Fundamentals of Biochemistry

Fundamentals of
Biochemistry
Fourth Edition
Donald Voet • Judith G. Voet •
Charlotte W. Pratt
Chapter 5
Proteins: Primary Structure
Copyright © 2013 by John Wiley & Sons, Inc. All rights reserved.
Chapter 5
Polypeptide Diversity
Key Concepts 5.1
• In theory, the size and composition of a polypeptide chain
are unlimited.
• In cells, this potential variety is limited by the efficiency of
protein synthesis and by the ability of the polypeptide to fold
into a functional structure.
Bovine Insulin: 1° Structure
Most Proteins Have
100-1000 Amino Acids
Chapter 5
Polypeptide Diversity
Checkpoint 5.1
• Explain why polypeptides have such variable sequences.
• What factors limit the size and compositions of
polypeptides?
Chapter 5
Protein Purification & Analysis
Key Concepts 5.2
• Environmental conditions such as pH and temperature
affect a protein’s stability during purification.
• An assay based on a protein’s chemical or binding
properties may be used to quantify a protein during
purification.
• Fractionation procedures take advantage of a protein’s
unique structure and chemistry in order to separate it from
other molecules.
• Increasing the salt concentration causes selective “salting
out” (precipitation) of proteins with different solubilities.
Chapter 5
Protein Purification & Analysis
Key Concepts 5.2
• A protein’s ionic charge, polarity, size, and ligand-binding
ability influence its chromatographic behavior.
• Gel electrophoresis and its variations can separate proteins
according to charge, size, and isoelectric point.
• The overall size and shape of macromolecules and larger
assemblies can be assessed through ultracentrifugation.
Recombinant Proteins May Be Found in
Inclusion Bodies
Process Diagram: Enzyme-Linked
Immunosorbent Assay (ELISA)
Process Diagram: Enzyme-Linked
Immunosorbent Assay (ELISA)
Process Diagram: Enzyme-Linked
Immunosorbent Assay (ELISA)
Process Diagram: Enzyme-Linked
Immunosorbent Assay (ELISA)
Process Diagram: Enzyme-Linked
Immunosorbent Assay (ELISA)
Absorbance Spectra of
Aromatic Amino Acids
Protein Purification Procedures
Fractionation by Salting Out
Proteins are Least Soluble at
Their Isoeletric Point
Ion Exchange Chromatography
Gel Filtration Chromatography
Affinity Chromatography
SDS-PAGE Separates by Mass
SDS-PAGE Separates by Mass
2-D Gel Electrophoresis
Ultracentrifugation
Chapter 5
Protein Purification & Analysis
Checkpoint 5.2
• What are some environmental conditions that must be
controlled while purifying a protein?
• Describe how a protein may be quantified by an assay or by
absorbance spectroscopy.
• Explain how salting out is used in protein fractionation.
• Explain how an antibody could be useful for purifying a
protein and for determining its concentration.
• Describe the basis for separating proteins by ion exchange,
hydrophobic interaction, gel filtration, and affinity
chromatography.
• Describe the processes of gel electrophoresis, SDS-PAGE,
and 2D gel electrophoresis.
• What kinds of information can be gathered by
ultracentrifugation?
Chapter 5
Protein Sequencing
Key Concepts 5.3
• To be sequenced, a protein must be separated into
individual polypeptides that can be cleaved into sets of
overlapping fragments.
• The amino acid sequence can be determined by Edman
degradation, a procedure for removing N-terminal residues
one at a time.
• Mass spectrometry can identify amino acid sequences from
the mass-to-charge ratio of gas-phase protein fragments.
• Protein sequence data are deposited in online databases.
Process Diagram: Protein Sequencing
Process Diagram: Protein Sequencing
Process Diagram: Protein Sequencing
Box 5-1: Frederick Sanger
& Protein Sequencing
Dansyl Chloride for End Group Analysis
Dansyl Chloride for End Group Analysis
Dansyl Chloride for End Group Analysis
Disulfide Cleavage with Mercaptans
Iodoacetate Blocks Disulfide Formation
Endopeptidases Yield Small Protein
Fragments for Easier Sequencing
Chemical Cleavage Yields Small Protein
Fragments for Easier Sequencing
Chemical Cleavage Yields Small Protein
Fragments for Easier Sequencing
Chemical Cleavage Yields Small Protein
Fragments for Easier Sequencing
Chemical Cleavage Yields Small Protein
Fragments for Easier Sequencing
Endopeptidase Specificity
Edman Degradation
Edman Degradation
Edman Degradation
Electrospray Ionization (ESI)
Mass Spectrometry
Electrospray Ionization (ESI)
Mass Spectrum
Tandem Mass Spectrometry
Overlapping Fragments Allow
Determination of Amino Acid Sequence
Mapping Disulfide Bond Positions
Protein & DNA Sequence Data Banks
UniProt Database Entry
Chapter 5
Protein Sequencing
Checkpoint 5.3
• Summarize the steps involved in sequencing a protein.
• Why is it important to identify the N-terminal residue(s) of
a protein?
• What are some advantages of sequencing peptides by mass
spectrometry rather than by Edman degradation?
• Explain why long polypeptides must be broken into at least
two different sets of peptide fragments for sequencing.
• What types of information can be retrieved from a protein
sequence database?
Chapter 5
Protein Evolution
Key Concepts 5.4
• Sequence comparisons reveal the evolutionary
relationships between proteins.
• Protein families evolve by the duplication and divergence
of genes encoding protein domains.
• The rate of evolution varies from protein to protein.
1º Protein Structure From Related
Species Resemble One Another
Phylogenetic Tree Reveals
Evolutionary History
Genealogy of Globin Proteins
Sequence Divergence Rates Vary
Construction of Multidomain Proteins
Chapter 5
Protein Evolution
Checkpoint 5.4
• How can sequence comparisons reveal which amino acid
residues are essential for a protein’s function?
• Using Table 5-6, identify some invariant, conservative, and
hypervariable positions. What types of amino acids appear at
conservatively substituted sites?
• Explain how the number of amino acid differences
between homologous proteins can be used to construct a
phylogenetic tree.
• Explain the origin of orthologous proteins, paralogous
proteins, and multidomain proteins.
• Why do different proteins appear to evolve at different
rates?
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