid
MALDI is a pulsed ionization source
+/- 25 kV
 What is Time of Flight (TOF)?

Flight Tube

4-25 kV Detector

Ion Source

The ions enter the flight tube with the lighter ions
traveling faster than the heavier ions
ESI-MS/MS
m/z = (M+n)/n
Tandem MS or MS/MS for sequencing the short stretches of polypeptide
 MS/MS: Product Ion Scan
(most common MS/MS mode)
Select Scan
Precursor CAD Products

Q1 Q2 Q3
• The precursor ion is sent into the collision cell and fragmented
• Q1 is fixed, Q3 sweeps a given mass range
• Used for structural information such as peptide sequence
 Peptides
• (1) purification from tissue, a task often
made difficult by the vanishingly low
concentrations of some peptides
• (2) genetic engineering
• (3) direct chemical synthesis
 The Nobel Prize in
Chemistry 1984
"for his
development of
methodology for
chemical synthesis
on a solid matrix“
(1962)

(USA)
Chemical synthesis of a peptide on an insoluble
polymer support
Chemical synthesis of a peptide on an
insoluble polymer support
Chemical synthesis of a peptide on an
insoluble polymer support
 Protein Sequences and Evolution

Aligning protein sequences with the use of gaps

Sequence in the EF-1/EF-Tu protein family.
Certain segments of a protein sequence may be found in the
organisms of one taxonomic group but not in other groups; these
segments can be used as signature sequences for the group in
which they are found.
 Evolutionary tree derived from amino acid sequence
comparisons

A bacterial evolutionary tree, based on the sequence divergence observed in

the GroEL family of proteins