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 20 100 = 1.27 x 10 130 9 x 10 78 possible combinations 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 A 280 – 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 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.