voet 05 - 5 Proteins Primary Structure This chapter covers...

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5 Proteins: Primary Structure This chapter covers protein purification and primary structure. In this chapter you will learn how a protein’s size, charge, and general shape can be analyzed and used to develop procedures to purify the protein. Many of the principles you learned in the preceding chapters about thermodynamics, aqueous solutions, and acid–base chemistry can be applied to the isolation of proteins. This chapter also includes a discussion of the strategies and chemical methods for determining the primary structure of proteins, focusing on Edman degradation and introducing mass spectrometry as the latest technique for determining polypeptide mass and sequence. The chapter concludes with a section discussing protein evolution. Here you will see how comparisons of primary structures have led biochemists to categorize proteins into families and identify specific modules or motifs involved in specific functions. Essential Concepts Polypeptide Diversity 1. The primary structure of a protein is the amino acid sequence of its polypeptide chain (or chains if the protein contains more than one polypeptide). 2. Proteins are synthesized in cells by the stepwise polymerization of amino acids in the order specified by the sequence of nucleotides in its gene. The 5 end of the messenger RNA corresponds to the amino terminus of the polypeptide, which is the end that contains a free amino group bound to the α carbon. 3. Although the theoretical possibilities for polypeptide composition and length are unlimited, polypeptides found in nature are limited somewhat in size and composition. Most polypeptides contain between 100 and 1000 residues and do not necessarily include all 20 genetically encoded amino acids. Leu, Ala, Gly, Ser, Val, and Glu are the most abundant amino acids in proteins, while Trp, Cys, Met, and His are the least common. Protein Purification and Analysis 4. A general approach to protein purification requires the following: (a) A rapid and efficient method to disrupt cells so that the contents of the lysed cells (lysate) can be quickly stabilized in a buffer of appropriate pH and ionic strength.
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Chapter 5 Proteins: Primary Structure 54 (b) Consideration of factors that affect the stability of the desired protein, such as pH, temperature, presence of degradative enzymes, adsorption to surfaces, and solvent conditions for long term storage. (c) Appropriate separation techniques to effectively purify the desired protein from the total proteins of the lysate. (d) A test or assay to easily assess the activity of the desired protein at each step in the purification. 5. A protein contains multiple charged groups, so its solubility in aqueous solution varies with the concentrations of dissolved salts (ionic strength), pH, and temperature. When salt is added to a protein at low ionic strength, the protein’s solubility increases with increasing ionic strength (called salting in). At some point, the solubility of the protein decreases (called salting out). This variable solubility can be exploited to selectively precipitate
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This note was uploaded on 08/12/2009 for the course IQ 23123 taught by Professor Varios during the Spring '09 term at Universidade de Brasília.

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voet 05 - 5 Proteins Primary Structure This chapter covers...

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