protein chemistry, Biochemistry determination of the protein structure by the different mechanism.

1. By Dr Anurag Yadav
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2. • Biomedical importance.
• Steps in determining the amino acid sequence.
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3. Biomedical importance :
Proteins are physically and functionally complex macro-
molecules, that perform multiple critically important roles.
Proteins are subjected to physical and functional changes that
mirror the life cycle of the organism in which they reside.
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4. • Typically protein is born at translation,
-matured through post-translational processing events such as
partial proteolysis,
-alternate btwn working and resting state through the
interventions of regulatory factors,
-ages through oxidation, deaminations etc.,
-& dies when it is degraded to its amino acids.
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5. • Important goal is to :
-identification of the proteins
-& provides both a molecular finger print for its
identification & information that can be used to identify ,
clone gene/ genes that encodes it.
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6. In order to study the structure and function of
protein, the interest of protein molecule must be
isolated in a pure form.
Several methods are available:
a. Methods based on solubility properties.
b. Methods based on charge properties.
c. Methods based on molecular size.
d. Methods based on the affinity of protein
for other molecules.
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7. 1st – determine no of peptide chains
2nd – determine AA composition
3rd – identification of N and C terminal AA
4th – sequence edmans degradation(20-30 AA)
5th – very long chain proteins by hydrolysis
6th - position of disulphide bonds
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8. Determine AA composition
Complete hydrolysis
Chromatographic separation
identification
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9. Determine number of peptide chains
• Dansyl chloride + N terminal AA of the peptide
• No and nature of dansyl AA determined
• Indicated no of polypeptide chain in the protein
• 2 different peptide chains 2 different dansyl AA
complete hydrolysis by boiling with 6 N
HCl at 110 degree for 18-36 hours
under anaerobic conditions
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10. Identification of N and C terminal AA
• N terminal  Dansyl chloride and sanger reagent ( FDNB
– fluorodinitrobenzene )
• C terminal  carboxypeptidase A and B
• Continued action of A and B results in removal of AA
from C end
• A – not acts if C end  Arginine,proline/lysine
• B – acts only if penultimate residue is proline
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11. Edman degradation (20-30 AA )
• It is Series of reaction which involves stepwise labeling,
removal & identification of AA from N terminal residue
of an peptide, leaving all other peptide intact is called as
Edman degradation.
• Stepwise removal of AA
• Start from N terminal.
• Determination of AA sequencing of protein.
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14. • Coupling reaction
• Cleavage reaction
• Conversion reaction
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16. Coupling reaction
Phenylisothiocyanate ( PITC ) + N terminal amino group of
peptide chain
Phenylthiocarbamyl (PTC) derivative of the peptide
PTC washed with organic solvent ( benzene ) to extract
excess PITC and side products
Dried under vaccum
inert atmosphere ( to avoid
oxidation of sulphur atom of
PITC)
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17. Cleavage reaction
Dried PTC derivative + anhydrous acid
( trifluroacetic acid )
Cleavage of PTC polypeptide ( peptide bond
near PTC )
release original N terminal AA residue (2-anilino-
5-thiazoline derivative)
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18. Conversion reaction
unstable Thiazolinone ( derivative of N terminal
AA )
more stable derivative PTH
( isomeric 3 phenyl 2 thiohydantoin )
heating thiazolinone in 1 M
HCl at 80 degree for 10 min
PTH - AA is end product of one cycle of
edmans degradation
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20. • Degraded AA  identified by reverse HPLC
• A new amino terminal is exposed,
• Procedure is repeated until the entire sequence is
determined.
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21. • All steps + injecting into HPLC column + identification of
PTH derivative
Sequencing  automated analyzer (sequenator)
that mixes reagent in proper proportion,
separate the products, identify them & record results.
run for overnight
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22. Very long chain proteins
• Very long chain proteins
• small peptides of overlapping sequence
• Purified and Edmans degradation
Hydrolysis by 2 or more different site specific
enzymes (partial hydrolysis )
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23. Protein cleavage and peptide
formation
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• Two methods of cleavage
– Chemical method
– Enzymatic method

24. • ENZYMATIC METHOD: this is done by subjecting the
polypeptide chain to hydrolysis by 2/more different site
specific enzymes.
- Trypsin : hydrolyses peptide bond formed by the carboxyl
group of Lysine/Arginine.
- Chymotrypsin: preferntially acts on the peptide bonds formed
by the carboxyl group of the aa phenylalanine, tyrosine,
tryptophan or leucine.
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25. CHEMICAL METHOD: cyanogen bromide is used for
cleave peptide bond on the carboxyl end of the
methionine.
• N – bromosuccinimide cleaves tryptophan
• these AA rarely occurs in protein structure
• tends to produce large peptides
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27. • Native protein cleaved by proteolytic enzymes
• Intact disulphide bridges + small peptides
• Dithiothreitol (DTT) cleaves disulphide bridge
• Cleaved peptides disappears (disulphide
bond)
• Reappear as peptides of lower mass
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29. Ordering an Peptide fragment
• The sequenced peptides may be arranged into complete
sequence of original peptide by generating the overlapping
fragments;
- overlapping fragments are generated.
- overlapping fragments are sequenced.
- overlapping sequence are matched.
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31. Higher levels of protein structure
• X ray diffraction
• Nuclear magnetic resonance
• Ultraviolet light spectroscopy
• Optical rotatory dispersion
• Circular dichroism
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32. X ray crystallography
• Crystal of protein is first
produced ( represents 3
dimensional lattice of that
molecule )
• Crystal is mounted inside the
capillary tube with solution
from which crystallized ( to
prevent drying )
• Seal the tube and allow to
pass x ray beams
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33. • Diffraction data and phase information is collected.
• Using computer electron density map is plotted.
• Using computer graphics known protein sequence is
fitted into electron density map.
• Produce the three dimensional model of a protein.
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34. NMR spectroscopy
• Measures absorbance of radiofrequency of atomic nuclei
• By knowing the frequency at which particular nucleus
absorbs energy
• We can identify functional group available in the
molecule
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37. Mass spectrometry
• MS applicable  small nonpolar molecules
• Absolute requirement ions in gas phase
• Fast atom bombardment (FAB) , ESI ,MALDI methods
development in ionization technology by introduction
of these methods  analysis of large charged
molecules such as proteins and peptides possible
• Ions corresponding to one peptide is selected in the
first analyser  colloided with argon gas in a collison
cell to generate fragment ions  fragment ions thus
generated separated according to mass , in a second
analyser  identified and sequence determined
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38. • Structure determined  only in pure form
• Chromatographic techniques Ion exchange , adsorption ,
partition , size exclusion , affinity , HPLC
• Purity of protein  electrophoresis
• Molecular weight  mass spectrometry
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39. Hydrophobicity profile
• Average hydrophobicity per residue against sequence
number
• Averaging is achieved by evaluating and using prediction
algorithms
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