•Biochemistry

Course
Chapter 1
Protein Structure and
analysis

Weihong Hou, Ph.D., Associate Prof.

Department of Biochemistry
Section 1 Chemical Composition of Protein
Amino Acids
Section 2 Protein structure:
Structure: primary→secondary → tertiary →
quaternary
Domains, motif
Section 3 Protein analysis
Purification, sequence, mass, and
structure analysis
 1. 20 standard amino acids (except for proline)

COO-

H3N+ Cα H

1) α -carbon is chiral (asymmetric) except in glycine (R is H)

2) Amino acids can exit in both D- and L- stereoisomers, but only L-
isomers are found in proteins
3) Amino acids are dipolar ions (zwitterions in aqueous solution )
4) The side chains (R) differ in size, shape, charge and chemical
reactivity
5) A few proteins contain nonstandard amino acids that are formed
by post-translational modification of the parent amino acids.
2. Classification of Amino Acids

1) Amino acids with charged side chains (salt bridges, hydrophilic )

“Acidic” amino acids: containing additional carboxyl groups which are usually
ionized

aspartic acid (Asp, D) glutamic acid (Glu, E)

“Basic” amino acids: containing positively charged groups
Lysine (Lys, K): a second amino group attached to the ε -carbon atom

Arginine (arg, R): a guanidine group attached to the δ -carbon atom

Histidine (His, H): a imidazole group has a pKa near neutrality. This group
can be reversibly protonated under physiological conditions, which contribute
to the catalytic mechanism of many enzymes.
(2) Amino acids with polar uncharged side chains (hydrophilic)
--containing groups that form hydrogen bonds with water
Serine (Ser, S) & threonine (Thr, T) have hydroxyl groups.

Asparagine (Asn, N) & Glutamine (Gln, Q) are the amide derivatives of

Asp and Glu

Cysteine (Cys, C) has a thiol group which is often oxidizes to cystine

(3) Amino acids with nonpolar side chains
aliphatic side chains

Glycine (Gly, G)

Proline (Pro, P): imino acid

Methionine (Met, M): contains a sulfur atom

Alkyl side chains

Alanine (Ala, A)

Valine (Val, V)

Leucine (Leu, L)

Isoleucine (Ile, I)
aromatic side chains
--aromatic structure accounts for most of UV absorbance of proteins at 280 nm

Phenylalanine (Phe, F)

Tyrosine (Tyr, Y)

Tryptophan (Trp,
W)
 Section 2 Protein structure
1. Shapes:
(1) Globular proteins( enzymes)

Complementary fit of a substrate molecule to the catalytic site

on an enzyme molecule.
(2) Fibrous proteins: important structural
proteins (silk fibroin, keratin in hair and wools )

Protofibril ( 初原纤维 ) microfibril ( 微管 )

Keratin ( 角蛋白 )

keratin in hair
2. Protein structure
(1) Primary structure
Polypeptides contain N- and C- termini and are
directional, usually ranging from 100-1500 AAs

Formation of a peptide bond (shaded in gray) in a dipeptide.

N terminus

C terminus

Structure of the pentapeptide Ser-Gly-Tyr-Ala-Leu.

(2) Secondary structure
α -helix
• right-handed
• 3.6 aa per
turn
• hydrogen
bond
N-H···O=C

A stereo, space-filling representation Collagen triple helix:

three polypeptide
intertwined
Model of the α helix. The
polypeptide backbone is folded
into a spiral that is held in place by
hydrogen bonds (black dots)
between backbone oxygen atoms
and hydrogen atoms. All the
hydrogen bonds have the same
polarity. The outer surface of the
helix is covered by the side-chain
R groups.
 β -sheet: hydrogen bonding of the pepetide bond
N-H and C=O groups to the complementary groups
of another section of the polypeptide chain

Parallel β sheet: sections run A stereo, space-filling

in the same direction representation of the six-
Antiparallel β sheet: sections stranded antiparallel β sheet.
run in the opposite direction
A two-stranded β sheet
β SHEETS. (a) A simple two-stranded β sheet with antiparallel β
strands. A sheet is stabilized by hydrogen bonds (black dots) between
the β strands. The planarity of the peptide bond forces a β sheet to
be pleated; hence, this structure is also called a β pleated sheet, or
simply a pleated sheet. (b) Side view of a β sheet showing how the
R groups protrude above and below the plane of the sheet. (c) Model
of binding site in class I MHC (major histocompatibility complex)
molecules, which are involved in graft rejection. A sheet comprising
eight antiparallel β strands (green) forms the bottom of the binding
cleft, which is lined by a pair of α helices (blue). A disulfide bond is
shown as two connected yellow spheres. The MHC binding cleft is
large enough to bind a peptide 8 10 residues long.
(3) Tertiary structure

The different sections of secondary structure and connecting regions fold into a
well-defined tertiary structure, with hydrophilic amino acids mostly on the
surface and hydrophobic ones in the interior. The structure is stabilized by
noncovalent interactions and sometimes disulfide bonds. Denaturation leads to
loss of secondary and tertiary structure.

Noncovalent interaction between side chains that hold the tertiary structure
together: van der Waals forces, hydrogen bonds, electrostatic salt bridges, hydrophobic
interactions
Covalent interaction: disulfide bonds
 The different sections of α -helix, β -sheet, other minor
secondary structure and connecting loops of a polypeptide
fold in three dimensions
Noncovalent interaction between side chains that hold the
tertiary structure together: van der Waals forces,
hydrogen bonds, electrostatic salt bridges, hydrophobic
interactions
Covalent interaction: disulfide bonds

Denaturation of protein by disruption of its 2o and 3o

structure will lead to a random coil conformation
(4) Quaternary structure
Many proteins are composed of two or more polypeptide chains
(subunits). These subunits may be identical or different. The same
forces which stabilize tertiary structure hold these subunits together.
This level of organization called quaternary structure.

The quaternary structure of hemoglobin 。  1-yellow; β 1-light

blue; α 2-green; β 2-dark blue; heme-red
back
 Domains, motifs

Domains: structurally independent units

(tertiary) of a protein, being connected by
sections with limited higher order structure
within the same polypeptide. (Figure)

They can also have specific function such as

substrate binding
•Protein
structure
Structural motifs:
• Groupings of secondary structural elements that
frequently occur in globular proteins
• Often have functional significance and represent the
essential parts of binding or catalytic sites conserved
among a protein family

β α β motif
•Protein
structure
Domain

The tertiary structures of the above c-type cytochromes

Cys, Met and His side chains covalently linked to the heme to
the protein
Back
Section 3 Protein analysis
1. Purification: to obtain enough pure sample for study

2. Sequencing: determine the primary structure of a pure

protein sample
3. Mass determination: determine the molecular weight
(MW) of an interested protein.

4. Determine the tertiary structure:X-ray crystallography

and NMR.
1. Protein purification

• To purify the interested protein from

other proteins and nonprotein molecules
existing in the cells

• An essential experimental step prior to study

of any individual protein
 The principal properties of proteins
used for purification

Size: gel filtration chromatography

Charge: ion-exchange chromatography, isoelectric
focusing, electrophoresis
Affinity: affinity chromatography
Recombinant techniques: involving DNA
manipulation and making protein purification so easy
(1) Gel filtration chromatography
Bead: the matrix
determine the size of the
pore
(2)Ion-exchange chromatography
Sample mixture
++
+

Protein Ion
binding displacing

Column + anions Column + proteins Column + anions

Purified protein
(3) Electrophoresis

Protein migrate at
different position
depending on their net
charge

+
(4) Affinity
chromatography
• Enzyme-substrate
binding
• Receptor-ligand binding

• Antibody-antigen binding
(5) Recombinant techniques

•Clone the interested protein encoding gene in an

expression vector with a purification tag added at
the 5’- or 3’ end of the gene

•Protein overexpression in a cell

•Protein purification with affinity chromatography.

2. Mass Determination

Gel filtration chromatography and SDS-PAGE

•Comparing of the unknown protein with a proper standard

•Popular SDS-PAGE: cheap and easy with a 5-10% error
•SDS: sodium dodecyl sulfate, makes the proteins
negatively charged and the overall charge of a protein is
dependent on its mass.
Mass spectrometry:

•Molecules are vaporized and ionized, and the

degree of deflection (mass-dependent) of the ions in
an electromagnetic field is measured
•extremely accurate, but expensive
•MALDI can measure the mass of proteins smaller than
100 KDa

•Helpful to detect post-translational modification

•Protein sequencing: relying on the protein data base
 3. Protein sequencing : Determine the
primary structure of a protein

Specific enzyme/chemical cleavage:

•Trypsin cleaves after lusine(K) or arginine (R)
•V8 proteease cleaves after glutamic acid (E)
• Cyanogen bromide cleaves after methionine (M)

Edaman degradation:
•Performed in an automated protein sequencer
•Determine the sequence of a polypeptide from N-
terminal amino acid one by one.
•Expensive and laborious
Most protein sequences are deduced from the DNA/cDNA
sequence

Direct sequencing: determine the N-terminal

sequences or some limited internal sequence 
construction of an oligonulceotide or antibody probe
fishing the gene or cDNA
4. X-ray crystallography and NMR
Determing the tertiary structure (3-D) of a
protein
(1) X-ray crystallography:
•Measuring the pattern of diffraction of a beam of X-rays
as it pass through a crystal. The first hand data obtained
is electron density map, the crystal structure is then
deduced.
•A very powerful tool in understanding protein tertiary
structure

•Many proteins have been crystallized and analyzed

(2) NMR
•Measuring the relaxation of protons after they
have been excited by radio frequencies in a strong
magnetic field

•Measure protein structure in liquid but not in crystal

•Protein measured can not be larger than 30

KDa