Protein Notes

Chapter 5: Proteins

  • Chapter 5: Proteins

Part 1: Polypeptide Diversity

  • Part 1: Polypeptide Diversity

Peptides: A Variety of Functions

  • Peptides have a variety of functions.

  • Hormones and pheromones:

    • Insulin (think sugar)

    • Oxytocin (think childbirth): stimulates the muscles of the uterus to contract

  • Neuropeptides

    • Substance P (pain mediator)

  • Antibiotics

    • Viomycin: used in a drug cocktail against M. tuberculosis

  • Protection, e.g., toxins

    • Amanitin (mushrooms)

    • Conotoxin (cone snails)

    • Chlorotoxin (scorpions)

Levels of Structure in Proteins

  • Four levels:

    • Primary structure = covalent bonds linking amino acid residues in a polypeptide chain

    • Secondary structure = recurring structural patterns

    • Tertiary structure = 3D folding of polypeptide

    • Quaternary structure = 2+ polypeptide subunits

Peptide Subunits

  • Some proteins consist of a single polypeptide chain

  • Multisubunit protein: have two or more polypeptides associated noncovalently.

    • However, disulfide bonds can be made

  • The individual polypeptide chains in a multisubunit protein may be identical or different.

  • Oligomeric protein: If at least two identical subunits.

    • Identical units = protomers

    • hydrophobic interxn VdW Hbond salt bridge

Amino acid sequence of bovine insulin

  • The primary structure of bovine insulin, protein hormone discovered by Frederick Sanger in 1953

  • Two polypeptide chains are joined by disulfide cross linkage.

    • Intrachain disulfide bond

    • Interchain Disulfide bonds

  • Watson crick DNA double helix

Sanger's Nobel Prize and Sequencing

  • 1980: 2nd Nobel Prize for Sequencing

  • 500 sp virus genome chain terminating method

  • Later this tech was used for human genome project

Protein Function and Amino Acid Sequence

  • The Function of a Protein Depends on Its Amino Acid Sequence

  • Escherichia coli produces more than 3,000 different proteins.

  • Human has ~25,000 genes encoding much larger number of proteins

  • Each type of protein has unique 3D structure.

  • Amino acid sequence confers 3D structure

  • 3D structure confers function

Protein Size Variation

  • Proteins Vary in Size

  • 40 residues: minimum to fold into a stable shape to carry out a specific function

  • More than 1000 residues: the longer the polypeptide, the greater the likelihood of introducing errors during and transcription and translation

  • Most Proteins Have 100-1000 Amino Acids

Other

  • Chymotrypsin

  • Figure 4-1

  • Glycine

Amino Acid Composition of Proteins

  • Amino Acid Composition of Proteins

  • Varying proportions of amino acids of proteins with different functions

  • Variability in use of additional compounds

Part 2: Protein Purification and Analysis

  • Part 2: Protein Purification and Analysis

Isolating Proteins from Cells

  • Isolating Proteins from Cells

    1. Get protein out of the cell and into solution

      • Mechanical disruption to release contents (“lyse” cells)

    2. Filter or centrifuge to remove large, insoluble particles

      • If membrane bound, add detergent to recover protein from lipids

Keeping Proteins Stable

  • Keeping Proteins Stable

    • pH – use of buffer

    • Temperature – thermal stability of proteins

    • Inhibition of degradative enzymes – proteases, nucleases

    • Surfaces and air/water interface – avoid foaming and keep concentrated.

    • Storage – prevent oxidation, microbial contamination, -80∘^{\circ}C

Protein Dialysis - Buffer Exchange

  • Protein Dialysis - Buffer Exchange

  • Diagram illustrating the process of dialysis to exchange buffers.

Proteins Separated and Characterized by Electrophoresis

  • Proteins Can Be Separated and Characterized by Electrophoresis

  • Electrophoresis = visualize and characterize purified proteins

  • Electric field causes proteins to migrate according to their size and charge

  • Gel matrix hinders mobility of proteins according to their size and shape

  • Can be used to estimate (It is an analytical method):

    • number of different proteins in a mixture

    • degree of purity

    • isoelectric point

    • approximate molecular weight

Electrophoresis: SDS-Polyacrylamide Gel Electrophoresis (SDS PAGE)

  • Electrophoresis: SDS-Polyacrylamide Gel Electrophoresis (SDS PAGE)

  • Diagram of SDS-PAGE setup and results.

Sodium Dodecyl Sulfate (SDS)

  • Sodium Dodecyl Sulfate (SDS)

  • Sodium dodecyl sulfate (SDS) = a detergent

    • binds and partially unfolds proteins

    • gives all proteins a similar charge-to - mass ratio

    • electrophoresis in the presence of SDS separates proteins by molecular weight

  • smaller proteins migrate more rapidly I Toner it

β-mercaptoethanol

  • β-mercaptoethanol

  • Breaks disulfide bonds

    • Intrachain disulfide bond

    • Interchain Disulfide bonds

  • Gel filtration us SDS PAGE inducing agent Girone in its native state possible quarter nary struct I B mercaptoethanol will separate complex start into individua subunit lead MW I more accurate MW

Electrophoresis: SDS-Polyacrylamide Gel Electrophoresis (SDS PAGE)

  • Electrophoresis: SDS-Polyacrylamide Gel Electrophoresis (SDS PAGE)

  • pl = ?

Electrophoresis for Protein Analysis – Separates by Mass

  • Electrophoresis for Protein Analysis – Separates by Mass

  • Uses cross-linked polymer polyacrylamide gels

  • Use standards to estimate molecular weight

  • Visualization = Coomassie blue dye binds to proteins

Estimating the Molecular Weight of a Protein

  • Estimating the Molecular Weight of a Protein

  • Plot of log Mr of marker proteins vs. relative migration during electrophoresis = linear

  • If protein has subunits more than one band appear

Using Isoelectric Focusing to Determine the pI of a Protein

  • Using Isoelectric Focusing to Determine the pI of a Protein

  • Description of the isoelectric focusing process.

  • Table of Isoelectric Points of Some Proteins

2D Gel Electrophoresis

  • 2D Gel Electrophoresis

  • 2D Gel Electrophoresis: Combining isoelectric focusing and SDS electrophoresis sequentially in a process

2D Gel Electrophoresis

  • 2D Gel Electrophoresis

  • Horizontal separation reflects differences in pI

  • Vertical separation reflects differences in molecular weight.

2D Gel Electrophoresis

  • 2D Gel Electrophoresis

  • Permits resolution of complex protein mixtures of proteins

  • More sensitive than individual methods

2D Gel Electrophoresis

  • 2D Gel Electrophoresis: separates proteins with Similar pI but different MW