? Amino acids and
structure
? acids and bases
? Protein structure
? myoglobin and
hemoglobin
? collagen
? Protein purification
? Chromatography of
proteins
? Electrophoresis of proteins
? Protein sequencing
B Amino acids and protein
Amino Acid
Stereoisomers (stereo-isomer)
The 20 standard amino acids
Ionization
B1 Amino acids
Basic Structure of an amino acid
COO-
NH3+ C H
R
COO-
H C NH3+
R
L-Amino acid D-Amino acid
The two enantiomers
Enantiomers
? Enantiomers are molecules that have opposite
spatial configuration are said to be optically active,
? One enantiomer will rotate polarized light a set
number of degrees to the right,This is called the
dextrorotatory isomer or (+) isomer,
? The other enantiomer will rotate the plane polarized
light the same number of set degrees in the opposite
left direction,This isomer is said to be a levorotatory
isomer or (-) isomer,
The two enantiomers
The 20 standard amino acids
?Hydrophobic aliphatic amino acids
?Hydrophobic aromatic amino acids
?Polar charged amino acids
?Polar uncharged amino acids
Hydrophobic aliphatic amino acids
Gly,甘氨酸
H
H2N COOH
H
C
glycine
Hydrophobic aliphatic amino acids
Ala:丙氨酸
CH3
H2N COOH
H
C
alanine
Hydrophobic aliphatic amino acids
Val:缬氨酸
H
H3C C CH3
H2N C COOH
H
valine
Hydrophobic aliphatic amino acids
Leu:亮氨酸
H
H3C C CH3
CH2
H2N C COOH
H
leucine
Hydrophobic aliphatic amino acids
isoleucin Ile:异亮氨酸
CH3
CH2
H3C C H
H2N C COOH
H
Hydrophobic aliphatic amino acids
CH3
S
CH2
CH2
H2N C COOH
H
methionine Met,甲硫氨酸
Hydrophobic aliphatic amino acids
H
H2C
CH2
CH2
C N
COOH
H
proline
Pro,脯氨酸
Hydrophobic aliphatic amino acids
S-H
CH2
H2N C COOH
H
Cysteine
Cys,半胱氨酸
Cystine
Hydrophobic aromatic amino acids
Phe:苯丙氨酸
CH2
+H3N CH C O
O
Phenylalanine
Hydrophobic aromatic amino acids
Tyr:酪氨酸
CH2
+H3N CH C O
O
OH
Tyrosine
Hydrophobic aromatic amino acids
Trp:色氨酸
C O
O
NH
CH2
CH H3N
Trptophan
Polar charged amino acids
Arg:精氨酸
NH2
C NH2
NH
CH2
CH2
CH2
H3N CH C O
O
Arginine
Polar charged amino acids
Lys:赖氨酸
NH3+
CH2
CH2
CH2
CH2
H3N CH C O
O
Lysine
Polar charged amino acids
His:组氨酸
CH2
H3N CH C O
O
NH+
HN
Histidine
Polar charged amino acids
COO-
+H3N C H
CH2
C
O O-`
Aspartate
Asp,天冬氨酸
Polar charged amino acids
Glu:谷氨酸
O
+NH3 CH C O-
CH2
CH2
C O
O-
Glutamate
Polar uncharged amino acids
O H
CH2
H2N C COOH
H
Serine
Ser,丝氨酸
Polar uncharged amino acids
Thr:苏氨酸
+H3N C COO-
CH OH
CH3
Threonine
Polar uncharged amino acids
Asn:天冬酰胺
+H3N CH COO-
CH2
C O
NH2
Asparagine
Polar uncharged amino acids
O NH2
C
CH2
CH2
H2N C COOH
H
Glutamine
Gln,谷氨酰胺
The 20 standard amino acids
?acids, bases and pH
?Buffer
?Ionization of amino acids
B2 Acids and bases
acids,bases and pH
? An acid can be defined as a proton donor
? A base as a proton acceptor
? Acid<----->H+ +base
? E.g,CH3COOH< ------> H++CH3COO-
? NH4+ < ---- >H++NH3
? The pH of a solution is a measure of its
concentration of protons,pH=-lg[H+]
Buffers
Ionization
Nonionic
form
COOH
H2N C H
R
COO-
H3N+ C H
R
Zwitterionic
form
Dissociation of amino acid
Titration curve of glycine
Peptide bond
Primary structure
Secondary structure
Tertiary structure
Quaternary structure
B3 Protein Structure
Peptide bond
Peptide units
Resonance structure of the peptide bond
Primary structure
Disulfide bond
Secondary structure
? The sceondary structure in a protein is the
regular folding of region of the ploypeptide
chain,
? The two most common types of protein fold are
α–helix and the β–pleated sheet,
The folding of the peptide
chain into an α-helix
α-helix
α-helix
H-bond
β-sheet
Parallel and antiparallel β-sheet
β-sheet
The folding of the peptide
chain in a ?-turn
Super-secondary structure
? Rossman M,G,1973
? αα
? βαβ
? β β
Structural domain
Tertiary structure
? Tertiary structure,refers
to the spatial
arrangement of amino
acids that are far apart in
the linear sequence as
well as those residues
that are adjacent,
? Again,it is the sequence
of amino acids that
specifies this final three-
dimensional structure,
H-bond in tertiary structure
Quaternary structure
? Quaternary structure refers to the
spatial arrangement of the polypeptide
subunits and the nature of the
interactions between them,
? These interactions may be covalent
links or non-covalent interactions
Quaternary structure
Levels of structure in proteins
Protein stability
? Non-covalent interactions ( electrostatic
forces,hydrogen bonds,hydrophobic
forces )
? covalent interactions ( disulfide bonds )
? Peptide bonds
Protein folding
? Spontaneously fold
? Hydrophobic force
? Ordered set of pathways
? Accessory protein,
I,Protein disulfide isomerase
II,Peptidyl prolyl cis - tran isomerase
III,Molecular chaperone
B4 Myoglobin and hemoglobin
? Oxygen-binding proteins
? Myoglobin 肌红蛋白
? Hemoglobin 血红蛋白
? Binding of oxygen to heme
? Allostery 别构
? Mechanism of the allosteric change
? The bohr effect
? Fetal hemoglobin 胎儿血红蛋白
? Hemoglobinopathies 血红蛋白病
Oxygen-binding proteins
? Myoglobin and Hemoglobin are the two oxygen-
binding proteins present in large multicellular
organisms,
? Hemoglobin transports oxygen in the blood and is
located in the erythrocytes;
? Myoglobin stores the oxygen in the muscles,
Myoglobin
Myoglobin structure
Hemoglobin
Protoporphyprinx原卟啉
Heme血红素
Ferriheme高铁血红素
animation
hemo
Allostery变构
? Allosteric protein
? Cooperative
? Non-cooperative
? Oxygen dissociatio curves (Sigmoidal,
hyperbolic)
Oxygen dissocation curves for Hb and Mb
The bohr effect
The bohr effect
Fetal hemoglobin
Fetal hemo
B5 collagen
? Function and diversity
? Biosynthesis
? Composition and post-translational
modifications
? Structure
? Secretion and aggregation
? Cross-links
? Bone formation
collagen
Collagen
Collagen
Collgen synthiesis
Collegen bond
Collagen
B6 Protein Purification
Protein Solubility
Protein size
Protein charge
Protein specific binding affinity
Selection of a protein source
Homogenization and solubilization
Stabilization of proteins
Assay of proteins
Principles of protein purification
Gel filtration chromatography
Ion exchange chromatography
Affinity chromatography
B7 Chromatography of proteins
Gel filtration chromatography
Gel filtration chromatography
Gel filtration chromatography
Ion exchange chromatography
Affinity chromatography
Native PAGE
SDS-PAGE
Isoelectric focusing
B8 Electrophoresis of Protein
Visualization of proteins in gels
Electrophoresis
Native polyacrylamide gel
electropphoresis
Isoelectric focusing
Isoelectric focusing
Isoelectric focusing
Two-dimensional gel
electrophoresis
Appearance of protein after
electrophoresis
Amino acid composition analysis
Edman degradation
Sequencing strategy
B9 Protein sequencing and
peptide synthesis
Amino acid composition analysis
? Hydrolized into amino acids
? Ion exchange chromatography
? Colorimetric比色 reaction (ninhydrin茚
三酮,fluorescamine荧光胺 )
Edman degradation
Recombinant DNA technology
? The sequence of a protein can be determined
using recombinat DNA technology to identify
and sequence the piece of DNA encoding the
protein,
? The amino acid sequence of the protein can
then be deduced from its DNA sequencing
using the genetic code,
Information derived from
protein sequences
? The amino acid sequence of a protein not only
reveals the primary structure of the protein but
also information on possible protein families or
groups and evolutionary relationships,potential
gene duplication and possible post-translational
modifications,
? In addition,a knowledge of the amino acid
sequence can be used to generate specific
antibodies and DNA probes,