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.

Primary structure of proteins = amino acid sequence By convention, always written N (amine) terminus to C (carboxylic acid) terminus Bovine insulin: 1 protein, 2 chains (subunits) held together by two disulfide bridges between cysteine side chains (Sanger and Tuppy, Biochem. J. 49 (4): 463–81, 1951)

Table 5-1

Proteins function when their primary, secondary, tertiary and (if applicable) quaternary structures are intact. If any of these are lost, the protein is denatured. Denaturation is reversible, in most cases. Protein stability is dependent on:

Proteins function when their primary, secondary, tertiary and (if applicable) quaternary structures are intact. If any of these are lost, the protein is denatured. Denaturation is reversible, in most cases. Protein stability is dependent on: • pH • temperature • concentrations of ions in solution • absence of proteases and surfaces

Quantifying protein concentration ELISA: Enzyme-linked immunosorbent assay

Quantifying protein concentration Proteins absorb in the ultraviolet part of the EM spectrum. So, in principle, a spectrometer may be used to quantify protein concentration at 280 nm (aromatic rings) or 200 nm (peptide bond). Problem: nucleic acids absorb at 260 nm.

Purifying proteins

Purifying proteins: “salting out”

Purifying proteins: chromatography and the isoelectric point

Purifying proteins: ion exchange chromatography By changing the pH or ionic strength of the eluting solution, a mixture of proteins may be separated

Purifying proteins: gel filtration chromatography This separates by molecular size

Purifying proteins: affinity chromatography For instance, I used lentil lectin-infused beads as the stationary phase to purify a glycoprotein in a protein mixture, because of the lectin’s affinity for certain sugars.

Purifying proteins: SDS-PAGE electrophoresis SDS = sodium dodecyl sulfate (a surfactant that denatures proteins into long strands) PAGE = polyacrylamide (the matrix material) gel electrophoresis

Purifying proteins: electrophoresis

Purifying proteins: electrophoresis AND isoelectric focusing: 2D gel electrophoresis

Purifying proteins: ultracentrifugation By spinning a protein mixture in an ultracentrifuge (>100,000 g), you can get proteins to settle at the bottom of a centrifuge tube. The rate at which it does so is quantified by the sedimentation coefficient, which is measured in Svedbergs (S), which is a unit equal to 10–13 seconds.

Purifying proteins: ultracentrifugation By spinning a protein mixture in an ultracentrifuge (>100,000 g), you can get proteins to settle at the bottom of a centrifuge tube. The rate at which it does so is quantified by the sedimentation coefficient, which is measured in Svedbergs (S), which is a unit equal to 10–13 seconds. Yes, this is the same “S” found in, say, organelle subunits, like the 16S subunit of the bacterial ribosome.

Protein sequencing

Protein sequencing: Sanger method Upon hydrolysis, the N-terminal amino acid has a chromophore attached, which can be separated and identified Box 5-1

Protein sequencing: Dansyl chloride Similar to the Sanger method – N-terminus residue

Protein sequencing: Cleave the disulfide bridge to free sub-units

Protein sequencing: Prevent the disulfide bridge from reforming by preventing oxidation of thiol

Protein sequencing: Selective cleaving of peptide bonds Trypsin is an enzyme that cleaves the peptide bond at the C-terminus of lysine or arginine (if the N-terminus is not a proline)

endo- = within; exo- = outside of

Protein sequencing: Edman degradation Can remove up to 100 residues from the N-terminus, one at a time Protein sequencing: Edman degradation Pehr Edman (1950)

Protein sequencing: Mass spectrometry After selective cleavage of proteins, electrospray ionization mass spectrometry allows for the rapid calculation of fragment size

Protein sequencing: Mass spectrometry sample output Sample calculation 5-1 shows the original protein to have a mass of 16,975 D

Protein sequencing: Tandem mass spectrometry

Protein sequencing: Aligning the fragments

Protein sequencing: The placement of disulfide bridges can be determined

Proteomics Also, the BioSample database at ncbi.nlm.nih.gov

Figure 5-20

Table 5-5 part 1

Table 5-5 part 2

Cytochrome c in different organisms: numbers indicate differences per 100 residues

Human hemoglobin = α2 β2

What does a steep slope mean?

Proteins evolve by repeating or adding domains