Section B Protein Structure

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B2 Protein structure -Secondary

• a-helix
• right-handed
• 3.6 aa per turn
• hydrogen bond
• N-HOC

A stereo, space-filling representation
Collagen triple helix three polypeptide
intertwined
2
b-sheet hydrogen bonding of the peptide bond N-H
and CO groups to the complementary groups of
another section of the polypeptide chainx
Parallel b sheet sections run in the same
direction Antiparallel b sheet sections run in
the opposite direction
A stereo, space-filling representation of the
six-stranded antiparallel b sheet.
fibroin
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B2 Protein structure and function
A two-stranded b sheet
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B2 Protein structure -Tertiary
The different sections of a-helix, b-sheet, other
minor secondary structure and connecting loops of
a polypeptide fold in three dimensions
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B2 Protein structure and function
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
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B2 Protein structure -Quaternary
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.
A stereo, space-filling drawing showing the
quaternary structure of hemoglobin
a1-yellow b1-light blue a2-green b2-dark blue
heme-red
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• Advantages of the quaternary structure
• It allows very large protein molecules to be
  made, such as tubulin?
• It can provide greater functionality to a protein
  by combining different activities into a single
  entity.
• The interactions between the subunits can often
  be modified by binding of small molecules and
  lead to the allosteric effects seen in enzyme
  regulation

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B2 Protein structure and function
Protein Folding chaperones are involved in vivo

1. The rapidly reversible formation of local
   secondary structure
2. Formation of domains through the cooperative
   aggregation of folding nuclei
3. Assembly of domains into a molten globule
4. Conformational adjustment of the monomer
5. Final conformational adjustment of the dimeric
   protein to form the native structure.

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B2 Protein structure and function
Prosthetic groups (??) covalently or
noncovalently attached to many conjugated
proteins, and give the proteins chemical
functionality. Many are co-factors in enzyme
reactions. Apoprotein (?????) Examples heme
groups in hemogobin (Figure)
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B2 Protein structure and function

• Biological functions of proteins
• Enzymes substrate binding, side chain in
  catalysis
• Signaling cell membrane
• Transport and storage hemoglobin transports
  oxygen
• Structure and movement collagen, keratin,
  tubulin in cytoskeleton, actin and myosin for
  muscle contraction
• Nutrition casein (???) and ovalbumin(????)
• Immunity antibodies
• Regulation transcription factors

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B2 Protein structure - Domains, motifs and
families
Domains structurally independent units of many
proteins, connected by sections with limited
higher order structure within the same
polypeptide. (Figure) They can also have
specific function such as substrate binding
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• 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

bab motif
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Protein families structurally and functionally
related proteins from different sources
Motif
The primary structures of c-type cytochromes from
different organisms
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Protein purification

• An essential experimental step when study any
  individual protein

• An experimental step to purify the interested
  protein from other proteins and nonprotein
  molecules existing in the cells

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The principal properties of proteins used for
purification

1. Size gel filtration chromatography

2. Charge ion-exchange chromatography,
isoelectric focusing electrophoresis
3. Hydrophobicity hydrophobic interaction
chromatography
4. Affinity affinity chromatography
5. Recombinant techniques involving DNA
manipulation and making protein purification so
easy
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1. gel filtration chromatography

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2. Charge ion-exchange chromatography,
isoelectric focusing, electrophoresis
Isoelectric point (pI) the pH at which the net
surface charge of a protein is zero
-

-

-

-

-

-
-


-

pHpI
pHltpI
pHgtpI
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Ion-exchange chromatography
Sample mixture



Ion displacing
Protein binding
Column anions
Column anions
Column proteins
Purified protein
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Electrophoresis
Protein migrate at different position depending
on their net charge
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Isoelectric focusing
A protein will stop moving at position
corresponding to its isoelectric point (pI) in a
pH gradient gel.
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3. Hydrophobicity hydrophobic interaction
chromatography
Similar to ion-exchange chromatography except
that column material contains aromatic or
aliphatic alkyl groups
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4. Affinity chromatography

• Enzyme-substrate binding

Substrate analogs competitive inhibitors
ding

• Receptor-ligand binding

d

• Antibody-antigen binding

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5. Recombinant techniques

• Clone the protein encoding gene of interest 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.

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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.

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Mass Determination
Mass spectrometry

• Molecules are vaporized and ionized (by Xe/Ar
  beam), and the degree of deflection
  (mass-dependent) of the ions in an
  electromagnetic field is measured

• Extremely accurate (0.01 error), but expensive

• ESI (electrospray ionization) and MALDI
  (matrix-assisted laser desorption/ionization) can
  measure the mass of proteins smaller than 100 KDa

• Protein sequencing relying on the protein data
  base

• Helpful to detect post-translational modification

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Determine the primary structure of a protein p

• Amino acid composition
• Acid treatment to hydrolyze peptide bonds 6M
  HCl, 110C for 24 hrs.
• Chromatographic analysis
• However, you cannot get the sequence!

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Determine the primary structure of a protein
protein sequencing

• Specific enzyme/chemical cleavage
• Pepsin cleaves after lysine(K) or arginine (R)
• V8 protease cleaves after glutamic acid (E)
• Cyanogen bromide cleaves after methionine (M)

• Edman degradation
• Performed in an automated protein sequencer
• Determine the sequence of a polypeptide from
  N-terminal amino acid one by one.
  Phenylisothiocyanate ????? reaction)
• Expensive and laborious

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Protein sequence analysis (1) Sequence
HLMGSHLVDALELVMGDRGFEYTPKAWLV Pepsin T1
HLMGSHLVDALELVMGDR T2
GFEYTPK T3
AWLV V8 V1 HLMGSHLVDALE V2
LVMGDRGFE V3
YTPKAWLV
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Protein sequence analysis (2) Computer
analysis T1 HLMGSHLVDALELVMGDR
V1 HLMGSHLVDALE V2 LVMGDRGFE
T2 GFEYTPK V3
YTPKAWLV T3
AWLV Sequence HLMGSHLVDALELVMGDRGFE
YTPKAWLV
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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
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X-ray crystallography and NMR Determing the 3-D
structure of a protein
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
  3-D structure

• Many proteins have been crystallized and analyzed

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X-ray crystallography and NMR Determing the 3-D
structure of a protein
NMR Nuclear magnetic resonance (NMR) spectroscopy

• 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

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• Summary
• 20 Amino acids D E (-), H, K, R (), S, T, C,
  N, Q (polar), G,A,V,L, I, M, P (nonpolar), F, Y,W
  (aromatic)
• Protein structure primary, secondary, tertiary,
  quaternary
• Protein analysis purification, sequencing, mass
  determination, X-ray crystallography and NMR