Download B2 Protein structure and function

Section B - Protein
Structure
Contents
B1 Amino acids
Structure, Charged side chains, Polar uncharged side
chains, Nonpolar aliphatic side chains, Aromatic side
chains
B2 Protein structure and function
Size and shapes, Primary structure, Secondary structure,
Tertiary structure, Quaternary structure, Prosthetic groups,
What do proteins do, Domains, motifs, Families and
evolution
B3 Protein analysis
Protein Purification, Protein sequencing, Mass
determination, X-ray crystallography and NMR, Functional
analysis, Proteomics
B1 Amino acids—Structure
1. a-carbon is chiral (手性；asymmetric) except in glycine
(R is H)
2. Amino acids are dipolar ions (zwitterions ,兼性离子) in
aqueous solution and are amphoteric.
3. The side chains (R) differ in size,
shape, charge and chemical reactivity
4. A few proteins contain
nonstandard amino acids that are
formed by post-translational
modification of proline and lysine.
B1 Amino acids—Charged
side chains
• “Acidic” amino acids: containing additional carboxyl
groups which are usually ionized
Asp
Glu
• “Basic” amino acids: containing positively
charged groups
His
Arg
Lys
B1 Amino acids—
Polar uncharged side chains
•Contain groups that form hydrogen bonds with
water, hydrophilic.
• Ser
•
Thr
Gln
Asp
Cys
B1 Amino acids—
Nonpolar aliphatic side chains
• Gly
•
Ala
Ile
Val
Met
Leu
Pro
B1 Amino acids—
Aromatic side chains
•Accounts for most of UV absorbance of proteins at
280 nm hydrophobic （疏水的)
Phe
Tyr
Trp
B2 Protein structure and function—
Size and shapes
1. A few thousands Daltons (x 103) to more
than 5 million Daltons (x 106)
2. Some proteins contain bound nonprotein
materials (prosthetic groups or other
macromolecules), which accounts for the
increased sizes and functionalities of the
protein complexes.
• Two broad classes of protein may be
distinguished:
① Globular
( most enzymes)
② Fibrous protein
( silk protein, 角蛋白keratin)
B2 Protein structure and function—
Primary structure
• A peptide bond is a chemical bond formed between two
molecules when the carboxyl group of one molecule reacts
with the amino group of the other molecule, thereby releasing a
molecule of water (H2O).
N terminus
C terminus
Structure of the pentapeptide (胸腺喷丁,促胸腺生成素
32-36五肽[免疫调节剂])Ser-Gly-Tyr-Ala-Leu.
B2 Protein structure and function—
Secondary structure
•a-helix:
right-handed 3.6 aa per turn
hydrogen bond N-H···O=C
•b-sheet:
hydrogen bonding of the
pepetide bond N-H and C=O
groups to the complementary
groups of another section of
the polypeptide chain
B2 Protein structure and function—
Tertiary structure
• The different sections of a-helix, b-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 by heat and extremes of pH will lead to a
random coil conformation
B2 Protein structure and function—
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.
CAM kinase II
Many proteins are composed of two or more polypeptide chains (subunits)
（a）Primary stucture
Advantages of the quaternary structure:
1. It allows very large protein molecules to be
made, such as tubulin.
2. It can provide greater functionality to a protein by
combining different activities into a single entity.
3. The interactions between the subunits can often
be modified by binding of small molecules and
lead to the allosteric effects seen in enzyme
regulation.
B2 Protein structure and function—
Prosthetic groups
Coenzymes
• vitamins: NAD+ (B3) · NADP+ (B3) · Coenzyme A (B5) · THF / H4F
(B9), DHF, MTHF · Ascorbic acid (C) · Menaquinone
(K) · Coenzyme F420
• non-vitamins: ATP · CTP · SAM · PAPS · GSH · Coenzyme
B · Coenzyme M · Coenzyme Q · Methanofuran · BH4 · H4MPT
Organic prosthetic groups
• vitamins: TPP / ThDP (B1) · FMN, FAD (B2) · PLP / P5P
(B6) · Biotin (B7) · Methylcobalamin, Cobamamide (B12)
• non-vitamins: Haem / Heme · Lipoic acid · Molybdopterin · PQQ
Metal prosthetic groups
• Ca2+ · Cu2+ · Fe2+, Fe3+ · Mg2+ · Mn2+ · Mo · Ni2+ · Se · Zn2+
•Covalently or noncovalently attached to many
conjugated proteins （结合、共轭蛋白）, and give
the proteins chemical functionality. Many are cofactors in enzyme reactions.
Fig. Since not enough energy is
liberated in oxidation of succinate
and transfer of e to ubiquinon no
free hence no gain in free energy
results in no pumping of protons
in complex II.
B2 Protein structure and function—
What do proteins do
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Enzymes
Signaling
Transport and storage
Structure and movemrnt
Nutrition
Immunity
Regulation
B2 Protein structure and function—
Domains, motifs, families and evolution
• A protein domain is a part of protein sequence and
structure that can evolve, function, and exist
independently of the rest of the protein chain.
• Structural motif, a
pattern in a protein
structure formed by the
spatial arrangement of
amino acids
• A protein family is a
group of evolutionarily
related proteins, and is
often nearly
synonymous with gene
family.
Pyruvate kinase, a protein
from three domains
B3 Protein analysis—
•
•
•
•
•
•
•
Protein Purification
Gel filtration chromatography
Ion-exchange chromatography
Isoelectric focusing
Electrophoresis
Hydrophobic interaction chromatography
Affinity chromatography
Overexpression
<size> Gel filtration chromatography
<size and charge>
Ion-exchange
chromatography
<charge>
Isoelectric focusing
<charge>
Electrophoresis
<hudrophpbicity>
Hydrophobic interaction
chromatography
< affinity>
Affinity
chromatography
Overexpression
• In the laboratory, the protein encoded by a gene is sometimes
expressed in increased quantity. This can come about by increasing
the number of copies of the gene or increasing the binding strength
of the promoter region.
Example：
His · tag
T7 promoter
lac operator
Escherichia coli
T7 promoter
Vector lac operator
lacI coding sequence
His · tag
IPTG (a lactose analog)
B3 Protein analysis—
Protein sequencing
• Protein sequencing :
Determine the primary structure of a protein
Peptide sequencing by tandem mass spectrometry - backbone cleavages
• The two major direct methods of protein sequencing
are mass spectrometry and the Edman
degradation reaction.
• Edaman degradation:
1. Performed in an automated protein sequencer
2. Determine the sequence of a polypeptide from
N-terminal amino acid one by one.
3. Expensive and laborious
Edman
degradation
Mechanism
Specific enzyme/ chemical cleavage:
• Trypsin cleaves after lysine( K) or arginine ( R)
• V8 protease cleaves after glutamic acid (E)
• Cyanogen bromide cleaves after methionine
(M)
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
B3 Protein analysis—
• Gel filtration
chromatography
• Electrophoresis
( SDS-PAGE)
• Mass
spectrometry
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 510% 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
B3 Protein analysis—
X-ray crystallography and NMR
X-ray crystallography
•Measuring the pattern of diffraction of a beam of Xrays 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.
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.
B3 Protein analysis—
Functional analysis
• Tertiary and quaternary structural determination is still a
relatively cost and laborious procedure.
• Computational methods will allow the prediction of both
structure and possible function from simple amino acid
sequence information.
• Understanding of the true function of a protein still
requires its isolation and biochemical and structure
characterization.
• Identification of all the other proteins with which a protein
interacts in the cell is another important aspect of
functional analysis.
• If the gene foe the protein can be inactivated by
mutagenesis or deleted be recombinant DNA techniques,
then the phenotype of the resulting mutant can be
studied.
B3 Protein analysis—
Proteomics
• Proteomics is the identification and analysis of
the total protein complement expressed by any
given cell type under defined conditions.
• Two-dimensional Protein Electrophoresis
• peptide mass fingerprint
Two-dimensional Protein Electrophoresis (2DE)
Analysis of peptide mass
fingerprint of the tryptic digest of
P. falciparum actin I by MALDI
mass spectrometry. a. Spectrum
(500-3500 Da) with zoom-scan
(1850-1990 Da) showing Nterminal fragment at 1872 Da
and fragment containing
histidine 73 at 1947 Da. b. The
table shows selected peptide
matches from three
experiments. N-terminal
fragment and fragment
containing histidine 73 are in
bold.
Multiple choice questions
1. Which of the following is an imino acid?
A proline.
B hydroxy lysine.
C tryptophan.
D histidine.
2． Protein family members in different species that
carry out the same biochemical role are described
as
.
A paralogs.
B structural analogs.
C heterologs.
D orthologs.
3. Which of the following is not a protein secondary
structure?
A α-helix.
B triple helix.
C double helix.
D ß-pleated sheet.
4． In isoelectric focusing, proteins are separated
.
A in a pH gradient.
B in a salt gradient.
C in a density gradient.
D in a temperature gradient.
5．Edman degradation sequences
peptides . . .
A using a cDNA sequence.
B according to their masses.
C From the C-terminus to the N-terminus.
D from the N-terminus to the C-terminus.
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