principles of keeping proteins in their native state Flashcards
what is the primary protein structure?
the sequence of amino acids in a polypeptide chain held together with peptide bonds.
what is the secondary protein structure?
alpha-helix, beta-pleated sheets held together by hydrogen bonds.
what is the tertiary protein structure?
interactions between R-groups that give the protein its 3D arrangement.
what bonds are involved in tertiary protein structure?
- van der waals
- ionic bonds
- hydrophobic interaction between R-groups
- hydrogen bonds
- disulphide bridges between cystine and methionine.
what is the quaternary protein structure?
more than one polypeptide subunit exhibiting all previous bonds.
what is denaturation?
denaturation is the major change in a proteins structure by application of an external stress or compound.
when a protein is denatured the shape of the active site is altered which prevents a substrate from binding and completing its function, rendering the protein non-functional.
what happens to quaternary protein structure during denaturation?
quaternary structure proteins break up into individual polypeptide subunits.
what happens to tertiary protein structure during denaturation?
tertiary structure is disrupted by the breaking of bonds: ionic interactions, hydrogen bonds, van der waals, and hydrophobic interactions between R-groups.
what happens to secondary protein structure during denaturation?
secondary structure proteins unravel from alpha-helix or beta-sheets, as the hydrogen bonds vibrate and break due to an increase in kinetic energy.
what happens to primary protein structure during denaturation?
primary protein structure is not affected.
how is renaturation possible?
denaturation can be reversible. renaturation is possible as all the information for the protein is contained in the primary structure of protein.
what conditions can result in denaturation?
- increase in temperature
- change in pH
- use of chemical denaturants.
what is the effect of temperature in denaturation.
proteins have an optimum temperature of 37ºc - 40ºc. an increase in temperature above the optimum will provide too much kinetic energy, which causes the atoms to vibrate making them thermally unstable, this increase in energy overcomes the hydrogen bonds holding the proteins active site together, changing its shape therefore rendering the protein non-functional.
what is the effect of pH in denaturation?
a change in pH will alter the acidic and basic groups on amino acid side chains in a proteins active site, by altering the equilibrium point of ionisation, this renders the protein inactive but maintains its 3D arrangement.
what are proteases?
proteases breakdown proteins into short peptide fragments. (chemical denaturants)
give an example of chemical denaturants.
- trypsin
- urea
- SDS
how does trypsin work as a chemical denaturant?
trypsin cleaves the polypeptide chain at the carboxyl side of lysine and argenine, except when either is followed by a proline.
trypsin should be stored at very low temperatures to prevent autolysis.
how does urea work as a chemical denaturant?
urea denatures proteins by decreasing the hydrophobic effect, and by directly bonding to the amide groups of the protein via hydrogen bonding.
how does SDS work as a chemical denaturant?
SDS (sodium dodecyl sulphate) is a detergent (soap) with a hydrophobic tail which will dissolve hydrophobic molecules, and a negatively charged hydrophilic head.
if a cell is incubated with SDS, the membranes will be dissolved, all the proteins will be solubilised by the detergent, and all of the protein will be given a negative charge. the proteins will retain only their primary structure and will have a negative charge, which means they will all migrate towards the positive pole when placed in an electric field.
what is proteolysis?
proteolysis is the directed degradation of a protein by proteases or by intermolecular digestion which will decrease the amount of protein present in a sample.
what can be used to prevent proteolysis?
- protease inhibitor cocktail
- DTT and 2ME
what is a protease inhibitor cocktail?
a protease inhibitor cocktail is designed to inhibit all protease enzymes to limit degradation.
how are cysteine residues preserved?
DTT and 2ME are used to preserve cysteine residues. DTT and 2ME are reducing agents that reduce oxidation of cysteine residues, they affect the tertiary structure of a protein.
what are common methods of storage?
- solution at 4ºc
- solution in 50% glycerol at -20ºc
- frozen at -20ºc or -80ºc, or in liquid nitrogen
- lyphophilized.
