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Fundamentals of
Biochemistry
Third Edition
Donald Voet • Judith G. Voet •
Charlotte W. Pratt
Chapter 5
Proteins: Primary Structure
Copyright © 2008 by John Wiley & Sons, Inc.
Section 1 – Polypeptide Diversity
Insulin primary structure
Primary Structure – linear sequence of amino acids in a polypeptide
chain
20100 = 1.27 x 10130 possible combinations
9 x 1078 atoms estimated in universe
Section 2 – Protein Purification
• Purification was difficult for a endogenous
protein
– First proteins studies were very abundant
• Modern cloning techniques all for
production of large quantities of specific
proteins
– This process still requires that the protein be
isolated from a cell, and purified from the other
cellular components
Conditions affect protein
Stability
• pH
– The wrong pH causes denaturation
• Temperature
– The wrong temperature can cause denaturation
• Presence of other proteins
– Proteases can destroy proteins
• Adsorption to surfaces
– Some proteins can be denatured upon exposure to air
• Long term storage
– Most proteins should be stored at -20°C or lower to minimize
degradation and denaturation
ELISA
Enzyme linked immunosorbent
assay
Used to determine (quantify) the
amount of protein present
Animation
Spectroscopic method for
determining protein
concentration
Beer-Lambert law
A=εcl
A280 – absorbance of F, Y, W
Colorimetric method for
determining protein concentration
Bradford assay
Salting Out
Ion Exchange Chromatography
Animation
Gel Filtration Chromatography
Animation
Affinity Chromatography
Immunoaffinity
Metal chelate
SDS-PAGE
Sodium-dodecyl sulfate – Poly acrylamide gel electrophoresis
Capillary Electrophoresis
2D Gel electrophoresis
Section 3 – Protein Sequencing
• Important to know the sequence of a protein
– Primary structure dictates shape
– Evolutionary comparisons can be made
– Diseases arise from mutations of primary
structure
Step 1.
Step 2
Step 3
Step 4
Edman
Degradation
Animation
Section 4 – Protein Evolution
Protein Evolution
• Homologue – evolutionarily similar
proteins within the same species
– Invariant residue – identical aa among
homologues
– Conserved residue – similar (class) aa among
homologues
– Hypervariable residue – no similarity among
homologues
Protein Evolution
• Domain – region of proteins that have very
similar folding patterns (40-200 aa)
• Orthologues – homologous proteins in
different species
• Paralogues – independently evolving genes
in the same species
• Pseudogenes – duplicated gene that are not
expressed
All problems at end of
chapter except 6, 13, and
19
You have isolated a decapeptide (10 residues) called FP, which has
anticancer activity. Determine the sequence of the peptide from
the following information.
1. One cycle fo Edman degradation of intact FP yields 2 mole of
PTH-aspartate per mole of FP.
2. Treatment of a solution of FP with 2-ME followed by the
addition of trypsin yields three peptides
(Ala, Cys, Phe) (Arg, Asp) (Asp, Cys, Gly, Met, Phe)
The intact (Ala, Cys, Phe) peptide yields PTH-cysteine in the
first cycle of Edman degradation.
3. Treatment of 1 mol of FP with carboxypeptidase (cleaves at Cterminus of all residues) yields 2 mol of phenylalanine.
4. Treatment of intact pentapeptide (Asp, Cys, Gly, Met, Phe) with
BrCN yields two peptides with composition (homoserine lactone,
Asp) and (Cys, Gly, Phe) The (Cys, Gly, Phe) peptide yields PTHglycine in the first cycle of Edman degration.