Levels of protein structure Flashcards
- What is the primary structure?
- sequence of amino acids along a protein chain is called its primary structure
How is primary structure represented?
sequence of three later names of the relevant amino acids
How is the primary structure held together
- covalent bond
- peptide linkage
- relatively stable
- requires harsh condition - e.g. boiling with 6 mol dm-3 hydrochloric acid - to break the amino acids apart
What is the secondary structure?
- hled in place by hydrogen bonds
- between - for example - C=O and -N-H groups
WHat are teh structures formed from the secondary structure?
- protein chain may form a helix (alpha helix)
- or a folded sheet (beta pleated sheet
What are the characteristics of the secondary structure?
- hydrogen bonds are much weaker than covalent bonds
- can be relatively easily be disrupted
- e.g. by gentle heating or changes in pH
What is the tertiary structure?
- the alpha-helix or beta-pleated sheet can itself be folded into a three dimensional structure
- this is called the tertiary structure
How is the teritiary structure held in place?
mixture of:
- hydrogen bonding
- ionic interiactions
- disulfide briges
- (van der Waals exist between all molecules
How are proteins determind?
- for secondary and tertiary - X-ray diffraction
- for primary
- find out the number of each type of amino acid present
- reflux with HCl (yina the craic ) hydrolysis and that
- breaks the amide bonds
- results in a mixture containing all the individual acids in the original protein
What is thin-layer chromotography?
- similar to p[aper chromotography but the paper is replaced by a chromotography plate
- consists of a thin, flexible plastic sheet coated with a thin layer of silica (silicon dioxide)
- this white poweder is called the stationary phase
How is TLC began?
- small spot containing the mixture of amino acids to be seperated is placed on a line about 1cm
- plate is placed in a tank with suitable solvent of depth 0.5cm
- the starting line must be above the initial level of the solvent
- the solvent (or mixture) is called the mobile phase or the eluent
What is done with the tank in TLC?
- a lid is placed on the tank so that the inside of it is saturated with solvent vapour
- and the solvent is allowed to rise up the place
- as it does so, it carries the amino acids with it
- each amino acids lages behind the solvent from to an extent that depends on its affinity for the solvent compared with its affinity of the affinity with the stationary phase
- this depends on the intermolecular forces that act between the amino acid and the solvent
- the stronger they are, the closer the amino acid is to the solvent from
What happens when the solvent has almost reached the top of the plate?
- the plate is removed from the tank
- the position to which the solvent front has moved is marked
- amino acids are colourless. so the positions they have reached need to be made visible
How are the amino acids made visible?
- plate is sprayed with a develop[ing agent, such as ninhydring
- this reacts with amino acids to from a purple compound
- or by shing uv light on the plate
- if the solvent is suitable, the amino acids will be completly sparated
How are Rf values calculated
distanced moved by the spot / distance moved by solvent
How are amino acids identified?
comparing Rf values of each spot with the values obtained by known pure amino acids run in the same solvent mixture
What is 2 dimensional TLC?
- plate turned 90
- chromatogram run again with different solvent
- makes it easier to see the separation between spots and gives two Rf values
- if these 2 values match those for a known amino acid you can be more confident in your identification
