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Fundamentals of Biochemistry
Third Edition
Fundamentals of Biochemistry
Third Edition
Chapter 5Proteins: Primary Structure
Chapter 5Proteins: Primary Structure
Copyright © 2008 by John Wiley & Sons, Inc.Copyright © 2008 by John Wiley & Sons, Inc.
Donald Voet • Judith G. Voet • Charlotte W. Pratt
Donald Voet • Judith G. Voet • Charlotte W. Pratt
Section 1 – Polypeptide Diversity
Insulin primary structure
Primary Structure – linear sequence of amino acids in a polypeptide chain
20100 = 1.27 x 10130 possible combinations9 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=εclA280 – 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-PAGESodium-dodecyl sulfate – Poly acrylamide gel electrophoresis
Capillary Electrophoresis2D 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
EdmanDegradation
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 C-terminus 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 PTH-glycine in the first cycle of Edman degration.