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Chapter 5 Proteins: Their Primary Structure and Biological Function. Essential Question. What structural forms do polypeptide chains assume, how can the sequence of amino acids in a protein be determined, and what are the biological roles played by proteins?. Outline.
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- Primary (1°) - sequence
- Secondary (2°) - local structures - H-bonds
- Tertiary (3°) - overall 3-dimensional shape
- Quaternary (4°) - subunit organization
(a) Proteins having structural roles in cells are typically fibrous and often water insoluble. (b) Myoglobin is a globular protein. (c) Membrane proteins fold so that hydrophobic amino acid side chains are exposed in their membrane-associated regions.
Bovine pancreatic ribbonuclease A contains 124 amino acid residues, none of which are Trp. Four disulfide bridges are indicated in gold.
Secondary structures in proteins
The α-helix and the β-pleated strand are the two principal secondary structures found in proteins.
Folding of the polypeptide into a compact, roughly spherical conformation creates the tertiary (3°)level of protein structure.
Hemoglobin is a tetramer consisting of two α and two β polypeptide chains.
Imagine the conformational possibilities for a protein in which two of every three bonds along its backbone are freely rotating single bonds.
Enzyme linked immunosorbent assay
Used to determine (quantify) the amount of protein present
Spectroscopic method for determining protein concentration
A280 – absorbance of F, Y, W
Colorimetric method for determining protein concentration
A typical protein purification scheme uses a series of separation methods. Note the dramatic increase in specific activity* of the enzyme through a series of five different purification procedures.
*The term “specific activity” refers to the activity of the enzyme per mg of protein.
Techniques, Figure 1.
A dialysis experiment. The solution of macromolecules is placed in a semipermeable membrane bag, and the bag is immersed in a bathing solution. Diffusible solutes in the dialysis bag equilibrate across the membrane.
Sodium-dodecyl sulfate – Poly acrylamide gel electrophoresis
Techniques, Figure 6.
A plot of protein mobility versus log of molecular weight of individual peptides.
Techniques, Figure 7.
A two-dimensional electrophoresis separation. Macromolecules are first separated according to charge by isoelectric focusing in a tube gel. The gel containing separated molecules is then place on top of an SDS-PAGE slab, and the molecules are electrophoresed into the SDS-PAGE gel, where they are separated according to size.
- real amino acid sequencing
- sequencing the corresponding DNA in the gene
The hormone insulin consists of two polypeptide chains, A and B, held together by two disulfide (S-S) cross-bridges. The A chain has 21 amino acid residues and an intrachain disulfide; the B polypeptide contains 30 amino acids.
1. If more than one polypeptide chain, the chains are separated and purified.
2. Intrachain S-S (disulfide) cross-bridges are cleaved.
3. The N-terminal and C-terminal residues are identified.
4. Each polypeptide chain is cleaved into smaller fragments, and the composition and sequence of each fragment is determined.
5. Step 4 is repeated, using a different cleavage procedure to generate a different and overlapping set of peptide fragments.
6. The overall amino acid sequence of the protein is reconstructed from the sequences in overlapping fragments.
Separation of chains
- extreme pH
- 8M urea
- 6M guanidine HCl
- high salt concentration (usually ammonium sulfate)
Cleavage of Disulfide bridges
- dithiothreitol or dithioerythritol
- to prevent recombination, follow with an alkylating agent like iodoacetate
Identify N- and C-terminal residues
Identify N- and C-terminal residues
Fragmentation of the chains
The products of the reaction with trypsin are a mixture of peptide fragments with C-terminal Arg or Lys residues and a single peptide derived from the C-terminal end of the polypeptide.
Compare cleavage by trypsin and staphylococcal protease on an unknown peptide:
Overlap of the two sets of fragments:
e.g., cytochrome c is highly conserved
Frequencies of amino acids in the proteins of the SWISS-PROT database.
Alignment of the amino acid sequences of two protein homologs using gaps. Shown are parts of the amino acid sequences of the catalytic subunits from the major ATP-synthesizing enzyme (ATP synthase) in a representative archaea and a bacterium. These protein segments encompass the nucleotide-binding site of these enzymes.
Identical residues in the two sequences are shown in red. Introduction of a three-residue-long gap in the archaeal sequence optimizes the alignment of the two sequences.
The BLOSUM62 substitution matrix provides scores for all possible exchanges of one amino acid with another.
The sequence of cytochrome c from more than 40 different species reveals that 28 residues are invariant. When the sequences of a given protein from multiple organisms are homologous, they are said to be “orthologous”.
This phylogenetic tree depicts the evolutionary relationships among organisms as determined by the similarity of their cytochrome c sequences.
The sequence of cytochrome c is compared with an inferred ancestral sequence represented by the base of the tree on the previous slide. Uncertainties are denoted by question marks.
The amino acid sequences of the globin chains of human hemoglobin and myoglobin show a strong degree of homology.
Compare this structure with the structures of the β-chain of horse methemoglobin and that of sperm whale myoglobin.
Figure 5.21 Compare this structure with the structures of the α-chain of horse methemoglobin and that of sperm whale myoglobin.
This evolutionary tree is inferred from the homology between the amino acid sequences of the α–globin, β-globin, and myoglobin chains.
The tertiary structures of hen egg white lysozyme and human α-lactalbumin are very similar.
The tertiary structure of hexokinase. Compare this structure with that of G-actin These two proteins have different sequences and different functions, but similar tertiary structures.
Proteins may be "conjugated" with other chemical groups